SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FROM TARGETS TO THERAPEUTICS
  5. CAV2.2 IN PAIN
  6. CAV1.3 IN PARKINSON'S DISEASE
  7. CAV1.2 IN SCHIZOAFFECTIVE SPECTRUM DISORDERS
  8. CAV2.1: MIGRAINE AND CANCER
  9. CAV3 CHANNELS: SLEEP, PAIN, SEIZURE, AND OBESITY
  10. ANCILLARY SUBUNITS AND PROTEIN–PROTEIN INTERACTIONS
  11. OTHER CAV CHANNELS
  12. OFF-TARGET LIABILITIES
  13. IN VITRO ASSAYS AND PRACTICALITIES
  14. CONCLUSIONS
  15. REFERENCES

There are both opportunities and challenges in developing improved or novel therapeutics based on targeting voltage-gated calcium channels. Calcium channels are technically difficult with respect to target-based drug discovery but unquestionably are key points for influencing human physiology and pathophysiology. Many marketed drugs, including nifedipine, nimodipine, ethosuximide, pregabalin, and ziconotide, exert therapeutic efficacy via inhibition of calcium channels. Although there is no obvious path to improving these existing drugs, recent clinical results with dihydropyridine drugs point to testable strategies for treatment of Parkinson's disease and possibly other CNS disorders by inhibiting L-type calcium channels. Roles of T-type calcium channels in pain and in abnormal sleep are plausible and may soon be tested with clinical trials of novel therapeutics. Success with any of these trials certainly will be followed by intensive efforts for further refinements around the target class. WIREs Membr Transp Signal 2013, 2:85–104. doi: 10.1002/wmts.71

For further resources related to this article, please visit the WIREs website.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FROM TARGETS TO THERAPEUTICS
  5. CAV2.2 IN PAIN
  6. CAV1.3 IN PARKINSON'S DISEASE
  7. CAV1.2 IN SCHIZOAFFECTIVE SPECTRUM DISORDERS
  8. CAV2.1: MIGRAINE AND CANCER
  9. CAV3 CHANNELS: SLEEP, PAIN, SEIZURE, AND OBESITY
  10. ANCILLARY SUBUNITS AND PROTEIN–PROTEIN INTERACTIONS
  11. OTHER CAV CHANNELS
  12. OFF-TARGET LIABILITIES
  13. IN VITRO ASSAYS AND PRACTICALITIES
  14. CONCLUSIONS
  15. REFERENCES

Voltage-gated calcium channels are control points for an unusually large diversity of physiological functions. This reflects the key cellular role of calcium ions in linking electrical and biochemical signaling and the ubiquitous role of intracellular calcium ions in second-messenger pathways. Members of the 10 major calcium channel subtypes (α1 subunits Cav1.1–1.4, Cav2.1–2.3, and Cav3.1–3.3, plus ancillary β, α2δ, and possibly γ subunits1) are involved in critical systems such as cardiac pacing and contractility, neuronal signaling and disorders, and insulin release and metabolic regulation. From a drug discovery point of view, then, this diversity of function makes calcium channels both a significant opportunity and a steep challenge: calcium channels govern functions critical to disease but also govern vital physiological functions that can present safety liabilities if disrupted. That is, any new therapeutic must have some degree of selectivity within the calcium channel family, or else side effects must be tolerable within the overall context of the disease. A surprising number of clinical drugs, some widely used, already target calcium channels, giving proof-of-concept for the target class in general and some guideposts for development of future drugs. The purpose of this review is not to review physiological roles of individual calcium channels but to look at calcium channels as targets for further therapeutic innovation in light of the practicalities of making a drug.

FROM TARGETS TO THERAPEUTICS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FROM TARGETS TO THERAPEUTICS
  5. CAV2.2 IN PAIN
  6. CAV1.3 IN PARKINSON'S DISEASE
  7. CAV1.2 IN SCHIZOAFFECTIVE SPECTRUM DISORDERS
  8. CAV2.1: MIGRAINE AND CANCER
  9. CAV3 CHANNELS: SLEEP, PAIN, SEIZURE, AND OBESITY
  10. ANCILLARY SUBUNITS AND PROTEIN–PROTEIN INTERACTIONS
  11. OTHER CAV CHANNELS
  12. OFF-TARGET LIABILITIES
  13. IN VITRO ASSAYS AND PRACTICALITIES
  14. CONCLUSIONS
  15. REFERENCES

Most published research (not just calcium channel research) aimed at finding drug targets focuses on finding physiological roles and mechanisms of action for individual proteins or genes. Commonly this is done with behavioral pharmacology, particularly by studies of knockout mice, and the gene of interest is then inferred to be a target for pharmacological inhibition. Studies carried out individually by many groups often lead to more proposed targets than likely could all be valuable. For example, probably somewhere on the order of 400 individual genes have been proposed as potential targets for new analgesic drugs to treat pain.2 This large number is not altogether surprising, given that any gene that affects the survival or excitability of any neuron in the pain pathway could reasonably be expected to alter pain transmission when deleted genetically or inhibited pharmacologically. Yet with pain, as with other diseases, it is seldom that published research takes a comparative slant to determine which targets among many proposed are the best. This constitutes a major problem for the drug discovery world. In this respect, the many calcium channel inhibitors with good in vivo properties already available as tool compounds are a great asset for target validation.

Another largely unavoidable concern with target selection in general is translation of animal circuits and behavioral models to human disease. For some indications, again using pain as an example, the translation is reasonable if challenging—animals unquestionably do experience pain, and a number of quantitative endpoints validated with existing clinical drugs are available.3,4 Other diseases, such as diabetes, neuropsychiatric or neurodegenerative disorders, or the exceptionally diverse forms of cancer, present very steep challenges. Here again, existing calcium channel pharmacology gives key information for the target class, in that epidemiological studies of the many calcium channel inhibitors used clinically can be used to predict efficacy or side effects in humans for a novel indication. Some calcium channels have on-target liability, and all calcium channels have class liability that may necessitate selectivity within the calcium channel family for any novel inhibitor. Existing drugs give some guide to the selectivity required for any future drugs.

Despite these uncertainties and difficulties, clearly in some cases calcium channel pharmacology is the key to successful clinical therapeutics. Calcium channel inhibitors are used for a wide variety of indications, consistent with the many cellular roles for calcium channels. Dihydropyridines (DHPs) such as Norvasc® (amlodipine) and Procardia® (nifedipine) lower blood pressure by specific inhibition of Cav1.2, and these are widely used. Prialt® (ziconotide) is effective against multiple forms of pain, via specific inhibition of Cav2.2 N-type channels. Zarontin® (ethosuximide) has efficacy against some forms of epilepsy either wholly or in part by inhibition of Cav3 T-type channels. Lyrica® (pregabalin), a next-generation version of Neurontin® (gabapentin), is first-line therapy for chronic pain, with a label for treatment of fibromyalgia, diabetic nerve pain, and pain after shingles. In addition to these drugs, many nonselective sodium channel inhibitors (e.g., phenytoin and lamotrigine) given for epilepsy (and sometimes off-label for pain) may inhibit calcium channels as part of their clinical profile.

In many cases, a key to therapeutic success of a given compound is the biophysical property of state dependence.5 Physiologically, calcium channel proteins, like all ion channels, alternate among physically distinct conformations—e.g., closed, open, and inactivated. Changes in these states are often accompanied by dramatic changes in drug affinity. Most small-molecule inhibitors of calcium channels bind with highest affinity under conditions that inactivate the channel, and this often is of therapeutic benefit. For example, the state dependence of DHP drugs enables them to inhibit Cav1.2 in smooth muscle, where channels are mostly inactivated, without inhibition of Cav1.2 in working myocardium, where channels are mostly non-inactivated because of the intrinsic electrical properties of the tissue. On the problematic side, state dependence can lead to great uncertainty over whether a given plasma or brain exposure of a strongly state-dependent compound would be expected to actually inhibit the target, as precise gating states of calcium channels in physiological tissues are rarely known.

It is a bit sobering that, Prialt® excepted, all drugs targeting calcium channels were developed without knowledge of the molecular target or in some cases even of the existence of calcium channels, which dates from the 1980s. Whether de novo target-based drug discovery can be done on a calcium channel is, remarkably, an open question. As detailed below, calcium channels in general are quite difficult drug targets from a feasibility standpoint. Their strength as drug targets is that they unquestionably play a role in disease, as shown by existing drugs, by clinical genetics, and by epidemiology. The remainder of this review assesses how new drugs might come from targeting the most-validated calcium channel subtypes (Table 1).

Table 1. Some Roles of Calcium Channel α1 Subtypes in Diseased and Normal Physiological Processes
α1 Subunit GeneClinically Validated IndicationPossible New IndicationPossible On-Target Liability
  1. Clinically validated indications are those treated with current drugs, and possible new indications are those therapeutic areas that are current possibilities for drug discovery. On-target liabilities represent physiological functions controlled by individual α1 subunits that might represent safety or toxicology concerns for inhibitors of each subtype.

Cav1.1  Muscle contractility
Cav1.2Hypertension, angina, neurological damage from subarachnoid hemorrhageParkinson's, schizoaffective disordersBlood pressure, cardiac contractility
Cav1.3 Parkinson'sBradycardia, hearing
Cav1.4  Vision
Cav2.1 Migraine, oncologyMovement/Ataxia
Cav2.2Severe chronic refractory pain Blood pressure
Cav2.3   
Cav3.1 Sleep, obesity, epilepsy 
Cav3.2 Pain control 
Cav3.3 Sleep

CAV2.2 IN PAIN

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FROM TARGETS TO THERAPEUTICS
  5. CAV2.2 IN PAIN
  6. CAV1.3 IN PARKINSON'S DISEASE
  7. CAV1.2 IN SCHIZOAFFECTIVE SPECTRUM DISORDERS
  8. CAV2.1: MIGRAINE AND CANCER
  9. CAV3 CHANNELS: SLEEP, PAIN, SEIZURE, AND OBESITY
  10. ANCILLARY SUBUNITS AND PROTEIN–PROTEIN INTERACTIONS
  11. OTHER CAV CHANNELS
  12. OFF-TARGET LIABILITIES
  13. IN VITRO ASSAYS AND PRACTICALITIES
  14. CONCLUSIONS
  15. REFERENCES

Cav2.2 N-type calcium channels clearly are a key control point in pathways for pain, an area of great unmet medical need. (Cav2.2 is also a potential target for stroke6–9 or seizure,10 but any novel Cav2.2-targeting drugs likely will be developed for pain, a more straightforward clinical and regulatory path.) Evidence is preclinical and clinical. Several strains of Cav2.2 knockout mice profiled by separate laboratories all show large deficits in multiple pain-related behaviors11–14; intrathecal injection of Cav2.2 antagonists produces analgesia in animals15–21; and, most convincingly, Prialt® (ziconotide, synthetic ω-CTx-MVIIA) delivered intrathecally relieves pain in humans. Ziconotide has been found effective in several human pain trials: postsurgical pain, neuropathic pain, non-malignant lower back pain, and failed back surgery syndrome, and in a mixed population of patients with opiate-resistant pain from cancer/chemotherapy and AIDS-related neuropathy.22–25 Efficacy is undoubtedly via inhibition of Cav2.2 in the spinal cord: Cmax and AUC in cerebrospinal fluid (CSF) of clinically effective doses (manufacturer's information) are within the tens of nanomolar-range in vitro affinity of ziconotide for Cav2.2,26–30 and ziconotide is not known to inhibit any other target at even nearly the same affinity. Prialt®, approved in December 2004 and now marketed by Jazz Pharmaceuticals, is delivered intrathecally via continuous delivery from a surgically implanted pump. Its label is for ‘management of severe chronic pain in patients for whom intrathecal therapy is warranted, and who are intolerant of or refractory to other treatment, such as systemic analgesics, adjunctive therapies, or IT morphine’. Additional proposed trials, both from public groups, include one for neuropathic cancer pain (www.clinicaltrials.gov, identifier NCT00996983) and one for bolus instead of continuous administration (www.clinicaltrials.gov, identifier NCT01373983). Given that Prialt® is an FDA-approved therapeutic, this seems to be a very modest level of interest in further development. Despite its impressive efficacy, uptake of Prialt® has been light, with manufacturer-reported revenue of just $6.1 million for the full 2010 year (Elan pharmaceuticals press release). The sparse use despite impressive analgesic efficacy could indicate difficult dosing conditions, undesirable side effects, or still-developing delivery or reimbursement infrastructure. Of the side effects listed, perhaps one of the more worrisome is ‘severe psychiatric symptoms’ and that ‘patients with a pre-existing history of psychosis should not be treated with Prialt®’. Hallucinations were reported in 12% of patients taking Prialt® (all manufacturer's product information).

The obvious next step is an orally available state-dependent small-molecule inhibitor of Cav2.2 that could deliver efficacy comparable to Prialt® without the need for surgery to implant a delivery pump and with easier dose titration. The hope is that hyperexcitable nociceptors producing chronic pain produce an environment in which Cav2.2 transits inactivated states more than in normal tissue, so that a state-dependent Cav2.2 inhibitor would produce efficacy equivalent to ziconotide but with fewer central nervous system (CNS)-mediated side effects. Ziconotide is likely a pore blocker that inhibits Cav2.2 in closed as well as inactivated states.31 Systemic dosing of a small molecule likely would be easier than dosing of ziconotide: pharmacokinetics of i.t. ziconotide are determined by the high turnover rate of the CSF (CSF flow rate 0.4–0.6 mL/min, roughly 4 h for complete turnover32). This necessitates delivery of Prialt® via continuous pump infusion, which can make optimal dose titration difficult and likely precludes clinical trials for stroke or seizure. By contrast, CNS exposures of a small molecule are more likely to be determined by plasma pharmacokinetics, and engineering stable plasma exposures with small molecules is more or less well understood and can be fairly routine (depending on the scaffold).

None of this is new information, and small-molecule inhibitors of Cav2.2 from many companies including AstraZeneca, Ajinomoto, Warner-Lambert, Parke-Davis, SmithKline Beecham, Neurosearch, Vertex, and Neuromed have been published starting from the late 1990s,33–47 with a peak in patent publications around 2000. A wide range of small organic molecules from different scaffolds that inhibit Cav2.2 in vitro have been found, and many of these scaffolds support modifications without loss of potency, crucial for developing a drug from an initial hit. Analgesic efficacy in preclinical models upon oral administration of several small molecules that block Cav2.2 (and other targets) has been reported.48,49 Calcium channels have been shown tractable to uptake assays (e.g., 45Ca uptake into chick synaptosomes34,50), high-throughput fluorescence-based assays,51 and automated electrophysiology,52,53 meaning that in vitro driver assays should pose only routine throughput and precision challenges to developing inhibitors of Cav2.2. And yet despite the importance and feasibility of Cav2.2 as a target, no Cav2.2-specific small-molecule inhibitor has shown efficacy in a clinical trial. The lack of progress in this area despite unmet medical need suggests steep medical or technical challenges to developing a small-molecule inhibitor of Cav2.2. With very rare exceptions, whether from the academic or the industrial world, negative results unfortunately are not published, reported, or commented on. Accordingly, all that is possible is to speculate on some of the potential hurdles.

For a systemically delivered small-molecule Cav2.2 inhibitor, a major possible pitfall is on-target liability, prominently blood pressure. Pharmacological and genetic evidence from several preclinical species shows that Cav2.2 in peripheral sympathetic neurons governs arterial contraction, baroreceptor reflexes, and some cardiac responses.11,54–57 Here apparently the intrathecal delivery route of Prialt® is an advantage: ziconotide likely is cleared from the CSF straight to the lymph, and any ziconotide that reaches the bloodstream is degraded quickly by endogenous peptidases and so does not reach sympathetic neurons. A small-molecule inhibitor delivered p.o., however, would necessarily present itself to these peripheral neurons as well as to spinal cord neurons. A hope is that an inactivation-preferring small-molecule inhibitor would produce analgesia while exerting minimal inhibition of less-inactivated Cav2.2 in non-hyperexcitable sympathetic neurons, but this remains undemonstrated. Just as significant as blood pressure is possible on-target liability from inhibition of Cav2.2 in the brain. It certainly is possible that an inactivation-preferring small molecule Cav2.2 inhibitor could target nociceptors and spare central neurons, but this remains unaddressed by data. The exact in vitro biophysical properties that might produce such functional selectivity are unknown, and CNS readouts such as psychosis, dizziness, and nausea that can be translated to humans are well-nigh impossible to obtain from animals. Accordingly, dosing limits and proof-of-concept for a future Cav2.2 inhibitor likely would have to be set with direct clinical studies, not an optimal situation.

In addition to on-target liabilities, Cav2.2 is fairly close in primary sequence of critical functional areas to channels known to govern key physiological functions, particularly Cav1.2 and Cav2.1 (see below), and it is unknown how much in vitro selectivity is needed to produce an acceptable therapeutic window. To date, several small-molecule inhibitors of Cav2.2 are claimed to have some selectivity over these channels, but by and large these are early and proprietary compounds that have not yet been subject to testing by multiple laboratories using a range of protocols. Most Cav2.2 inhibitors claimed in the patent literature are fairly weak inhibitors of Cav2.2 in vitro, with nowhere near the potency (tens of nanomolar?) probably required for a molecule that could inhibit Cav2.2 without being expected to cause a wide range of off-target effects. Given the initial surge of interest in Cav2.2 shown by the patent literature, one possibility is that potency and selectivity are quite difficult to engineer. The only claimed Cav2.2 inhibitors in clinical trials now are Z-160, a reformulation of NMED-160/MK-6721, and CNV-2197944, both in Phase One trials (Thomson Reuters, February 2012) presumably to test pharmacokinetics and dosing.

CAV1.3 IN PARKINSON'S DISEASE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FROM TARGETS TO THERAPEUTICS
  5. CAV2.2 IN PAIN
  6. CAV1.3 IN PARKINSON'S DISEASE
  7. CAV1.2 IN SCHIZOAFFECTIVE SPECTRUM DISORDERS
  8. CAV2.1: MIGRAINE AND CANCER
  9. CAV3 CHANNELS: SLEEP, PAIN, SEIZURE, AND OBESITY
  10. ANCILLARY SUBUNITS AND PROTEIN–PROTEIN INTERACTIONS
  11. OTHER CAV CHANNELS
  12. OFF-TARGET LIABILITIES
  13. IN VITRO ASSAYS AND PRACTICALITIES
  14. CONCLUSIONS
  15. REFERENCES

Recent work has postulated that in the dopaminergic neurons of the substantia nigra pars compacta (SNc), whose degeneration is the likely causal basis of Parkinson's disease, spontaneous spiking in older neurons is driven by the Cav1.3 calcium channel.58 Repetitive calcium influx through the Cav1.3 calcium channel is then theorized to cause excess calcium entry and neuronal death via high mitochondrial stress on the neurons59 and via increased cytoplasmic dopamine levels.60 Remarkably, in vitro, spontaneous spiking of SNc neurons could be converted from calcium channel-based to sodium channel-based by adding isradipine, a dihydropyridine (DHP) antagonist of both Cav1.2 and Cav1.3 channels.58 The theory makes sense biophysically—Cav1.3 is a low-voltage-activated L-type calcium channel61,62 ideally suited to drive electrical pacemaking in neurons,63–65 as it undoubtedly does in mouse sinoatrial node.66 As a therapeutic strategy, then, this suggests that isradipine could reduce calcium entry and lessen degeneration of SNc neurons while preserving spiking and maintaining the normal release of dopamine from SNc neurons into the striatum.

Epidemiological studies have given this theory a great boost67,68 (but see Ref 69). In particular, a retrospective study of the Danish health registry that included over 10,000 Parkinson's patients showed that those who had taken the L-type blocking DHP amlodipine for hypertension had unchanged risk of Parkinson's, whereas those who had taken other L-type blocking DHPs (including nifedipine, nicardipine, felodipine, and isradipine) for hypertension had as a group a ∼30% lower risk of Parkinson's (2.8% incidence vs 3.8% control incidence).68 This risk reduction was consistent with that of a separate study encompassing a separate population with over 3000 patients.67

Among DHPs, amlodipine is an outlier in that it does not bind to DHP receptors in the brain, as studied in rats and mice with a displacement assay (although it does access the brain in mice, with brain:plasma ratio equal to that of nifedipine).70 The inference is that inhibition of brain DHP receptors protects against Parkinsonian neurodegeneration of SNc neurons, by switching the spontaneous spiking of SNc neurons from calcium-based to sodium-based. The lack of protection with amlodipine shows that the protective effect is not indirectly via lowered blood pressure. The suggested target profile for a novel therapeutic, then, is a CNS-penetrant Cav1.3 inhibitor, possibly with selectivity over Cav1.2, because Cav1.2 governs blood pressure and cardiac contractility. No Cav1.3-selective compound has been reported. This almost certainly is through lack of attempt rather than lack of feasibility; even existing DHPs can have quite different inhibitory effects on Cav1.2 and Cav1.3.61 The 30% reduction in Parkinson's risk from those taking CNS-penetrant DHPs is all the more remarkable, because the different CNS-penetrant DHP drugs all pooled together in epidemiological studies were dosed for target coverage in the vasculature. That is, the protective effect of a dosing regimen or new therapeutic designed to give target coverage in the CNS might actually be a good deal larger than 30%.

There are a number of opportunities for further exploration of this intriguing hypothesis. Clinically, it is unclear why Parkinson's risk was not also lowered among those taking the nondihydropyridine CNS-penetrant L-type blockers diltiazem71 and verapamil.72 This suggests that the precise molecular selectivity and in vivo distribution of diltiazem and verapamil compared to different DHPs might be helpful in understanding a neuroprotective effect of L-type inhibition. Moreover, amlodipine is an outlier among DHPs in multiple ways. In addition to not binding in the brain, it has very high volume of distribution, tissue-specific accumulation, long half-life, and very slow receptor binding and unbinding.73–75 It is possible that one of these parameters contributes to anti-Parkinsonian efficacy (or inefficacy) of a given DHP. Mechanistically, it is somewhat of a mystery whether inhibition of Cav1.2, not Cav1.3, contributes to the neuroprotective effects of CNS-penetrant DHPs. The brain as a whole has far more DHPRs composed of Cav1.2 than Cav1.3,76 and several in vitro studies show that some DHPs inhibit Cav1.3 function only weakly and incompletely.77 In contrast, all DHPs exert strong and complete inhibition of Cav1.2 (when studied with conventional biophysical protocols), suggesting that damaging calcium entry in SNc neurons may be via Cav1.2. Adding to the complexity, given the very strong state dependence of DHP drugs, the exact resting membrane voltage, action potential width, and spiking frequency of the relevant neurons in vivo will affect strongly potency on either Cav1.2 or Cav1.3.58,78–80 Clearly, the clinical anti-Parkinsonian effect of DHPs shows that these drugs are doing something, with L-type channels the only feasible target. Ongoing clinical trials with isradipine should inform greatly future efforts to treat Parkinson's disease via DHP receptors.

As with Cav2.2, there are a number of cautions about clinical use of a putative Cav1.3 inhibitor, including possible on-target liabilities. Mice deficient in Cav1.3 are deaf and have bradycardia, because of missing Cav1.3 in inner-ear hair cells and sinoatrial node,66 and bradycardia via Cav1.3 inhibition has also been reproduced pharmacologically.81 If translatable to humans, hearing loss is a significant hurdle that might or might not be a tolerable side effect of an anti-Parkinson's drug. Note, however, that existing DHP drugs do not cause hearing loss as a side effect, suggesting a therapeutic window for Parkinson's prevention compared to hearing loss, regardless of the molecular mechanisms. Recently, bradycardia has been shown in humans as an effect of polymorphisms in CACNA1D gene encoding Cav1.3.82 This strongly suggests that Cav1.3 inhibition will cause some degree of bradycardia in humans. It is possible that asymptomatic bradycardia could be tolerated clinically as tradeoff for success in treating such a severe disease as Parkinson's—for example, amlodipine is listed as producing bradycardia in less than 1% but greater than 0.1% of patients in controlled or open clinical trials (manufacturer's information). Accordingly, defining safety hurdles is a matter for a direct trial with a particular molecule.

As far as making a new therapy, assuming that Cav1.3-specific inhibition is further validated as treatment for Parkinson's, the hurdle may be high. DHPs are available generically and have decades of safety data, so any novel Cav1.3-targeting drug would have to either provide greater than 30% protection or have sufficient selectivity over Cav1.2 so as to not affect blood pressure. It is more likely that existing DHPs would be reoriented to treat Parkinson's; dose-selection studies with isradipine already have been completed.83 More importantly, a Cav1.3 inhibitor might be more effective in preventing disease than in reversing the disease once established. It is difficult to see how any pharmacological treatment could ameliorate a disease whose hallmark is extensive neuronal death. Marking patients for treatment, then, would require an FDA-accepted measure of a prodromal stage of Parkinson's sufficient to initiate treatment. Interestingly, nicotine also shows a consistent and very strong (∼50%) epidemiological reduction in risk for Parkinson's, and it is possible that L-type inhibitors, whether novel or repurposed, would compete with (or, one would hope, add to) emerging nicotinic therapies.84,85 Whether or not an entirely new therapy for Parkinson's is developed, certainly the hypothesis of L-type channel involvement in Parkinson's soon will be tested clinically, rigorously, and quickly, with doses designed to produce appropriate brain exposures. If efficacy is confirmed, mechanistic efforts will further refine the target profile and may support extension to other neurodegenerative diseases, including Alzheimer's.86

CAV1.2 IN SCHIZOAFFECTIVE SPECTRUM DISORDERS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FROM TARGETS TO THERAPEUTICS
  5. CAV2.2 IN PAIN
  6. CAV1.3 IN PARKINSON'S DISEASE
  7. CAV1.2 IN SCHIZOAFFECTIVE SPECTRUM DISORDERS
  8. CAV2.1: MIGRAINE AND CANCER
  9. CAV3 CHANNELS: SLEEP, PAIN, SEIZURE, AND OBESITY
  10. ANCILLARY SUBUNITS AND PROTEIN–PROTEIN INTERACTIONS
  11. OTHER CAV CHANNELS
  12. OFF-TARGET LIABILITIES
  13. IN VITRO ASSAYS AND PRACTICALITIES
  14. CONCLUSIONS
  15. REFERENCES

Several recent genetic studies point to Cav1.2 as a control point for disorders of the CNS. Notably, genome-wide association screening has found and replicated that polymorphism in the Cav1.2 gene (CACNA1C) is linked to an increased risk of bipolar disorder.87–89 Strikingly, the same locus was found to be associated with depression and with schizophrenia in a separate study,90 highlighting both the importance of CACNA1C in neurological function and the close overlap between different forms of schizoaffective disorders. It seems to be a reasonable guess that Cav1.2 itself, and not another allele in linkage disequilibrium with Cav1.2, is causal for disease, given the density of the genome coverage in these studies. Work undoubtedly is forthcoming to determine whether the tag SNPs or linked sequences are casual for disease and to assess effects on Cav1.2 transcription, splicing, expression, trafficking, and function. Interestingly, recent work suggests involvement of the Cav1.4 (CACNA1F) L-type channel in autism91 and possibly schizophrenia.92 If schizoaffective disorders correlate with increased Cav1.2 expression or function, Cav1.2 may indeed be a possible point for therapeutic intervention with existing drugs that are Cav1.2 inhibitors. Note, however, that the low odds ratios described for the disease-associated polymorphisms may indicate a fairly subtle effect, and that these tag SNPs thus far are in non-coding regions of the channel, making mechanism difficult to decipher. If Cav1.2-related defects ultimately are developmental, pharmacological intervention might be less useful; but given the plasticity within even the adult nervous system93,94 it can be hoped that pharmacological intervention could help established disease. In parallel, it should be possible within existing epidemiological data to determine whether CNS-penetrant Cav1.2 inhibitors (again, using amlodipine and other therapies as controls for blood pressure) affect susceptibility to schizoaffective disorders or can even reverse disease. Pending positive results, the next challenge would be to determine if individuals with schizoaffective disorder but without disease-causing polymorphisms in Cav1.2 could also be treated with Cav1.2-inhibitory drugs—that is, to determine if L-type function is key for idiopathic schizophrenia. For manipulation of Cav1.2, existing drugs are much more likely to be useful than any new class of therapeutic, and DHPs with preferential brain accumulation probably could be engineered, given current knowledge on DHP SAR and on how to engineer CNS penetration and retention.

Another genetic study of note is of the rare monogenic disorder Timothy Syndrome (TS), a developmental disorder causing cardiac, musculoskeletal, and cognitive defects. An elegant study—including the discovery of mosaicism in an affected patient—has shown that overactive Cav1.2 is causal for disease in an autosomal dominant manner.95,96 Disease-causing mutations expressed in vitro form channels with defective voltage-dependent inactivation, presumably leading to uncontrolled and toxic concentrations of calcium ions in the many muscle, neuronal, and cardiac tissues in which Cav1.2 is expressed.97 Clinically, although TS features defects in cognition, it is important to note that TS encompasses cardiac abnormality, poor survival, and other features quite distinct from what is commonly referred to as autism spectrum disorder. It may or may not be justifiable to use TS as evidence for the role of Cav1.2 in common autism spectrum disorders, although note that mice engineered heterozygous for one TS allele do display behavioral defects that could be interpreted in light of human autism.98 In theory, the hypothesis of Cav1.2 involvement in common forms of autism might point toward clinical testing of existing DHPs, but safety concerns might well be prohibitive in administering a regimen of antihypertensives to even adult autism patients. Again, this points to a need for L-type blockers with either preferential selectivity for brain receptors (probably quite difficult, even if given appropriate tissue-specific channel variants) or preferential localization in the brain (possibly achievable).

CAV2.1: MIGRAINE AND CANCER

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FROM TARGETS TO THERAPEUTICS
  5. CAV2.2 IN PAIN
  6. CAV1.3 IN PARKINSON'S DISEASE
  7. CAV1.2 IN SCHIZOAFFECTIVE SPECTRUM DISORDERS
  8. CAV2.1: MIGRAINE AND CANCER
  9. CAV3 CHANNELS: SLEEP, PAIN, SEIZURE, AND OBESITY
  10. ANCILLARY SUBUNITS AND PROTEIN–PROTEIN INTERACTIONS
  11. OTHER CAV CHANNELS
  12. OFF-TARGET LIABILITIES
  13. IN VITRO ASSAYS AND PRACTICALITIES
  14. CONCLUSIONS
  15. REFERENCES

Genetic evidence shows clearly that one of three forms of familial hemiplegic migraine (migraine with aura) is due to overactive Cav2.1, in particular to channels that open in response to weaker depolarizations.99–102 Given the strong, even dominant, role of Cav2.1 in controlling neurotransmitter release and its wide expression in the brain,103,104 it is easy to envision that overactive Cav2.1 would lead to significant and fairly global defects. For this particular genetic form of migraine, a Cav2.1 blocker (if safe) likely would stop migraine, but it is unknown whether Cav2.1 inhibition would control common migraine. Cav2.1 does seem to control cortical spreading depression,105 a feature of some more general non-familial types of migraine, so the possibility for an effective antimigraine therapeutic via inhibition of Cav2.1 is real. Cav2.1 clearly is a major control point for regulation of CNS excitability, shown by the strong dependence on Cav2.1 of synaptic transmission at a variety of synapses, the serious neurological and motor deficits in the Cav2.1 knockout mouse,106,107 and the seizure and ataxia phenotype in mice carrying one of the tottering alleles.108–110 Unfortunately, the widespread functions of Cav2.1 mean that on-target side effects may prohibit development of any therapeutic. One can speculate that even severe on-target side effects might be tolerated in the very short term (hours, perhaps via a therapeutic delivered sublingually) to disrupt the onset or progression of a migraine. Migraines refractory to treatment with triptans can be sufficiently severe that patients may be administered i.v. ketamine (which clears rapidly111), so the precedent is there, but the engineering and toxicology challenges are steep to finding a CNS-penetrant, fast-clearance, selective Cav2.1 inhibitor. Theoretically, a Cav2.1 inhibitor with the correct degree of state dependence could avoid some of the safety liabilities associated with global Cav2.1 inhibition, but state dependence is difficult to engineer quantitatively, and it is unknown how much or what type of state dependence would quiet selectively the neurons mediating migraine. Perhaps a more promising angle is to determine whether specific subsets of neuronal pathways governed by Cav2.1 affect migraine initiation or progression, and to target other receptors controlling pathway excitability. Here, more basic research is needed to find tractable and safe molecular targets within migraine circuitry containing Cav2.1.

An interesting possibility for a Cav2.1 therapeutic comes from Lambert-Eaton myasthenic syndrome (LEMS).112,113 In this syndrome, autoantibodies, mostly against Cav2.1, cause muscle weakness and poor motor coordination because of functional inhibition of Cav2.1, likely at the neuromuscular junction.114–116 Antibody has been isolated from CSF and might inhibit Cav2.1 in cerebellar Purkinje neurons as well, thereby contributing to motor dysfunction.117 Several features are of note here. First, an antibody that binds to and inhibits calcium channels of intact live cells is remarkable proof-of-concept. Antibody therapeutics in general have higher potency and specificity than small molecules, an approach particularly important for a family such as calcium channels in which highly homologous genes govern therapeutic efficacy and undesirable side effects. Engineering inhibitory antibodies to members of the voltage-gated ion channel superfamily, however, has proved difficult. Despite attractive targets and mature technologies, few examples are available of inhibitory antibodies, particularly of monoclonal antibodies, although a polyclonal antibody has been described that inhibits Cav2.1 in vitro.118 Second, LEMS shows that an antibody can penetrate to the synapse in vivo and may also pass the blood–brain barrier (at least under the circumstances of this disease), as antibody has been isolated from CSF.117 Third, LEMS could give a clue toward oncology mechanism or treatment. LEMS is often associated with small-cell lung carcinoma (SCLC). Although the interplay between SCLC and the neurological deficits of LEMS is not yet fully understood, it is possible that anti-Cav2.1 autoantibodies arise not from defective self-tolerance, but from immune recognition of Cav2.1 antigen presented via the neoplasm. Several cases suggest that SCLC patients with LEMS have better prognosis than SCLC patients without LEMS.119,120 It is conceivable, if speculative, that for whatever reason Cav2.1 plays a role in tumor growth, and that an anti-Cav2.1 mAb that inhibits channel function could be a useful therapeutic. For obvious and sad reasons, side effects that would be prohibitive for a drug taken for a neurological indication like migraine might well be tolerated in oncology as a tradeoff for effective antitumor activity.

CAV3 CHANNELS: SLEEP, PAIN, SEIZURE, AND OBESITY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FROM TARGETS TO THERAPEUTICS
  5. CAV2.2 IN PAIN
  6. CAV1.3 IN PARKINSON'S DISEASE
  7. CAV1.2 IN SCHIZOAFFECTIVE SPECTRUM DISORDERS
  8. CAV2.1: MIGRAINE AND CANCER
  9. CAV3 CHANNELS: SLEEP, PAIN, SEIZURE, AND OBESITY
  10. ANCILLARY SUBUNITS AND PROTEIN–PROTEIN INTERACTIONS
  11. OTHER CAV CHANNELS
  12. OFF-TARGET LIABILITIES
  13. IN VITRO ASSAYS AND PRACTICALITIES
  14. CONCLUSIONS
  15. REFERENCES

Cav3 channels (Cav3.1, Cav3.2, and Cav3.3 low-voltage-activated T-type channels) are expressed in nervous tissue, in sinoatrial node in many species, and even in nonexcitable cells.121,122 Somewhat surprisingly, given this wide expression, Cav3 channels appear to be comparatively safe targets for an inhibitor. Mouse knockouts of Cav3.1, Cav3.2, and Cav3.3 all survive to adulthood and can be bred, arguing for overall safety of the target class.123–125 Pharmacologically, Posicor® (mibefradil, a T-type blocker) was used clinically to treat hypertension, before being withdrawn for harmful drug–drug interactions due to inhibition of the CYP3A metabolic enzyme.126–128 Mibefradil is a potent in vitro inhibitor of all three Cav3 channels (as well as sodium channels),129,130 yet Posicor® passed safety and regulatory hurdles, suggesting that no prohibitive safety problems result from pharmacological inhibition of Cav3 channels. Although studies of knockout mice imply that the antihypertensive effect of mibefradil is mediated through Cav1.2 and not Cav3 channels,131 mibefradil is equally or more potent on Cav3 in vitro—suggesting that if it inhibits Cav1.2 in vivo, it likely inhibits Cav3 in vivo, and that the safety profile of Posicor® reflects any adverse events that would result from Cav3 inhibition. Similarly, the safety profiles of drugs including Orap® (pimozide, antipsychotic132,133), Dilantin® (phenytoin, antiepileptic134,135), and DHPs including Nimotop® (nimodipine136) (for review see Ref 121) can be interpreted to suggest no prohibitive liability to inhibition of Cav3 channels, including those in the CNS. These drugs are given clinically and inhibit T-type channels with potency more or less comparable to their presumed targets, suggesting that T-type inhibition is safe. The antiepileptic Zarontin® (ethosuximide) in particular may inform as to the safety of T-type inhibition. The plasma concentrations produced by effective doses137 and the in vitro potency on T-type channels138 are consistent with antiepileptic clinical efficacy arising from inhibition of T-type channels.139–141 If so, the clinical safety of ethosuximide also reflects nonselective inhibition of T-type channels. This argument comes with the considerable caution that it is entirely plausible that low receptor occupancy of T-type channels produces efficacy, whereas higher receptor occupancy might lead to on-target toxicity.

Given clinical pharmacological evidence that inhibition of T-type channels carries no glaring liability, then presumably Cav3.1, Cav3.2, and Cav3.3 each are candidates for target-based drug discovery, although with the considerable caution that little data address the role of T-type channels in humans. Particularly strong evidence points to the role of Cav3.1 channels in generation of thalamocortical spike wave discharges (SWDs) that underlie absence seizures. The Cav3.1 knockout mouse is missing both SWDs and seizures produced by GABAB agonists, and when each of several strains of mutant mice with spontaneous SWDs and absence behaviors (leaner, tottering, and stargazer; each with intact Cav3.1) were crossed to Cav3.1 knockouts, the resulting double mutants were seizure-resistant.140 That is, removal of Cav3.1 function suppresses seizures resulting from many mechanisms, good evidence that Cav3.1 plays a critical role in absence seizures.

Cav3.1-generated thalamocortical oscillations also prominently figure in sleep cycles: oscillations are less prominent during non-rapid eye movement (NREM) sleep, and both global and thalamus-specific Cav3.1 knockouts spend less time in NREM sleep.141,142 It is not entirely clear how Cav3.1 function would promote longer or more restful sleep—for example, Cav3.1 function could either promote sleep, inhibit a transition from sleeping to waking states, or work in additional ways—but Cav3.1 certainly seems to be involved in sleep somehow. Cav3.3, with similar function and with expression in thalamus complementary to Cav3.1, may also be a player in sleep via control of sigma oscillations that occur before entry into REM sleep.125 For validating a role of T-type channels in sleep one place to look is clinical effects of ethosuximide. Unfortunately, however, reported effects of ethosuximide are not even informative as to the broad question of whether T-type inhibition promotes or disrupts sleep. Drug side effects listed on PubMed Health include both ‘drowsiness’ and ‘suddenly awakening from sleep in a frightened state’. In any case, ethosuximide is only given to humans afflicted with epilepsy, so conclusions on the role of T-type channels in normal sleep are doubly difficult to draw. Mibefradil likewise apparently produced no sleep-related side effects that could validate or disprove clinical possibilities for Cav3 antagonism in humans. In sum, certainly Cav3.1 and Cav3.3 seem to play a key role in sleep circuitry preclinically, but the precise effects of a Cav3.1- or Cav3.3-targeted drug on human sleep states, especially the abnormal sleep states that would be seeking pharmaceutical treatment, are difficult to anticipate. Sleep is a straightforward clinical trial; affected patients are easy to find and otherwise healthy; there is every reason that pan-Cav or inhibitors subtype selective Cav3 inhibitors can be found and optimized for human dosing (see below); and available information does not point toward on-target liability for Cav3.1 or Cav3.3. So, we can hope for a direct answer to the role of Cav3 channels or individual Cav3 subtypes in sleep via clinical trials before too much longer. Merck has both extensive chemical matter on Cav3 channels and interest in sleep disorders (company web site), but information on progression of targeted T-type therapies is not public.

One remarkable and new possibility for a Cav3.1 inhibitor is the treatment of metabolic disorders, particularly obesity. Mice deficient in Cav3.1 were resistant to diet-induced weight gain, and obese mice treated pharmacologically with fairly low doses of either of two novel pan-T-type inhibitors selective against other calcium channels lost weight and improved lean muscle composition.143 This observation is all the more convincing, as the Merck group reporting the results may have noticed the effects of T-type inhibition on weight and body mass type in pursuit of other indications for Cav3. Again, one can hope for additional evidence to emerge in coming years, given the significant patent activity from a leading company in this space.

Among T-type channels, Cav3.2 has also been suggested as a drug target for pain, with the hypothesis that a Cav3.2-specific inhibitor will inhibit the excitability of peripheral nociceptors. Cav3.2 is the major T-type channel expressed in the cell bodies of peripheral sensory neurons within dorsal root ganglia,144 and published pharmacological,145–147 antisense,144,148 and knockout149 work in rodent models of pain suggest that inhibition of Cav3.2 reduces pain. It remains to be addressed to what extent Cav3.2 expression is in nociceptors, as opposed to non-nociceptive sensory neurons within the dorsal root ganglia. Several studies show that Cav3.2 is expressed in a subpopulation cell bodies from neurons specialized to detect not pain but light touch stimuli.150,151 A conjecture uniting the two hypotheses is that in a neuropathic pain case these mechanosensitive neurons conduct painful signals due to sensitization of the spinal cord or to changes in gene expression. Also as yet unaddressed is whether specific inhibition of Cav3.2 could override spiking driven by the strong and diverse sodium channel expression in these neurons, particularly axonal spiking. T-type channels tend not to couple directly to neurotransmitter release, so it seems most likely that a T-type inhibitor would have to reduce spiking in the entire neuron to lessen pain. It is a bit troubling when thinking of Cav3.2 as a target for pain that ethosuximide is not used clinically to treat pain, although a study is underway testing ethosuximide as a prophylactic for migraine (www.clinicaltrials.gov, identifier NCT001122381). Note also the evidence for involvement of Cav3.1 in pain circuitry: the Cav3.1 knockout is more sensitive to pain in the acid-induced writhing test, possibly via removal of sensory inhibition normally provided by thalamocortical oscillations152 or by control of descending inhibition.153,154

As with Cav2.2, the role of Cav3.2 in pain has been suggested for nearing 10 years, fluorescence-based and electrophysiology-based driver assays are available, chemical matter abounds, and pain has been one of the strongest areas for pharmaceutical investment. Unfortunately, however, no Cav3.2-selective inhibitors have been reported. Has Cav3.2 been underinvestigated? Or have unpublished investigations found minimal efficacy, or the target proven intractable due to on-target safety liability or to lack of chemical matter? As mentioned above, with appropriate caveats, pan-T-type inhibitors may be safe, and if this is the case then a Cav3.2 inhibitor should also be safe as far as the target goes. Chemical intractability of Cav3.2 also seems unlikely, as T-type channels are inhibited in the low micromolar or better range by molecules representing an unusually diverse number of chemical scaffolds,155 including mibefradil, phenytoin, clozapine, pimozide, bepridil, fluspirilene, zonisamide, valproate, 3- and 4-fluoro-piperidines, pyridyl amides, 1,3-dioxoisoindoles, and quinazolines. Many of these inhibitors are quite low-molecular-weight molecules with good human pharmacokinetic properties and CNS penetrance that potentially could serve as excellent starting points for optimization. Moreover, Cav3.2 is already known to be pharmacologically distinct from Cav3.1 and Cav3.3, because of an external metal ion-binding site that governs inhibition by nickel or zinc ions.156 As described above, a pan-T-type inhibitor that does not access the CNS might well serve the same purpose as a truly Cav3.2-selective molecule. Again, the most active prospect based on patent literature and in vivo proof-of-concept molecules seems to be Merck, with extensive and recent patent publications (inter alia157–161). One hopes that following clinical success for one indication, the role of T-type channels and Cav3 subtypes as targets for treatment of many neurological disorders can be explored.

ANCILLARY SUBUNITS AND PROTEIN–PROTEIN INTERACTIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FROM TARGETS TO THERAPEUTICS
  5. CAV2.2 IN PAIN
  6. CAV1.3 IN PARKINSON'S DISEASE
  7. CAV1.2 IN SCHIZOAFFECTIVE SPECTRUM DISORDERS
  8. CAV2.1: MIGRAINE AND CANCER
  9. CAV3 CHANNELS: SLEEP, PAIN, SEIZURE, AND OBESITY
  10. ANCILLARY SUBUNITS AND PROTEIN–PROTEIN INTERACTIONS
  11. OTHER CAV CHANNELS
  12. OFF-TARGET LIABILITIES
  13. IN VITRO ASSAYS AND PRACTICALITIES
  14. CONCLUSIONS
  15. REFERENCES

The undoubted success story of calcium channel ancillary subunits for clinical therapeutics is Neurontin® (gabapentin) and its next generation, Lyrica® (pregabalin). Neurontin® is indicated ‘for the management of postherpetic neuralgia in adults’ and ‘as adjunctive therapy in the treatment of partial seizures in patients over 12 years of age with epilepsy’. The mechanistic action of gabapentin and its newer congener pregabalin, both structurally close to GABA, long was thought to be within the GABAergic system, e.g., through GABAB receptor agonism or inhibition of GABA uptake. Recent work, however, has shown unequivocally that gabapentin and pregabalin interact with calcium channels. Gabapentin binds reasonably tightly to calcium channel subunits α2δ1 and α2δ2162–165; pregabalin loses its analgesic effect in transgenic mice with α2δ1 subunits that do not bind pregabalin166; and therapeutic concentrations of gabapentin lower functional calcium channel expression and depress synaptic transmission in the spinal cord167,168 (for review see Refs 169 and 170). It seems most likely that in binding to α2δ subunits, gabapentin and pregabalin reduce trafficking of correctly folded calcium channels to the membrane, thereby inhibiting excitation and reducing pain or seizures. Another parallel possibility is that gabapentin binding to α2δ1 disrupts direct protein–protein interaction between α2δ1 and thrombospondin and so inhibits formation of new excitatory synapses.171

The clinical safety of gabapentin is notable. The α2δ1 and α2δ2 subunits are widespread and interact promiscuously with all calcium channel α1 subunits in vitro, not just with the Cav2.2 α1 subunit that is unequivocally linked to pain.172 So whether gabapentin blocks synapse formation, reduces neurotransmitter release, or has these plus additional effects,169 one might expect widespread CNS disturbances from gabapentin. And indeed, BOLD-fMRI studies in human subjects show that gabapentin depresses background network activity in the brain,173 an action that may be linked to therapeutic efficacy and/or to side effects (or to neither). Yet, gabapentin has sufficient safety to be used as first-line therapy. Pregabalin has the same potency as gabapentin in vitro,174 but it reaches peak exposure more quickly and, crucially, has linear plasma exposure as a function of oral dose.175 This is a great advantage for a clinical drug, because it eases dose escalation or de-escalation as needed. Lyrica® is approved for fibromyalgia, painful diabetic neuropathy, and postherpetic neuralgia. The side effects of Lyrica® are generally the same as Neurontin®. Assuming the drugs decrease calcium channel expression and/or inhibit synapse formation, it is quite plausible that on-target action would produce the noted common side effects of dizziness, nausea, and somnolence. (On the other hand, pregabalin and gabapentin both are dosed to fairly high exposures, and high CNS exposures of any drug might well lead to these side effects.)

Can the mechanistic knowledge of α2δ1 subunit binding serve as the basis for a next generation of therapeutics with either improved efficacy, improved therapeutic window, or fewer side effects? Pregabalin and gabapentin already are widely used, suggesting good safety and efficacy, and thus a higher bar for a next-generation compound. Receptor occupancy is unknown; that is, it is not formally known whether a novel α2δ binder could be dosed higher, inhibit a higher fraction of α2δ, and so give greater efficacy. In clinical trials, however, efficacy saturates with dose/exposure, suggesting that maximal benefit through α2δ occupancy has been reached. It would be difficult to assess preclinically whether a new α2δ binder was superior to pregabalin, because it is not known at what synapses pregabalin works. A final caution to efforts for an improved drug via the α2δ pathway is that the listed CNS side effects of gabapentin and pregabalin—e.g., dizziness, somnolence, sedation, and nausea—are extremely difficult to track with preclinical species. Again, this means that it would be difficult to compare efficacy and therapeutic window of a new compound to pregabalin without direct comparative phase II clinical trials. One speculation for improvement is that a molecule that binds to α2δ1 without binding to α2δ2 (pregabalin and gabapentin bind to both) might retain efficacy while reducing some side effects. Although appealing, this is not directly addressed by data. However, the severe neurological phenotype of the ducky mouse, which carries a truncation mutation in the α2δ2 gene,176 suggests liability to α2δ2 inhibition, and that an α2δ1-selective ligand could have an improved therapeutic window. Another intriguing possibility is that inhibitors of function of the Cavα2δ3 gene might be novel analgesics with strong effects. This gene has been shown to govern pain in Drosophila and mice, and polymorphisms in the human gene correlate with reduced experimental and chronic pain.177

OTHER CAV CHANNELS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FROM TARGETS TO THERAPEUTICS
  5. CAV2.2 IN PAIN
  6. CAV1.3 IN PARKINSON'S DISEASE
  7. CAV1.2 IN SCHIZOAFFECTIVE SPECTRUM DISORDERS
  8. CAV2.1: MIGRAINE AND CANCER
  9. CAV3 CHANNELS: SLEEP, PAIN, SEIZURE, AND OBESITY
  10. ANCILLARY SUBUNITS AND PROTEIN–PROTEIN INTERACTIONS
  11. OTHER CAV CHANNELS
  12. OFF-TARGET LIABILITIES
  13. IN VITRO ASSAYS AND PRACTICALITIES
  14. CONCLUSIONS
  15. REFERENCES

Individual calcium channels in addition to those explored in detail here are known to govern important physiological roles and could be targets for therapeutic intervention. One possibility is Cav2.3 in insulin release and metabolic disorders.178,179 Calcium channel γ subunits could be a target for pain and other neuronal disorders via control of AMPA receptor trafficking,180–183 and some preliminary evidence associates Cav3 channels with cell growth.184 The Cav1.4 α1 subunit operates in the vision system, as originally discovered from study of rare genetic night blindness syndromes,185–188 and Cav1.4 is also expressed in T-cells, meaning that its manipulation could affect inflammatory and immune responses.189 Calcium channel β subunits may offer a way to manipulate indirectly expression levels of the α1 subunit with small molecules that would influence the protein–protein interaction between the α1 and the β subunits, but molecules have not been available to test the hypothesis. The most-often cited company specializing in accessory proteins to calcium channels, Lectus Therapeutics, was acquired by UCB in November 2011 (UCB press release). These mechanisms are still emerging and are not dealt with further here with respect to drug discovery.

OFF-TARGET LIABILITIES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FROM TARGETS TO THERAPEUTICS
  5. CAV2.2 IN PAIN
  6. CAV1.3 IN PARKINSON'S DISEASE
  7. CAV1.2 IN SCHIZOAFFECTIVE SPECTRUM DISORDERS
  8. CAV2.1: MIGRAINE AND CANCER
  9. CAV3 CHANNELS: SLEEP, PAIN, SEIZURE, AND OBESITY
  10. ANCILLARY SUBUNITS AND PROTEIN–PROTEIN INTERACTIONS
  11. OTHER CAV CHANNELS
  12. OFF-TARGET LIABILITIES
  13. IN VITRO ASSAYS AND PRACTICALITIES
  14. CONCLUSIONS
  15. REFERENCES

Any calcium channel target can reasonably be depicted as one with a class liability—that is, a target closely related to genes governing crucial physiological processes that could prove a toxicology liability (Table 1). Cav1.2 inhibition causes a loss in blood pressure in hypertensive patients, and so any experimental therapeutic calcium channel inhibitor may need selectivity against Cav1.2 else target a patient population for which blood pressure reduction can be dealt with clinically. Given the target validation for other diseases above, particularly the role of anti-Cav2.1 antibodies in producing LEMS, it seems clear that Cav2.1 inhibition carries risk of movement disorders and for seizure. As described, Cav1.3 inhibition may produce bradycardia and hearing loss. Expression patterns and clinical genetic data suggest that Cav1.4 may play a role in vision, particularly in low-light vision, although whether Cav1.4 inhibition would produce significant vision defects is unknown. Preclinical evidence from rat, rabbit, and mouse all show that inhibition of Cav2.2 in peripheral neurons will also likely alter blood pressure through impaired baroreceptor reflexes, and the clinical experience with Prialt® suggests that inhibition of Cav2.2 in the brain will produce similar side effects. Moreover, in many cases—e.g., phenytoin, lamotrigine, and mibefradil—calcium channel inhibitors are also broad-spectrum sodium channel inhibitors, suggesting that future experimental calcium channel inhibitors may need to be optimized against sodium channels of heart, brain, or skeletal muscle190 as well as hERG and other cardiac channels.191

Nevertheless, the picture is not entirely grim. Side-effect profiles are always a tradeoff against efficacy, and it is possible that molecule- or scaffold-specific parameters such as state dependence, binding kinetics, or in vivo tissue distribution may negate concerns about subtype selectivity. How much in vitro selectivity over an off-target calcium channel is needed to be reasonably confident of in vivo safety? The question is difficult to answer. For most compounds, inhibitory potency depends strongly on the gating state of the channel, and physiological contributions from resting, inactivated, and other gating states in precise tissues are unknown. Receptor occupancy necessary to produce toxicity is also unknown. As discussed above, existing L-type and T-type inhibitors give some guide to the selectivities that can be tolerated in a safe drug. (Ziconotide is not much guide to in vitro selectivity hurdles as it is so selective, approximately 10,000-fold selective for Cav2.2.28) In the case of Cav1.2 at least, in vitro selectivity measurements probably should be made on partially inactivated channels to reflect the situation in smooth muscle.192 That being said, the determination of therapeutic window for dose selection in humans is not in vitro selectivity but acute and extended preclinical toxicology studies, with comparison of the exposures producing efficacy to the exposures marking the no-observed-adverse-effects level. It may be considered somewhat of a surprise that existing drugs are as safe as they are: for example, DHPs and verapamil are not reported to cause significant cognitive liabilities despite the widespread and strong expression of Cav1.2 in the brain, nor metabolic liabilities despite the dependence of insulin release on Cav1.2.193,194 Similarly, Lyrica® has a clinically useful therapeutic window despite binding to α2δ subunits that interact promiscuously with all high-voltage-activated calcium channels. For de novo drug discovery against calcium channels—for example, small-molecule inhibitors of Cav1.3 or Cav2.2—probably the most important factor is to start with highly potent and selective chemical matter and work backward to define in vitro selectivity hurdles following measurements of in vivo efficacy and dosing limits.

IN VITRO ASSAYS AND PRACTICALITIES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FROM TARGETS TO THERAPEUTICS
  5. CAV2.2 IN PAIN
  6. CAV1.3 IN PARKINSON'S DISEASE
  7. CAV1.2 IN SCHIZOAFFECTIVE SPECTRUM DISORDERS
  8. CAV2.1: MIGRAINE AND CANCER
  9. CAV3 CHANNELS: SLEEP, PAIN, SEIZURE, AND OBESITY
  10. ANCILLARY SUBUNITS AND PROTEIN–PROTEIN INTERACTIONS
  11. OTHER CAV CHANNELS
  12. OFF-TARGET LIABILITIES
  13. IN VITRO ASSAYS AND PRACTICALITIES
  14. CONCLUSIONS
  15. REFERENCES

Feasibility as well as target validation influence drug discovery, and calcium channels are difficult targets in terms of having assays with the necessary precision, reproducibility, throughput, and physiological relevance to screen libraries or to carry out lead optimization. High-voltage-activated calcium channels are difficult to express and require coexpression of ancillary β and α2δ subunits in addition to the α1 subunit. Few cell lines are commercially available, particularly those expressing human rather than rat or rabbit clones, and native tissues or cell lines that might be a rich and uniform source of calcium channels generally are not available (possibly excepting smooth muscle tissue as a source of Cav1.2).

A variety of in vitro assays have been used for individual calcium channel subtypes (Table 2), none of them perfect. As with all assays, data content generally is inversely proportional to throughput. Ligand-binding displacement assays have high throughput, fidelity, and reproducibility, measure directly ligand–receptor interactions, and for Cav1.2 are available routinely via contract research organizations. Binding assays, however, measure ligand interaction with inactivated channels as membrane preparations do not preserve the electric field across the cell membrane. So, while sufficient for tracking structure–activity relations within a series, these assays are more difficult to correlate with activity that would produce in vivo efficacy or toxicology. More importantly, binding assays only measure drug interaction at a single predefined receptor site so may be subject to false negatives. Finally, good small-molecule ligands are not available for channels other than Cav1.2. Planar patch-clamp electrophysiology assays can be used on any calcium channel expressed at sufficient levels. These assays also measure receptor–ligand interactions directly and have the great advantage of allowing precise control of the voltage protocol and channel gating state. Electrophysiology-based assays, however, have medium to very low throughput and require significant capital and consumable resource investment. Biologically, they can be confounded by channel rundown or inactivation, which are easy to confuse with slow inhibition by a test compound. Fluorescence-based live-cell assays are adaptable to 96-well or 384-well plate-based formats and have sufficiently high throughput to screen hundreds of thousands of compounds. Disadvantages are that the ultimate readout is an indirect reporter of ligand–receptor interaction, so subject to false positives, and the lack of direct control over channel gating states. Depolarization-induced uptake of radiolabeled calcium can be used, particularly with chick synaptosomes, which express a nearly uniform population of Cav2.2. This assay offers high reproducibility and throughput, but does not allow control of channel gating state and is not available for other subtypes. Assays for ancillary subunits are quite specialized, and would need to reflect the ability of a small molecule to disrupt either downstream function of an ancillary subunit (e.g., calcium channel expression) or to disrupt protein–protein interaction with the α1 subunit. (As noted, gabapentin was found before its mechanism of action was known.)

Table 2. Common In Vitro Driver Assays for Optimizing Molecules That Inhibit Voltage-Gated Calcium Channels
Calcium Channel AssayAvailable for Many SubtypesThroughputPrecisionPhysiological
  1. ‘Availability’ refers to whether an assay of a given format is available for all individual calcium channel subtypes; ‘throughput’ to adaptability to many tens of thousands of compounds; ‘precision’ to how well an assay reflects direct receptor–ligand interactions; and ‘physiological’ to how well an assay reflects the actual physiological voltage and cellular milieu of the channel, which can greatly influence pharmacology.

Binding++++
45Ca uptake+++
Electrophysiology (automated)+++++++
Cell-based fluorescence++++++

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FROM TARGETS TO THERAPEUTICS
  5. CAV2.2 IN PAIN
  6. CAV1.3 IN PARKINSON'S DISEASE
  7. CAV1.2 IN SCHIZOAFFECTIVE SPECTRUM DISORDERS
  8. CAV2.1: MIGRAINE AND CANCER
  9. CAV3 CHANNELS: SLEEP, PAIN, SEIZURE, AND OBESITY
  10. ANCILLARY SUBUNITS AND PROTEIN–PROTEIN INTERACTIONS
  11. OTHER CAV CHANNELS
  12. OFF-TARGET LIABILITIES
  13. IN VITRO ASSAYS AND PRACTICALITIES
  14. CONCLUSIONS
  15. REFERENCES

The promise of new therapeutics targeting voltage-gated calcium channels generally has not been realized despite efforts beginning in some cases over a decade ago, and there is no obvious path to improving the existing drugs specific for calcium channels: pregabalin, ziconotide, and DHPs. Calcium channels hold the promise of being undoubted key players in many pathophysiological processes, and this is crucial information for an industry in which clinical failures due to the lack of efficacy are of great concern. However, the vital roles calcium channels play in the nervous, cardiovascular, and endocrine systems may necessitate that future drugs have selectivity among individual subtypes. This apparently has proven difficult to engineer for small-molecule calcium channel inhibitors, with the exception of DHPs, which were developed with ex vivo assays and not with target-based assays. Calcium channels are hard to express and overexpress, lack high-resolution crystal structures to aid the medicinal chemist in designing inhibitors, and are best suited to low-throughput electrophysiology-based assays that require specialized technical skill to carry out and interpret. On the brighter side, T-type calcium channels seem to be chemically tractable and almost surprisingly safe as drug targets, and we can hope soon for clinical trials addressing directly the roles of these channels in sleep, pain, and possibly epilepsy. Likewise, existing DHP drugs are safe and may see new application in Parkinson's disease. Other indications may emerge, as work continues to profile the roles of individual calcium channels in genetic disorders and to explore the role of calcium channels in the immune system.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. FROM TARGETS TO THERAPEUTICS
  5. CAV2.2 IN PAIN
  6. CAV1.3 IN PARKINSON'S DISEASE
  7. CAV1.2 IN SCHIZOAFFECTIVE SPECTRUM DISORDERS
  8. CAV2.1: MIGRAINE AND CANCER
  9. CAV3 CHANNELS: SLEEP, PAIN, SEIZURE, AND OBESITY
  10. ANCILLARY SUBUNITS AND PROTEIN–PROTEIN INTERACTIONS
  11. OTHER CAV CHANNELS
  12. OFF-TARGET LIABILITIES
  13. IN VITRO ASSAYS AND PRACTICALITIES
  14. CONCLUSIONS
  15. REFERENCES
  • 1
    Catterall WA. Voltage-gated calcium channels. Cold Spring Harb Perspect Biol 2011, 3:a003947.
  • 2
    Mogil J. What is the reason for lack of translation in the pain field? Pain Research Forum. 2011 Available at: http://www.painresearchforum.org/forums/discussion/4561-what-reason-lack-translation-pain-field. (Accessed May 2012).
  • 3
    Mogil JS. Animal models of pain: progress and challenges. Nat Rev Neurosci 2009, 10:283294.
  • 4
    Mogil JS, Davis KD, Derbyshire SW. The necessity of animal models in pain research. Pain 2010, 151:1217.
  • 5
    McDonough SI, Bean BP. Voltage-gated ion channels as drug targets. In: Triggle DJ, Gopalakrishnan M, Rampe D, Zheng W, eds. Voltage-Gated Ion Channels as Drug Targets. Weinheim: Wiley; 2006, 1936.
  • 6
    Valentino K, Newcomb R, Gadbois T, Singh T, Bowersox S, Bitner S, Justice A, Yamashiro D, Hoffman BB, Ciaranello R. A selective N-type calcium channel antagonist protects against neuronal loss after global cerebral ischemia. Proc Natl Acad Sci U S A 1993, 90:78947897.
  • 7
    Buchan AM, Gertler SZ, Li H, Xue D, Huang ZG, Chaundy KE, Barnes K, Lesiuk HJ. A selective N-type Ca(2+)-channel blocker prevents CA1 injury 24 h following severe forebrain ischemia and reduces infarction following focal ischemia. J Cereb Blood Flow Metab 1994, 14:903910.
  • 8
    Yenari MA, Palmer JT, Sun GH, de Crespigny A, Mosely ME, Steinberg GK. Time-course and treatment response with SNX-111, an N-type calcium channel blocker, in a rodent model of focal cerebral ischemia using diffusion-weighted MRI. Brain Res 1996, 739:3645.
  • 9
    Colbourne F, Li H, Buchan AM, Clemens JA. Continuing postischemic neuronal death in CA1: influence of ischemia duration and cytoprotective doses of NBQX and SNX-111 in rats. Stroke 1999, 30:662668.
  • 10
    Gasior M, White NA, Rogawski MA. Prolonged attenuation of amygdala-kindled seizure measures in rats by convection-enhanced delivery of the N-type calcium channel antagonists omega-conotoxin GVIA and omega-conotoxin MVIIA. J Pharmacol Exp Ther 2007, 323:458468.
  • 11
    Ino M, Yoshinaga T, Wakamori M, Miyamoto N, Takahashi E, Sonoda J, Kagaya T, Oki T, Nagasu T, Nishizawa Y, et al. Functional disorders of the sympathetic nervous system in mice lacking the α 1B subunit (Cav 2.2) of N-type calcium channels. Proc Natl Acad Sci U S A 2001, 98:53235328.
  • 12
    Kim C, Jun K, Lee T, Kim SS, McEnery MW, Chin H, Kim H-L, Park JM, Kim DK, Jung SJ, et al. Altered nociceptive response in mice deficient in the α(1B) subunit of the voltage-dependent calcium channel. Mol Cell Neurosci 2001, 18:235245.
  • 13
    Saegusa H, Kurihara T, Zong S, Kazuno A, Matsuda Y, Nonaka T, Han W, Toriyama H, Tanabe T. Suppression of inflammatory and neuropathic pain symptoms in mice lacking the N-type Ca2+ channel. EMBO J 2001, 20:23492356.
  • 14
    Hatakeyama S, Wakamori M, Ino M, Miyamoto N, Takahashi E, Yoahinaga T, Sawada K, Imoto K, Tanaka I, Yoshizawa T, et al. Differential nociceptive responses in mice lacking the α(1B) subunit of N-type Ca(2+) channels. Neuroreport 2001, 12:24232427.
  • 15
    Wang YX, Gao D, Pettus M, Phillips C, Bowersox SS. Interactions of intrathecally administered ziconotide, a selective blocker of neuronal N-type voltage-sensitive calcium channels, with morphine on nociception in rats. Pain 2000, 84:271281.
  • 16
    Bowersox SS, Gadbois T, Singh T, Pettus M, Wang YX, Luther RR. Selective N-type neuronal voltage-sensitive calcium channel blocker, SNX-111, produces spinal antinociception in rat models of acute, persistent and neuropathic pain. J Pharmacol Exp Ther 1996, 279:12431249.
  • 17
    Malmberg AB, Yaksh TL. Effect of continuous intrathecal infusion of omega-conopeptides, N-type calcium-channel blockers, on behavior and antinociception in the formalin and hot-plate tests in rats. Pain 1995, 60:8390.
  • 18
    Malmberg AB, Yaksh TL. Voltage-sensitive calcium channels in spinal nociceptive processing: blockade of N- and P-type channels inhibits formalin-induced nociception. J Neurosci 1994, 14:48824890.
  • 19
    Scott DA, Wright CE, Angus JA. Actions of intrathecal omega-conotoxins CVID, GVIA, MVIIA, and morphine in acute and neuropathic pain in the rat. Eur J Pharmacol 2002, 451:279286.
  • 20
    Sluka KA. Blockade of N- and P/Q-type calcium channels reduces the secondary heat hyperalgesia induced by acute inflammation. J Pharmacol Exp Ther 1998, 287:232237.
  • 21
    Chaplan SR, Pogrel JW, Yaksh TL. Role of voltage-dependent calcium channel subtypes in experimental tactile allodynia. J Pharmacol Exp Ther 1994, 269:11171123.
  • 22
    Atanassoff PG, Hartmannsgruber MW, Thrasher J, Wermeling D, Longton W, Gaeta R, Singh T, Mayo M, McGuire D, Luther RR. Ziconotide, a new N-type calcium channel blocker, administered intrathecally for acute postoperative pain. Reg Anesth Pain Med 2000, 25:274278.
  • 23
    Taqi D, Gunyea I, Bhakta B, Movva V, Ward S, Jenson M, Royal M. Intrathecal ziconotide effect on neuropathic symptoms and potential for adjunctive use with opiates: a retrospective review in 25 patients. Pain Med 2002, 3:180181.
  • 24
    Staats PS, Yearwood T, Charapata SG, Presley RW, Wallace MS, Byas-Smith M, Fisher R, Bryce DA, Mangieri EA, Luther RR, et al. Intrathecal ziconotide in the treatment of refractory pain in patients with cancer or AIDS: a randomized controlled trial. JAMA 2004, 291:6370.
  • 25
    Truman C, Barker T, Singh T. American Society of Pain Management Nurses' Annual Meeting, Houston, TX, 2001
  • 26
    Olivera BM, Cruz LJ, de Santos V, LeCheminant GW, Griffin D, Zeikus R, McIntosh JM, Galyean R, Varga J, Gray WR, et al. Neuronal calcium channel antagonists. Discrimination between calcium channel subtypes using omega-conotoxin from Conus magus venom. Biochemistry 1987, 26:20862090.
  • 27
    Wang YX, Bezprozvannaya S, Bowersox SS, Nadasdi L, Miljanich G, Mezo G, Silva D, Tarczy-Ho R. Peripheral versus central potencies of N-type voltage-sensitive calcium channel blockers. Naunyn Schmiedebergs Arch Pharmacol 1998, 357:159168.
  • 28
    Lewis RJ, Nielsen KJ, Craik DJ, Loughnan ML, Adams DA, Sharpe IA, Luchian T, Adams DJ, Bond T, Thomas L, et al. Novel omega-conotoxins from Conus catus discriminate among neuronal calcium channel subtypes. J Biol Chem 2000, 275:3533535344.
  • 29
    Sanger GJ, Ellis ES, Harries MH, Tilford NS, Wardle KA, Benham CD. Rank-order inhibition by omega-conotoxins in human and animal autonomic nerve preparations. Eur J Pharmacol 2000, 388:8995.
  • 30
    Kaneko S, Cooper CB, Nishioka N, Yamasaki H, Suzuki A, Jarvis SE, Akaike A, Satoh M, Zamponi GW. Identification and characterization of novel human Ca(v)2.2 (α 1B) calcium channel variants lacking the synaptic protein interaction site. J Neurosci 2002, 22:8292.
  • 31
    McDonough SI. Peptide inhibition of voltage-gated calcium channels: selectivity and mechanisms. In: McDonough SI, ed. Calcium Channel Pharmacology. New York: Kluwer Academic/Plenum; 2004, 95142.
  • 32
    Johanson CE, Duncan JA 3rd, Klinge PM, Brinker T, Stopa EG, Silverberg GD. Multiplicity of cerebrospinal fluid functions: new challenges in health and disease. Cerebrospinal Fluid Res 2008, 5:10.
  • 33
    Schelkun RM, Yuen P, Malone TC, Rock DM, Stoehr S, Szoke B, Tarczy-Hornoch K. Synthesis and biological activity of substituted bis-(4-hydroxyphenyl)methanes as N-type calcium channel blockers. Bioorg Med Chem Lett 1999, 9:24472452.
  • 34
    Axelsson O, Peters D, Ostergaard NE, Christophersen P. Piperidine compounds as calcium channel blockers. US Patent 5,981,539, 1999
  • 35
    Hu LY, Rafferty MF, Ryder TR. Aniline derivatives as calcium channel blockers. US Patent 6,251,918 B1, 2001
  • 36
    Hu LY, Rafferty MF, Ryder TR, Sercel AD, Song Y. Heteroaryl alkyl α substituted peptidylamine calcium channel blockers. US Patent 6,166,052, 2000
  • 37
    Hu LY, Malone TC, Nadasdi L, Rafferty MF, Ryder TR, Silva DF, Song Y, Szoke BG, Urge L. Substituted peptidylamine calcium channel blockers. US Patent 6,117,841, 2000
  • 38
    Hu LY, Rafferty MF, Ryder TR. Heterocyclic substituted aniline calcium channel blockers. US Patent 6,251,919 B1, 2001
  • 39
    Malone TC, Schelkun RM, Yuen P-W. Substituted phenols as novel calcium channel blockers. US Patent 6,124,280, 2000
  • 40
    Rafferty MF, Song Y. Tyrosine-derived compounds as calcium channel antagonists. US Patent 6,180,677 B1, 2001
  • 41
    Snutch TP, Zamponi GW. Calcium channel blockers. US Patent 6,011,035, 2000
  • 42
    Gonzales JEI, Wilson DM, Termin AP, Grootenhuis PDJ, Zhang Y, Petzoldt BJ, Fanning LTD, Neubert TD, Tung RD, Martinborough E, et al. Quinazolines useful as modulators of ion channels. WO 2004/078733 A1, 2004
  • 43
    Yuen P-W. Substituted quinolines and isoquinolines as calcium channel blockers, their preparation and the use thereof. US Patent 5,767,129, 1998
  • 44
    Zamponi GW. Farnesol-related calcium channel blockers. US Patent 6,267,945 B1, 2001
  • 45
    Harling JD, Orlek BS. Indane and tetrahydronaphthalene derivatives as calcium channel antagonists. US Patent 5,773,463, 1998
  • 46
    Chapdelaine M, Kemp L, McCauley J. N-type calcium channel antagonists for the treatment of pain. Patent WO 02/36567 A1, 2002.
  • 47
    Ohno S, Otani K, Niwa S, Iwayama S, Takahara A, Koganei H, Ono Y, Fujita S, Takeda T, Hagihara M, et al. Novel pyrimidine derivative and novel pyridine derivative. WO 02/22588 A1, 2002
  • 48
    McGivern JG, McDonough SI. Voltage-gated calcium channels as targets for the treatment of chronic pain. Curr Drug Targets CNS Neurol Disord 2004, 3:457478.
  • 49
    Snutch TP, Sutton KG, Zamponi GW. Voltage-dependent calcium channels–beyond dihydropyridine antagonists. Curr Opin Pharmacol 2001, 1:1116.
  • 50
    Lundy PM, Hamilton MG, Frew R. Pharmacological identification of a novel Ca2+ channel in chicken brain synaptosomes. Brain Res 1994, 643:204210.
  • 51
    Xia M, Imredy JP, Koblan KS, Bennett P, Connolly TM. State-dependent inhibition of L-type calcium channels: cell-based assay in high-throughput format. Anal Biochem 2004, 327:7481.
  • 52
    Huang CJ, Harootunian A, Maher MP, Quan C, Raj CD, McCormack K, Numann R, Negulescu PA, González JE. Characterization of voltage-gated sodium-channel blockers by electrical stimulation and fluorescence detection of membrane potential. Nat Biotechnol 2006, 24:439446.
  • 53
    Balasubramanian B, Imredy JP, Kim D, Penniman J, Lagrutta A, Salata JJ. Optimization of Ca(v)1.2 screening with an automated planar patch clamp platform. J Pharmacol Toxicol Methods 2009, 59:6272.
  • 54
    Wright CE, Angus JA. Effects of N-, P- and Q-type neuronal calcium channel antagonists on mammalian peripheral neurotransmission. Br J Pharmacol 1996, 119:4956.
  • 55
    Serone AP, Angus JA. Role of N-type calcium channels in autonomic neurotransmission in guinea-pig isolated left atria. Br J Pharmacol 1999, 127:927934.
  • 56
    Hawkes AL, Angus JA, Wright CE. omega-Conotoxin GVIA and prazosin, but not felodipine, cause postural hypotension in rabbits. Clin Exp Pharmacol Physiol 1995, 22:711716.
  • 57
    Wright CE, Angus JA. Hemodynamic and autonomic reflex effects of chronic N-type Ca2+ channel blockade with omega-conotoxin GVIA in conscious normotensive and hypertensive rabbits. J Cardiovasc Pharmacol 1995, 25:459468.
  • 58
    Chan CS, Guzman JN, Ilijic E, Mercer JN, Rick C, Tkatch T, Meredith GE, Surmeier DJ. ‘Rejuvenation’ protects neurons in mouse models of Parkinson's disease. Nature 2007, 447:10811086.
  • 59
    Guzman JN, Sanchez-Padilla J, Wokosin D, Kondapalli J, Ilijic E, Schumacker PT, Surmeier DJ. Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1. Nature 2010, 468:696700.
  • 60
    Mosharov EV, Larsen KE, Kanter E, Phillips KA, Wilson K, Schmitz Y, Krantz DE, Kobayashi K, Edwards RH, Sulzer D. Interplay between cytosolic dopamine, calcium, and α-synuclein causes selective death of substantia nigra neurons. Neuron 2009, 62:218229.
  • 61
    Xu W, Lipscombe D. Neuronal Ca(V)1.3α(1) L-type channels activate at relatively hyperpolarized membrane potentials and are incompletely inhibited by dihydropyridines. J Neurosci 2001, 21:59445951.
  • 62
    Koschak A, Reimer D, Huber I, Grabner M, Glossmann H, Engel J, Striessnig J. α 1D (Cav1.3) subunits can form l-type Ca2+ channels activating at negative voltages. J Biol Chem 2001, 276:2210022106.
  • 63
    Puopolo M, Raviola E, Bean BP. Roles of subthreshold calcium current and sodium current in spontaneous firing of mouse midbrain dopamine neurons. J Neurosci 2007, 27:645656.
  • 64
    Putzier I, Kullmann PH, Horn JP, Levitan ES. Cav1.3 channel voltage dependence, not Ca2+ selectivity, drives pacemaker activity and amplifies bursts in nigral dopamine neurons. J Neurosci 2009, 29:1541415419.
  • 65
    Wilson CJ, Callaway JC. Coupled oscillator model of the dopaminergic neuron of the substantia nigra. J Neurophysiol 2000, 83:30843100.
  • 66
    Platzer J, Engel J, Schrott-Fischer A, Stephan K, Bova S, Chen H, Zheng H, Striessnig J. Congenital deafness and sinoatrial node dysfunction in mice lacking class D L-type Ca2+ channels. Cell 2000, 102:8997.
  • 67
    Becker C, Jick SS, Meier CR. Use of antihypertensives and the risk of Parkinson disease. Neurology 2008, 70(16 Pt 2):14381444.
  • 68
    Ritz B, Rhodes SL, Qian L, Schernhammer E, Olsen JH, Friis S. L-type calcium channel blockers and Parkinson disease in Denmark. Ann Neurol 2010, 67:600606.
  • 69
    Simon KC, Gao X, Chen H, Schwarzschild MA, Ascherio A. Calcium channel blocker use and risk of Parkinson's disease. Mov Disord 2010, 25:18181822.
  • 70
    Uchida S, Yamada S, Nagai K, Deguchi Y, Kimura R. Brain pharmacokinetics and in vivo receptor binding of 1,4-dihydropyridine calcium channel antagonists. Life Sci 1997, 61:20832090.
  • 71
    McAuley BJ, Schroeder JS. The use of diltiazem hydrochloride in cardiovascular disorders. Pharmacotherapy 1982, 2:121133.
  • 72
    Brunner M, Langer O, Sunder-Plassmann R, Dobrozemsky G, Muller U, Wadsak W, Krcal A, Karch R, Mannhalter C, Dudczak R, et al. Influence of functional haplotypes in the drug transporter gene ABCB1 on central nervous system drug distribution in humans. Clin Pharmacol Ther 2005, 78:182190.
  • 73
    Burges RA, Dodd MG, Gardiner DG. Pharmacologic profile of amlodipine. Am J Cardiol 1989, 64:10I18I; discussion 18I–20I.
  • 74
    Mason RP, Campbell SF, Wang SD, Herbette LG. Comparison of location and binding for the positively charged 1,4-dihydropyridine calcium channel antagonist amlodipine with uncharged drugs of this class in cardiac membranes. Mol Pharmacol 1989, 36:634640.
  • 75
    Yamada S, Sugimoto N, Uchida S, Deguchi Y, Kimura R. Pharmacokinetics of amlodipine and its occupancy of calcium antagonist receptors. J Cardiovasc Pharmacol 1994, 23:466472.
  • 76
    Sinnegger-Brauns MJ, Huber IG, Koschak A, Wild C, Obermair GJ, Einzinger U, Hoda JC, Sartori SR, Striessnig J. Expression and 1,4-dihydropyridine-binding properties of brain L-type calcium channel isoforms. Mol Pharmacol 2009, 75:407414.
  • 77
    Lipscombe D, Helton TD, Xu W. L-type calcium channels: the low down. J Neurophysiol 2004, 92:26332641.
  • 78
    Helton TD, Xu W, Lipscombe D. Neuronal L-type calcium channels open quickly and are inhibited slowly. J Neurosci 2005, 25:1024710251.
  • 79
    Guzman JN, Sanchez-Padilla J, Chan CS, Surmeier DJ. Robust pacemaking in substantia nigra dopaminergic neurons. J Neurosci 2009, 29:1101111019.
  • 80
    Khaliq ZM, Bean BP. Pacemaking in dopaminergic ventral tegmental area neurons: depolarizing drive from background and voltage-dependent sodium conductances. J Neurosci 2010, 30:74017413.
  • 81
    Rose RA, Sellan M, Simpson JA, Izaddoustdar F, Cifelli C, Panama BK, Davis M, Zhao D, Markhani M, Murphy GG, et al. Iron overload decreases CaV1.3-dependent L-type Ca2+ currents leading to bradycardia, altered electrical conduction, and atrial fibrillation. Circ Arrhythm Electrophysiol 2011, 4:733742.
  • 82
    Baig SM, Koschak A, Lieb A, Gebhart M, Dafinger C, Nürnberg G, Ali A, Ahmad I, Sinnegger-Brauns MJ, Brandt N, et al. Loss of Ca(v)1.3 (CACNA1D) function in a human channelopathy with bradycardia and congenital deafness. Nat Neurosci 2011, 14:7784.
  • 83
    Simuni T, Borushko E, Avram MJ, Miskevics S, Martel A, Zadikoff C, Videnovic A, Weaver FM, Williams K, Surmeier DJ. Tolerability of isradipine in early Parkinson's disease: a pilot dose escalation study. Mov Disord 2010, 25:28632866.
  • 84
    Quik M. Smoking, nicotine and Parkinson's disease. Trends Neurosci 2004, 27:561568.
  • 85
    Quik M, Bordia T, Huang L, Perez X. Targeting nicotinic receptors for Parkinson's disease therapy. CNS Neurol Disord Drug Targets 2011, 10:651658.
  • 86
    Anekonda TS, Quinn JF, Harris C, Frahler K, Wadsworth TL, Woltjer RL. L-type voltage-gated calcium channel blockade with isradipine as a therapeutic strategy for Alzheimer's disease. Neurobiol Dis 2011, 41:6270.
  • 87
    Ferreira MA, O'Donovan MC, Meng YA, Jones IR, Ruderfer DM, Jones L, Fan J, Kirov G, Perlis RH, Green EK, et al. Collaborative genome-wide association analysis supports a role for ANK3 and CACNA1C in bipolar disorder. Nat Genet 2008, 40:10561058.
  • 88
    Sklar P, Smoller JW, Fan J, Ferreira MA, Perlis RH, Chambert K, Nimgaonkar VL, McQueen MB, Faraone SV, Kirby A, et al. Whole-genome association study of bipolar disorder. Mol Psychiatry 2008, 13:558569.
  • 89
    Sklar P, Ripke S, Scott LJ, Andreassen OA, Cichon S, Craddock N, Edenberg HJ, Nurnberger JI Jr, Rietschel M, Blackwood D, et al. Large-scale genome-wide association analysis of bipolar disorder identifies a new susceptibility locus near ODZ4. Nat Genet 2011, 43:977983.
  • 90
    Ripke S, Sanders AR, Kendler KS, Levinson DF, Sklar P, Holmans PA, Lin D-Y, Duan J, Ophoff RA, Andreassen OA, et al. Genome-wide association study identifies five new schizophrenia loci. Nat Genet 2011, 43:969976.
  • 91
    Myers RA, Casals F, Gauthier J, Hamdan FF, Keebler J, Boyko AR, Bustamente CD, Piton AM, Spiegelman D, Henrion E, et al. A population genetic approach to mapping neurological disorder genes using deep resequencing. PLoS Genet 2011, 7:e1001318.
  • 92
    Wei J, Hemmings GP. A further study of a possible locus for schizophrenia on the X chromosome. Biochem Biophys Res Commun 2006, 344:12411245.
  • 93
    Nader K, Schafe GE, LeDoux JE. The labile nature of consolidation theory. Nat Rev Neurosci 2000, 1:216219.
  • 94
    Hardt O, Einarsson EO, Nader K. A bridge over troubled water: reconsolidation as a link between cognitive and neuroscientific memory research traditions. Annu Rev Psychol 2010, 61:141167.
  • 95
    Splawski I, Timothy KW, Sharpe LM, Decher N, Kumar P, Bloise R, Napolitano C, Schwartz PJ, Joseph RM, Condouris K, et al. Ca(V)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. Cell 2004, 119:1931.
  • 96
    Splawski I, Timothy KW, Decher N, Kumar P, Sachse FB, Beggs AH, Sanguinetti MC, Keating MT. Severe arrhythmia disorder caused by cardiac L-type calcium channel mutations. Proc Natl Acad Sci U S A 2005, 102:80898096; discussion 8086–8088.
  • 97
    Liao P, Soong TW. CaV1.2 channelopathies: from arrhythmias to autism, bipolar disorder, and immunodeficiency. Pflugers Arch 2010, 460:353359.
  • 98
    Bader PL, Faizi M, Kim LH, Owen SF, Tadross MR, Alfa RW, Bett GCL, Tsien RW, Rasmusson RL, Shamloo M. Mouse model of Timothy syndrome recapitulates triad of autistic traits. Proc Natl Acad Sci U S A 2011, 108:1543215437.
  • 99
    Pietrobon D. Biological science of headache channels. Handb Clin Neurol (edited by Vinken PJ, Bruyn GW) 2010, 97:7383.
  • 100
    Pietrobon D. CaV2.1 channelopathies. Pflugers Arch 2010, 460:375393.
  • 101
    de Vries B, Frants RR, Ferrari MD, van den Maagdenberg AM. Molecular genetics of migraine. Hum Genet 2009, 126:115132.
  • 102
    Ophoff RA, Terwindt GM, Vergouwe MN, van Eijk R, Oefner PJ, Hoffman SMG, Lamerdin JE, Mohrenweiser HW, Bulman DE, Ferrari M, et al. Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+ channel gene CACNL1A4. Cell 1996, 87:543552.
  • 103
    Starr TV, Prystay W, Snutch TP. Primary structure of a calcium channel that is highly expressed in the rat cerebellum. Proc Natl Acad Sci U S A 1991, 88:56215625.
  • 104
    Mori Y, Friedrich T, Kim MS, Mikami A, Nakai J, Ruth P, Bosse E, Hoffman F, Flockerzi V, Furuichi T, et al. Primary structure and functional expression from complementary DNA of a brain calcium channel. Nature 1991, 350:398402.
  • 105
    van den Maagdenberg AM, Pietrobon D, Pizzorusso T, Kaja S, Broos LA, Cesetti T, van de Ven RCG, Tottene A, van der Kaa J, Plomp JJ, et al. A Cacna1a knockin migraine mouse model with increased susceptibility to cortical spreading depression. Neuron 2004, 41:701710.
  • 106
    Jun K, Piedras-Renteria ES, Smith SM, Wheeler DB, Lee SB, Lee TG, chin H, Adams ME, Scheller RH, Tsien RW, et al. Ablation of P/Q-type Ca(2+) channel currents, altered synaptic transmission, and progressive ataxia in mice lacking the α(1A)-subunit. Proc Natl Acad Sci U S A 1999, 96:1524515250.
  • 107
    Fletcher CF, Tottene A, Lennon VA, Wilson SM, Dubel SJ, Paylor R, Hosford DA, Tessarollo L, McEnery MW, Pietrobon D, et al. Dystonia and cerebellar atrophy in Cacna1a null mice lacking P/Q calcium channel activity. FASEB J 2001, 15:12881290.
  • 108
    Fletcher CF, Lutz CM, O'Sullivan TN, Shaughnessy JD Jr, Hawkes R, Frankel WN, Copeland NG, Jenkins NA. Absence epilepsy in tottering mutant mice is associated with calcium channel defects. Cell 1996, 87:607617.
  • 109
    Oda S. A new allele of the tottering locus, rolling mouse Nagoya, on chromosome no. 8 in the mouse. Jpn J Genet 1981, 56:295.
  • 110
    Mori Y, Wakamori M, Oda S, Fletcher CF, Sekiguchi N, Mori E, Copeland NG, Jenkins NA, Matsushita K, Matsuyama Z, et al. Reduced voltage sensitivity of activation of P/Q-type Ca2+ channels is associated with the ataxic mouse mutation rolling Nagoya (tg(rol)). J Neurosci 2000, 20:56545662.
  • 111
    Gan TJ. Pharmacokinetic and pharmacodynamic characteristics of medications used for moderate sedation. Clin Pharmacokinet 2006, 45:855869.
  • 112
    Titulaer MJ, Lang B, Verschuuren JJ. Lambert-Eaton myasthenic syndrome: from clinical characteristics to therapeutic strategies. Lancet Neurol 2011, 10:10981107.
  • 113
    Lennon VA, Kryzer TJ, Griesmann GE, O'Suilleabhain PE, Windebank AJ, Woppmann A, Miljanich GP, Lambert EH. Calcium-channel antibodies in the Lambert-Eaton syndrome and other paraneoplastic syndromes. N Engl J Med 1995, 332:14671474.
  • 114
    Pinto A, Gillard S, Moss F, Whyte K, Brust P, Williams M, Stauderman K, Harpold M, Lang B, Newsome-Davis J, et al. Human autoantibodies specific for the α1A calcium channel subunit reduce both P-type and Q-type calcium currents in cerebellar neurons. Proc Natl Acad Sci U S A 1998, 95:83288333.
  • 115
    Giovannini F, Sher E, Webster R, Boot J, Lang B. Calcium channel subtypes contributing to acetylcholine release from normal, 4-aminopyridine-treated and myasthenic syndrome auto-antibodies-affected neuromuscular junctions. Br J Pharmacol 2002, 136:11351145.
  • 116
    Engisch KL, Rich MM, Cook N, Nowycky MC. Lambert-Eaton antibodies inhibit Ca2+ currents but paradoxically increase exocytosis during stimulus trains in bovine adrenal chromaffin cells. J Neurosci 1999, 19:33843395.
  • 117
    Graus F, Lang B, Pozo-Rosich P, Saiz A, Casamitjana R, Vincent A. P/Q type calcium-channel antibodies in paraneoplastic cerebellar degeneration with lung cancer. Neurology 2002, 59:764766.
  • 118
    Liao YJ, Safa P, Chen YR, Sobel RA, Boyden ES, Tsien RW. Anti-Ca2+ channel antibody attenuates Ca2+ currents and mimics cerebellar ataxia in vivo. Proc Natl Acad Sci U S A 2008, 105:27052710.
  • 119
    Maddison P, Lang B. Paraneoplastic neurological autoimmunity and survival in small-cell lung cancer. J Neuroimmunol 2008, 201–202:159162.
  • 120
    Wirtz PW, Lang B, Graus F, van den Maagdenberg AM, Saiz A, Twijnstra A, Verschuuren JJGM. P/Q-type calcium channel antibodies, Lambert-Eaton myasthenic syndrome and survival in small cell lung cancer. J Neuroimmunol 2005, 164:161165.
  • 121
    Ertel EA. Pharmacology of Cav3 (T-Type) channels. In: McDonough SI, ed. Calcium Channel Pharmacology. New York: Kluwer Academic; 2004, 183236.
  • 122
    Perez-Reyes E. Molecular physiology of low-voltage-activated t-type calcium channels. Physiol Rev 2003, 83:117161.
  • 123
    Kim D, Song I, Keum S, Lee T, Jeong MJ, Kim SS, McEnery MW, Shin HS. Lack of the burst firing of thalamocortical relay neurons and resistance to absence seizures in mice lacking α(1G) T-type Ca(2+) channels. Neuron 2001, 31:3545.
  • 124
    Chen CC, Lamping KG, Nuno DW, Barresi R, Prouty SJ, Lavoie JL, Cribbs LL, England SK, Sigmund KD, Weiss RM, et al. Abnormal coronary function in mice deficient in α1H T-type Ca2+channels. Science 2003, 302:14161418.
  • 125
    Astori S, Wimmer RD, Prosser HM, Corti C, Corsi M, Liaudet N, Volterra A, Franken P, Adelman JP, Lüthi A. The Ca(V)3.3 calcium channel is the major sleep spindle pacemaker in thalamus. Proc Natl Acad Sci U S A 2011, 108:1382313828.
  • 126
    Veronese ML, Gillen LP, Dorval EP, Hauck WW, Waldman SA, Greenberg HE. Effect of mibefradil on CYP3A4 in vivo. J Clin Pharmacol 2003, 43:10911100.
  • 127
    Mullins ME, Horowitz BZ, Linden DH, Smith GW, Norton RL, Stump J. Life-threatening interaction of mibefradil and β-blockers with dihydropyridine calcium channel blockers. JAMA 1998, 280:157158.
  • 128
    Jacobson TA. Comparative pharmacokinetic interaction profiles of pravastatin, simvastatin, and atorvastatin when coadministered with cytochrome P450 inhibitors. Am J Cardiol 2004, 94:11401146.
  • 129
    Martin RL, Lee JH, Cribbs LL, Perez-Reyes E, Hanck DA. Mibefradil block of cloned T-type calcium channels. J Pharmacol Exp Ther 2000, 295:302308.
  • 130
    McNulty MM, Hanck DA. State-dependent mibefradil block of Na+ channels. Mol Pharmacol 2004, 66:16521661.
  • 131
    Moosmang S, Haider N, Bruderl B, Welling A, Hofmann F. Antihypertensive effects of the putative T-type calcium channel antagonist mibefradil are mediated by the L-type calcium channel Cav1.2. Circ Res 2006, 98:105110.
  • 132
    Richelson E, Souder T. Binding of antipsychotic drugs to human brain receptors focus on newer generation compounds. Life Sci 2000, 68:2939.
  • 133
    Santi CM, Cayabyab FS, Sutton KG, McRory JE, Mezeyova J, Hamming KS, Parker D, Stea A, Snutch TP. Differential inhibition of T-type calcium channels by neuroleptics. J Neurosci 2002, 22:396403.
  • 134
    Todorovic SM, Perez-Reyes E, Lingle CJ. Anticonvulsants but not general anesthetics have differential blocking effects on different T-type current variants. Mol Pharmacol 2000, 58:98108.
  • 135
    Todorovic SM, Lingle CJ. Pharmacological properties of T-type Ca2+ current in adult rat sensory neurons: effects of anticonvulsant and anesthetic agents. J Neurophysiol 1998, 79:240252.
  • 136
    Furukawa T, Nukada T, Namiki Y, Miyashita Y, Hatsuno K, Ueno Y, Yamakawa T, Isshiki T. Five different profiles of dihydropyridines in blocking T-type Ca(2+) channel subtypes (Ca(v)3.1 (α(1G)), Ca(v)3.2 (α(1H)), and Ca(v)3.3 (α(1I))) expressed in Xenopus oocytes. Eur J Pharmacol 2009, 613:100107.
  • 137
    Hvidberg EF, Dam M. Clinical pharmacokinetics of anticonvulsants. Clin Pharmacokinet 1976, 1:161188.
  • 138
    Coulter DA, Huguenard JR, Prince DA. Characterization of ethosuximide reduction of low-threshold calcium current in thalamic neurons. Ann Neurol 1989, 25:582593.
  • 139
    Huguenard JR. Neuronal circuitry of thalamocortical epilepsy and mechanisms of antiabsence drug action. Adv Neurol 1999, 79:991999.
  • 140
    Shin HS, Cheong EJ, Choi S, Lee J, Na HS. T-type Ca2+ channels as therapeutic targets in the nervous system. Curr Opin Pharmacol 2008, 8:3341.
  • 141
    Shin HS, Lee J, Song I. Genetic studies on the role of T-type Ca2+ channels in sleep and absence epilepsy. CNS Neurol Disord Drug Targets 2006, 5:629638.
  • 142
    Lee J, Kim D, Shin HS. Lack of δ waves and sleep disturbances during non-rapid eye movement sleep in mice lacking α1G-subunit of T-type calcium channels. Proc Natl Acad Sci U S A 2004, 101:1819518199.
  • 143
    Uebele VN, Gotter AL, Nuss CE, Kraus RL, Doran SM, Garson SL, Reiss DR, Li Y, Barrow JC, Reger TS, et al. Antagonism of T-type calcium channels inhibits high-fat diet-induced weight gain in mice. J Clin Invest 2009, 119:16591667.
  • 144
    Bourinet E, Alloui A, Monteil A, Barrere C, Couette B, Poirot O, Pages A, McRory J, Snutch TP, Eschalier A, et al. Silencing of the Cav3.2 T-type calcium channel gene in sensory neurons demonstrates its major role in nociception. EMBO J 2005, 24:315324.
  • 145
    Marger F, Gelot A, Alloui A, Matricon J, Ferrer JF, Barrère C, Pizzoccaro A, Muller E, Nargeot J, Snutch TP, et al. T-type calcium channels contribute to colonic hypersensitivity in a rat model of irritable bowel syndrome. Proc Natl Acad Sci U S A 2011, 108:1126811273.
  • 146
    Nelson MT, Joksovic PM, Perez-Reyes E, Todorovic SM. The endogenous redox agent L-cysteine induces T-type Ca2+ channel-dependent sensitization of a novel subpopulation of rat peripheral nociceptors. J Neurosci 2005, 25:87668775.
  • 147
    Choe W, Messinger RB, Leach E, Eckle VS, Obradovic A, Salajegheh R, Jevtovic-Todorovic V, Todorovic SM. TTA-P2 is a potent and selective blocker of T-type calcium channels in rat sensory neurons and a novel antinociceptive agent. Mol Pharmacol 2011, 80:900910.
  • 148
    Messinger RB, Naik AK, Jagodic MM, Nelson MT, Lee WY, Choe WJ, Orestes P, Latham JR, Todorovic SM, Jevtovic-Todorovic. In vivo silencing of the Ca(V)3.2 T-type calcium channels in sensory neurons alleviates hyperalgesia in rats with streptozocin-induced diabetic neuropathy. Pain 2009, 145:184195.
  • 149
    Choi S, Na HS, Kim J, Lee J, Lee S, Kim D, Park J, Chen C-C, Campbell KP, Shin H-S. Attenuated pain responses in mice lacking Ca(V)3.2 T-type channels. Genes Brain Behav 2007, 6:425431.
  • 150
    Shin JB, Martinez-Salgado C, Heppenstall PA, Lewin GR. A T-type calcium channel required for normal function of a mammalian mechanoreceptor. Nat Neurosci 2003, 6:724730.
  • 151
    Dubreuil AS, Boukhaddaoui H, Desmadryl G, Martinez-Salgado C, Moshourab R, Lewin GR, Carroll P, Valmier J, Scamps F. Role of T-type calcium current in identified D-hair mechanoreceptor neurons studied in vitro. J Neurosci 2004, 24:84808484.
  • 152
    Kim D, Park D, Choi S, Lee S, Sun M, Kim C, Shin H-S. Thalamic control of visceral nociception mediated by T-type Ca2+ channels. Science 2003, 302:117119.
  • 153
    Park C, Kim JH, Yoon BE, Choi EJ, Lee CJ, Shin HS. T-type channels control the opioidergic descending analgesia at the low threshold-spiking GABAergic neurons in the periaqueductal gray. Proc Natl Acad Sci U S A 2010, 107:1485714862.
  • 154
    Zambreanu L, Wise RG, Brooks JC, Iannetti GD, Tracey I. A role for the brainstem in central sensitisation in humans. Evidence from functional magnetic resonance imaging. Pain 2005, 114:397407.
  • 155
    Giordanetto F, Knerr L, Wallberg A. T-type calcium channels inhibitors: a patent review. Expert Opin Ther Pat 2011, 21:85101.
  • 156
    Kang HW, Park JY, Jeong SW, Kim JA, Moon HJ, Perez-Reyes E, Lee J-H. A molecular determinant of nickel inhibition in Cav3.2 T-type calcium channels. J Biol Chem 2006, 281:48234830.
  • 157
    Barrow JC, Reger TS, Yang Z-Q. Pyridyl amide T-type calcium channel antagonists. US Patent 7,875,636, 2007
  • 158
    Barrow JC, Cube RV, Ngo PL, Rittle KE, Yang Z, Young SD. Quinazolinone T-type calcium channel antagonists. US Patent 7,745,452, 2010
  • 159
    Schlegel KA, Shu Y, Yang Z-Q, Barrow J. Heterocycle amide T-type calcium channel antagonists. WO2011053542A1, 2011
  • 160
    Barrow JC, Yang Z-Q. Pyrazinyl phenyl amide T-type calcium channel antagonists. WO2011022315A1, 2011
  • 161
    Barrow JC, Lindsley CW, Shipe WD, Yang Z-Q. 3-Fluoro-piperidine T-type calcium channel antagonists. US20100222387A1, 2010
  • 162
    Gee NS, Brown JP, Dissanayake VU, Offord J, Thurlow R, Woodruff GN. The novel anticonvulsant drug, gabapentin (Neurontin), binds to the α2δ subunit of a calcium channel. J Biol Chem 1996, 271:57685776.
  • 163
    Brown JP, Gee NS. Cloning and deletion mutagenesis of the α2 δ calcium channel subunit from porcine cerebral cortex. Expression of a soluble form of the protein that retains [3H]gabapentin binding activity. J Biol Chem 1998, 273:2545825465.
  • 164
    Marais E, Klugbauer N, Hofmann F. Calcium channel α(2)δ subunits-structure and Gabapentin binding. Mol Pharmacol 2001, 59:12431248.
  • 165
    Qin N, Yagel S, Momplaisir ML, Codd EE, D'Andrea MR. Molecular cloning and characterization of the human voltage-gated calcium channel α(2)δ-4 subunit. Mol Pharmacol 2002, 62:485496.
  • 166
    Field MJ, Cox PJ, Stott E, Melrose H, Offord J, Su T-Z, Bramwell S, Corradini L, England S, Winks J. Identification of the α2-δ-1 subunit of voltage-dependent calcium channels as a molecular target for pain mediating the analgesic actions of pregabalin. Proc Natl Acad Sci U S A 2006, 103:1753717542.
  • 167
    Hendrich J, Van Minh AT, Heblich F, Nieto-Rostro M, Watschinger K, Streissnig J, Wratten J, Davies A, Dolphin AC. Pharmacological disruption of calcium channel trafficking by the α2δ ligand gabapentin. Proc Natl Acad Sci U S A 2008, 105:36283633.
  • 168
    Tran-Van-Minh A, Dolphin AC. The α2δ ligand gabapentin inhibits the Rab11-dependent recycling of the calcium channel subunit α2δ-2. J Neurosci 2010, 30:1285612867.
  • 169
    Bauer CS, Tran-Van-Minh A, Kadurin I, Dolphin AC. A new look at calcium channel α2δ subunits. Curr Opin Neurobiol 2010, 20:563571.
  • 170
    Bauer CS, Rahman W, Tran-van-Minh A, Lujan R, Dickenson AH, Dolphin AC. The anti-allodynic α(2)δ ligand pregabalin inhibits the trafficking of the calcium channel α(2)δ-1 subunit to presynaptic terminals in vivo. Biochem Soc Trans 2010, 38:525528.
  • 171
    Eroglu C, Allen NJ, Susman MW, O'Rourke NA, Park CY, Özkan E, Chakraborty C, Mulinyawe SB, Annis DS, Huberman AD, et al. The Gabapentin receptor α2δ-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis. Cell 2009, 139:380392.
  • 172
    Davies A, Hendrich J, Van Minh AT, Wratten J, Douglas L, Dolphin AC. Functional biology of the α(2)δ subunits of voltage-gated calcium channels. Trends Pharmacol Sci 2007, 28:220228.
  • 173
    Iannetti GD, Zambreanu L, Wise RG, Buchanan TJ, Huggins JP, Smart TS, Vennart W, Tracey I. Pharmacological modulation of pain-related brain activity during normal and central sensitization states in humans. Proc Natl Acad Sci U S A 2005, 102:1819518200.
  • 174
    Li Z, Taylor CP, Weber M, Piechan J, Prior F, Bian F, Cui M, Hoffman D, Donevan S. Pregabalin is a potent and selective ligand for α(2)δ-1 and α(2)δ-2 calcium channel subunits. Eur J Pharmacol 2011, 667:8090.
  • 175
    Bockbrader HN, Wesche D, Miller R, Chapel S, Janiczek N, Burger P. A comparison of the pharmacokinetics and pharmacodynamics of pregabalin and gabapentin. Clin Pharmacokinet 2010, 49:661669.
  • 176
    Barclay J, Balaguero N, Mione M, Ackerman SL, Letts VA, Brodbeck J, Canti C, Meir A, Page KM, Kusumi K, et al. Ducky mouse phenotype of epilepsy and ataxia is associated with mutations in the Cacna2d2 gene and decreased calcium channel current in cerebellar Purkinje cells. J Neurosci 2001, 21:60956104.
  • 177
    Neely GG, Hess A, Costigan M, Keene AC, Goulas S, Langeslag M, Griffin RS, Belfer I, Dai F, Smith SB, et al. A genome-wide Drosophila screen for heat nociception identifies α2δ3 as an evolutionarily conserved pain gene. Cell 2010, 143:628638.
  • 178
    Jing X, Li DQ, Olofsson CS, Salehi A, Surve VV, Caballero J, Ivarsson R, Lundquist I, Pereverez A, Schneider T, et al. CaV2.3 calcium channels control second-phase insulin release. J Clin Invest 2005, 115:146154.
  • 179
    Zhang Q, Bengtsson M, Partridge C, Salehi A, Braun M, Cox R, Eliasson L, Johnson PRV, Renström E, Schneider T, et al. R-type Ca(2+)-channel-evoked CICR regulates glucose-induced somatostatin secretion. Nat Cell Biol 2007, 9:453460.
  • 180
    Nissenbaum J, Devor M, Seltzer Z, Gebauer M, Michaelis M, Tal M, Dorfman R, Abitbul-Yarkoni M, Lu Y, Elahipanah T, et al. Susceptibility to chronic pain following nerve injury is genetically affected by CACNG2. Genome Res 2010, 20:11801190.
  • 181
    Payne HL. The role of transmembrane AMPA receptor regulatory proteins (TARPs) in neurotransmission and receptor trafficking (Review). Mol Membr Biol 2008, 25:353362.
  • 182
    Hashimoto K, Fukaya M, Qiao X, Sakimura K, Watanabe M, Kano M. Impairment of AMPA receptor function in cerebellar granule cells of ataxic mutant mouse stargazer. J Neurosci 1999, 19:60276036.
  • 183
    Chen L, Chetkovich DM, Petralia RS, Sweeney NT, Kawasaki Y, Wenthold RJ, Bredt DS, Nicoll RA. Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature 2000, 408:936943.
  • 184
    Panner A, Wurster RD. T-type calcium channels and tumor proliferation. Cell Calcium 2006, 40:253259.
  • 185
    Vincent A, Wright T, Day MA, Westall CA, Heon E. A novel p.Gly603Arg mutation in CACNA1F causes Aland island eye disease and incomplete congenital stationary night blindness phenotypes in a family. Mol Vis 2011, 17:32623270.
  • 186
    Strom TM, Nyakatura G, Apfelstedt-Sylla E, Hellebrand H, Lorenz B, Weber BHF, Wutz K, Gutwillinger N, Rüther K, Drescher B, et al. An L-type calcium-channel gene mutated in incomplete X-linked congenital stationary night blindness. Nat Genet 1998, 19:260263.
  • 187
    Bech-Hansen NT, Boycott KM, Gratton KJ, Ross DA, Field LL, Pearce WG. Localization of a gene for incomplete X-linked congenital stationary night blindness to the interval between DXS6849 and DXS8023 in Xp11.23. Hum Genet 1998, 103:124130.
  • 188
    Bech-Hansen NT, Naylor MJ, Maybaum TA, Pearce WG, Koop B, Fishman GA, Mets M, Musarella MA, Boycott KM. Loss-of-function mutations in a calcium-channel α1-subunit gene in Xp11.23 cause incomplete X-linked congenital stationary night blindness. Nat Genet 1998, 19:264267.
  • 189
    Omilusik K, Priatel JJ, Chen X, Wang YT, Xu H, Choi KB, Gopaul R, McIntyre-Smith A, Bech-Hansen NT, Waterfield D, et al. The Ca(v)1.4 calcium channel is a critical regulator of T cell receptor signaling and naive T cell homeostasis. Immunity 2011, 35:349360.
  • 190
    Gintant GA, Gallacher DJ, Pugsley MK. The ‘overly-sensitive’ heart: sodium channel block and QRS interval prolongation. Br J Pharmacol 2011, 164:254259.
  • 191
    Gintant G. An evaluation of hERG current assay performance: translating preclinical safety studies to clinical QT prolongation. Pharmacol Ther 2011, 129:109119.
  • 192
    Bean BP. Nitrendipine block of cardiac calcium channels: high-affinity binding to the inactivated state. Proc Natl Acad Sci U S A 1984, 81:63886392.
  • 193
    Sinnegger-Brauns MJ, Hetzenauer A, Huber IG, Renstrom E, Wietzorrek G, Berjukov S, Cavalli M, Walter D, Koshak A, Waldschütz R, et al. Isoform-specific regulation of mood behavior and pancreatic β cell and cardiovascular function by L-type Ca2+ channels. J Clin Invest 2004, 113:14301439.
  • 194
    Schulla V, Renstrom E, Feil R, Feil S, Franklin I, Gjinovci A, Jing X-J, Laux D, Lundquist I, Magnuson MA, et al. Impaired insulin secretion and glucose tolerance in β cell-selective Ca(v)1.2 Ca2+ channel null mice. EMBO J 2003, 22:38443854.