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Keywords:

  • afterhyperpolarization;
  • afterhyperpolarizing neurone;
  • calcium-activated potassium;
  • enteric nervous system;
  • channel;
  • intermediate-conductance potassium channel

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression and characterization of IK channel by RT–PCR and western blotting
  6. IK channel immunohistochemistry in the mouse distal colon
  7. Electrophysiological identification of IK channels
  8. Discussion
  9. General overview
  10. Biochemical characterization
  11. Subcellular localization
  12. Pharmacological characterization
  13. Correlation of the AHP with IK channel immunoreactivity
  14. Dogiel type I neurones
  15. Re-evaluation of AHPs in other neurones
  16. Potential clinical uses of IK channel blockers
  17. Acknowledgements
  18. References

Calcium-activated potassium channels are critically important in modulating neuronal cell excitability. One member of the family, the intermediate-conductance potassium (IK) channel, is not thought to play a role in neurones because of its predominant expression in non-excitable cells such as erythrocytes and lymphocytes, in smooth muscle tissues, and its lack of apparent expression in brain. In the present study, we demonstrate that IK channels are localized on specific neurones in the mouse enteric nervous system where they mediate the slow afterhyperpolarization following an action potential. IK channels were localized by immunohistochemistry on intrinsic primary afferent neurones, identified by their characteristic Dogiel type II morphology. The slow afterhyperpolarization recorded from these cells was abolished by the IK channel blocker clotrimazole. RT–PCR and western analysis of extracts from the colon revealed an IK channel transcript and protein identical to the IK channel expressed in other cell types. These results indicate that IK channels are expressed in neurones where they play an important role in modulating firing properties.

Abbreviations used
AH neurone

afterhyperpolarizing neurone

AHP

afterhyperpolarization

BK

large-conductance potassium channel

DMSO

dimethylsulfoxide

EM–MP

external muscle–myenteric plexus

IK

intermediate-conductance potassium channel

IPAN

intrinsic primary afferent neurone

PBS

phosphate-buffered saline

SDS–PAGE

sodium dodecyl sulphate–polyacrylamide gel electrophoresis

SK

small-conductance potassium channel

TEA

tetraethylammonium chloride

The slow afterhyperpolarization (AHP) that follows an action potential in many types of neurones is essential for normal functioning of nerve circuits because it controls the firing frequency of neurones. The channel type partly responsible for the AHP in many neurones is the small-conductance calcium-activated potassium channel (SK channel) which is activated by Ca2+ entry induced by depolarization of the membrane potential (Sah 1996). These channels exhibit a single-channel conductance of 4–14 pS in symmetrical K+, are voltage independent, highly sensitive to submicromolar concentrations of Ca2+ (EC50 0.5–0.7 µm), and are blocked by the bee venom toxin apamin. Typically, the AHP decays within 100–500 ms. In contrast, many neurones, including hippocampal pyramidal neurones (Lancaster and Adams 1986; Storm 1990; Sah 1996; Shah et al. 2001), sympathetic neurones (Martínez-Pinna et al. 2000) and enteric neurones (Hirst et al. 1985), exhibit an AHP with a much slower time course. The AHPs in enteric neurones last between 5 and 30 s, and are not blocked by selective large-conductance (BK) and SK channel blockers. Although SK channels are known to exhibit heterogeneous kinetic behaviour, with different sensitivities to apamin, no electrophysiological state of the SK channel has been described to date that is sufficient to explain the long-lasting AHP in these cells.

Intermediate-conductance (IK) calcium-activated potassium channels are closely related to the SK channel family but differ significantly in their pharmacology and single-channel conductance. IK channels are blocked by clotrimazole, are not sensitive to voltage and exhibit single-channel conductance of 30–40 pS. IK channels were first cloned in 1997 and are 42–44% identical to channels of the SK family (Ishii et al. 1997). Their distribution among various tissues is consistent with a role in non-excitable cells such as endothelial and epithelial cells, blood cells such as erythrocytes and lymphocytes, and also smooth muscle tissues. Furthermore, owing to the lack of mRNA expression in brain (Ishii et al. 1997), it is generally thought that IK channels are not expressed in neural cells (Jensen et al. 2001; Sah and Faber 2002).

Recent evidence, however, indicates that IK channels may play important roles in some types of neurones. Intrinsic primary afferent neurones (IPANs) of the enteric nervous system exhibit a pronounced long-lasting AHP following action potentials. The time course of this slow AHP is correlated with the ensemble-averaged current calculated from IK channel openings following the action potential (Vogalis et al. 2002a). The electrophysiological properties of IK channels in IPANs are similar to those described in other cells (Alvarez et al. 1992; Ishii et al. 1997; Dunn 1998; Vandorpe et al. 1998; Jensen et al. 1999; Neylon et al. 1999; Joiner et al. 2003; Wang et al. 2003). They are insensitive to apamin and low concentrations of tetraethylammonium chloride (TEA; 5 mm), display voltage independence, have flickery opening and closing behaviour, and are activated by submicromolar levels of cytoplasmic Ca2+ (Vogalis et al. 2002a). As in other cell types, these channels in the guinea pig duodenal afterhyperpolarizing (AH) neurones are blocked by the IK channel inhibitor clotrimazole (Vogalis et al. 2002b).

The biophysical and pharmacological data indicating that IK channels occur in enteric neurones, and that their opening is responsible for the AHP, comes entirely from studies in guinea pig small intestine (Vogalis et al. 2001, 2002a, 2002b, 2003). Although antisera to IK channels are available, these have proven ineffective in guinea pigs (Furness et al. 2003). In rat and human, immunohistochemical studies have revealed IK channel immunoreactivity of enteric neurones (Arnold et al. 2003; Furness et al. 2003), and western blots from extracts of the rat external muscle, which includes the myenteric neurones, reveals an IK-immunoreactive protein that runs at approximately the same place as the IK channel monomer (Furness et al. 2003). However, there is no direct evidence that the neurones that express a functional IK-like channel correspond to the neurones that exhibit IK channel-like immunoreactivity. Moreover, IK channel gene expression has not been demonstrated in the enteric nervous system. We have pursued these correlations in the mouse colon, because the amino acid sequence against which the antisera were raised corresponds to that of mouse, and because myenteric neurones in the mouse colon exhibit an AHP similar to that observed in guinea pig small intestine (Bian et al. 2003; Nurgali et al. 2004).

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression and characterization of IK channel by RT–PCR and western blotting
  6. IK channel immunohistochemistry in the mouse distal colon
  7. Electrophysiological identification of IK channels
  8. Discussion
  9. General overview
  10. Biochemical characterization
  11. Subcellular localization
  12. Pharmacological characterization
  13. Correlation of the AHP with IK channel immunoreactivity
  14. Dogiel type I neurones
  15. Re-evaluation of AHPs in other neurones
  16. Potential clinical uses of IK channel blockers
  17. Acknowledgements
  18. References

Experiments were performed on segments of distal colon removed from BALBc mice (20–25 g) after they had been stunned by a blow to the head and killed by cervical dislocation. All procedures were approved by the University of Melbourne Animal Experimentation Ethics Committee. Segments of colon were taken from the region just oral to the pelvic brim, and the segments were placed in phosphate-buffered saline (PBS; NaCl 118 mm, KCl 4.8 mm, NaHCO3 25 mm, NaH2PO4 1.0 mm, MgSO4 1.2 mm, glucose 11.1 mm, CaCl2 2.5 mm; equilibrated with 95% O2/5% CO2) and initially kept at room temperature (18–22°C). The solution contained 3 µm nicardipine and 1 µm hyoscine (both from Sigma-Aldrich, Sydney, New South Wales, Australia) to inhibit muscle movement. The mucosa and submucosa were removed, and the serosa and longitudinal muscle were carefully dissected away to expose the myenteric plexus adhering to the circular muscle (Nurgali et al. 2004). This preparation was pinned to the sylgard base of a recording dish (volume 1 mL) which was placed on the stage of an inverted microscope and continuously superfused (4 mL/min) with physiological saline that had been preheated to yield a bath temperature of 33–35°C.

Electrophysiology

Neurones were impaled with conventional borosilicate glass microelectrodes filled with 1% biocytin (Sigma-Aldrich) in 1 m KCl. Electrode resistances were 100–210 MΩ. Recordings were made using an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA, USA). Signals were digitized at 1 or 10 kHz, using a Digidata 1322 A interface (Axon Instruments) and stored using PC-based data acquisition software (Axoscope 8.2; Axon Instruments).

Measurements of electrophysiological properties were made after allowing the impalements to stabilize for at least 15 min without applying intracellular holding current. Action potentials were evoked by intracellular pulses of 0.1 ms duration and 0.1–0.2 nA intensity. Compound AHPs were elicited by three intracellular pulses at 10–20-ms intervals. Electrophysiological data are presented as mean ± SEM.

Hyoscine hydrobromide, nicardipine and clotrimazole were obtained from Sigma-Aldrich. All drugs were applied by addition to the superfusion solution. Evoked responses were measured before drug application and after at least 20 min in the presence of drug in the extracellular solution.

Neurone identification

Biocytin was passed from the recording electrodes into the neurones during impalement. Once a neurone in a ganglion had been injected with biocytin, the ganglion was drawn and the recording electrode was moved to a fresh ganglion to avoid ambiguity of cell identity. Tissues were processed as described previously (Clerc et al. 1998). At the end of each experiment, the tissue was fixed overnight in 2% formaldehyde plus 0.2% picric acid in 0.1 m sodium phosphate buffer (pH 7.0), cleared in three changes of dimethylsulfoxide (DMSO), and washed three times in PBS. The tissue was then reacted with streptavidin coupled to Texas Red to reveal the biocytin, the preparations were mounted on glass slides and the cells were located by fluorescence microscopy.

Preparations in which impaled nerve cells had been identified were removed from the slides and washed in PBS, before conversion of the streptavidin, bound to the biocytin, to a permanent deposit. This was achieved using goat anti-streptavidin antiserum coupled to biotin (Vector Laboratories, Burlingame, CA, USA), diluted 1 : 50 at room temperature. The biotin was in turn localized using an avidin–biotin–horseradish peroxidase kit (Vectastain; Vector Laboratories). The horseradish peroxidase was reacted with diaminobenzidine and hydrogen peroxide to yield a permanent deposit (Clerc et al. 1998).

Cell shapes, positions and projections were evaluated on an Olympus BH microscope (Melbourne, Australia) under positive-low phase contrast optics, and drawn with the aid of a camera lucida drawing tube at × 400 or × 1000 magnification.

Immunohistochemistry

For tissue to be examined in wholemount, segments of gut were opened along the mesenteric border, stretched and pinned on to balsa board, with the mucosal side down. Samples were fixed overnight at 4°C in fixative (see above). The fixative was removed from the tissue by washing three times for 10 min with DMSO and then three times for 10 min with PBS. When required, tissue was stored at 4°C in PBS to which sodium azide (0.1%) was added. After fixation and clearing, the tissue was dissected into layers. The mucosa, submucosa and circular muscle were removed to produce wholemounts of longitudinal muscle plus myenteric plexus. Before exposure to primary antibodies, wholemounts were in some cases exposed to 0.1% NaBH4 in 0.1 m phosphate buffer (pH 7.2) for 45 min in order to reduce free aldehydes and reduce non-specific binding, especially in cell nuclei (Furness et al. 2003). Tissue was agitated in NaBH4 at room temperature and then the NaBH4 was washed out by means of six 5-min washes in PBS.

Before incubation with antibody, preparations were exposed to 10% normal sheep serum plus 1% Triton X100 for 30 min at room temperature. Incubation in anti-IK channel antisera was for 48 h at 4°C in antibody diluent containing 10% normal sheep serum. IK 38/6 was used at a dilution of 1 : 2000 and M20 was at a dilution of 1 : 400. After incubation in primary antiserum, preparations were rinsed three times each for 10 min in PBS and then incubated for 1 h at room temperature with secondary antibody, goat anti-rabbit IgG coupled to Alexa 488 (Molecular Probes Inc., Eugene, OR, USA).

Double-labelling for calretinin and Hu antigen (human neuronal protein; Molecular Probes, Inc., Eugene, OR, USA) immunoreactivity was achieved using combinations of antibodies. After preincubation in 10% normal horse serum plus 1% Triton X100 for 30 min at room temperature, preparations were incubated in mixed primary antisera with 10% normal horse serum. After three 10-min washes in PBS they were incubated in a mixture of secondary antibodies. After a further three 10-min washes in PBS the tissue was mounted in glycerol buffered with 0.5 m sodium carbonate (pH 8.6) or in Dako fluorescence mounting medium (Dako Corp., Carpinteria, CA, USA).

Preparations were examined on a Zeiss Axioplan microscope (North Ryde, NSW, Australia) equipped with the appropriate filter cubes for discriminating between FITC, Alexa 488 and Alexa 594 fluorescence. We used filter set 10 for FITC and Alexa 488 (450–490 nm excitation filter and 515–565 nm emission filter), and filter set 00 for Alexa 594 (530–585 nm excitation filter and 615 nm emission filter). Images were recorded using a SpotRT cooled charge-coupled device camera and SpotRT 3.2 software (Diagnostic Instruments, Sterling Heights, MI, USA). Preparations were also analysed by confocal microscopy on a Biorad (Hercules, CA, USA) MRC1024 confocal scanning laser system installed on a Zeiss Axioplan 2 microscope. The images were 512 × 512 pixels and the thickness of each optical section was nominally 0.5 µm. Immunoreactive cells were scanned as a series of optical sections with a centre to centre spacing of 0.2 µm. The images were further processed using Confocal Assistant (Todd Clarke Brelje), Corel PhotoPaint and Corel Draw software programs (Corel Corporation, Sydney, Australia).

The proportions of neurones in which antigen immunoreactivity was co-localized were determined by examining fluorescently labelled, double-stained, preparations. Neurones were first located by the presence of a fluorophore that labelled one antigen, and then the filter was switched to determine whether or not the neurone was labelled for a second antigen, located with a fluorophore of a different colour. In this way, proportions of neurones labelled for pairs of antigens were determined. Cohort size was generally 100 neurones and data were collected from preparations obtained from at least three animals. The percentage of neurones immunoreactive for a particular marker that were also immunoreactive for a second neurochemical was calculated and expressed as mean ± SEM for n preparations counted.

Antibody characterization

Two rabbit antisera were used to localize the IK channel. The first antiserum (IK38/6) was raised against a 15-amino acid synthetic peptide corresponding to the N-terminus of the mouse and rat IK channel (Furness et al. 2003). Antiserum M20 was raised against a 15-amino acid sequence from the N-terminal of the human IK channel, which differs in only two amino acids from the mouse sequence, and was affinity purified (Boettger et al. 2002). Immunoreactivity for the M20 antiserum, which labels both the same cells in immunohistochemical studies and the same 49-kDa band on western blots of mouse colon external muscle as the IK38/6 antiserum, was blocked by preincubation with the peptide against which it was raised (Furness et al. 2003). On western blots of human embryonic kidney 293 cells transfected with rat IK channel cDNA plasmid, the M20 antiserum revealed a strong immunoreactive band which was only of low abundance in untransfected cells.

Western blotting

Protein was extracted from samples of mouse colonic external muscle including the myenteric plexus (EM–MP). Segments of the colon were placed in a solution of ice cold PBS containing 50 µm phenylmethylsulfonyl fluoride and 1 µm nicardipine, opened along the mesenteric border, and were stretched and pinned, mucosa side down, in a sylgard dish on ice, before being dissected into layers. Samples of the EM–MP were collected in T-PER Tissue Protein Extraction Reagent (Pierce, Rockford, IL, USA) containing 50 µm phenylmethylsulfonyl fluoride, 1 µg/µL leupeptin, 1 µg/µL pepstatin and 1 µg/µL aprotinin. Samples were then sonicated on ice, and frozen at − 20°C until used. Protein concentration was quantified using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA).

Protein lysate (10–30 µg) from each sample was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) through 10% gels under reducing conditions. Samples were run with broad-range biotinylated SDS–PAGE standards (Bio-Rad) in order to determine molecular mass. Samples were then electrophoretically transferred, overnight at 30 V to polyvinylidene difluoride membranes (Hybond-P, Amersham, Melbourne, Victoria, Australia). Non-specific sites on the membrane were blocked using PBS-T (PBS plus 0.1% (v/v) Tween 20) with either 5% skimmed milk or 10% normal horse serum. Membranes were washed three times in PBS-T (for 15, 5 and 5 min) and then exposed to diluted primary antisera for 1 h. Three antisera against the IK channel (38/6, M20 and 559/3) were used. The 559/3 antiserum was raised in sheep against the same 15-amino acid peptide (IK channel N-terminus) used to raise the 38/6 antisera in rabbits. Unbound antibodies were removed by further washing in PBS-T. Blots were exposed to an horseradish peroxidase-conjugated donkey anti-rabbit or anti-sheep antiserum (Amersham, Castle Hill, NSW, Australia) for 1 h. All incubations and washes were performed at room temperature with constant agitation. The portion of the blot containing the biotinylated protein standards was exposed to an avidin–horseradish peroxidase conjugate (Vectastain ABC kit) for 30 min. Membranes were then washed again in PBS-T and the horseradish peroxidase-labelled proteins detected using the enhanced chemiluminescence detection system (ECL; Amersham) in conjunction with hyperfilm ECL (Amersham).

IK channel mRNA expression

Expression of mRNA was determined by RT–PCR and DNA sequencing. Total RNA was prepared from mouse EM–MP using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). RNA was reverse transcribed in a 10-µL reaction volume containing 10 × buffer, 0.6 µL oligo d(T) primers (60 ng), 1 µL dNTP mix (40 mm), Rnase-free water and StrataScript RT (Stratagene, La Jolla, CA, USA). The resulting cDNA mix (2 µL) was then used for PCR (Platinum Taq; Invitrogen, Carlsbad, CA, USA) in a reaction volume of 50 µL. A range of PCR primers was designed on the basis of the mouse IK1 nucleotide sequence (GenBank accession number AF042487); degenerate primers were also designed based on the mouse (AF042487), human (AF022150) and rat (AF190458, AF156554) sequences. Touchdown PCR was performed using annealing temperatures ranging from 74°C to 57°C, decreasing 1°C per cycle, followed by 30 cycles at 57°C, in a Hybaid PCR Express (ThermoHybaid, Ashford, Middlesex, UK) thermocycler. In some cases, 10% Q solution (Qiagen, Hilden, Germany) was added to increase the amplification efficiency of long products. The PCR product was purified using the MinElute PCR purification kit (Qiagen) and then sequenced using Big Dye Terminator V3.1 (ABI). Gel separation of the sequencing product was performed by the Australian Genome Research Facility, Melbourne. Sequence analysis was performed using programs provided by the Australian National Genomic Information Service (ANGIS).

Expression and characterization of IK channel by RT–PCR and western blotting

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression and characterization of IK channel by RT–PCR and western blotting
  6. IK channel immunohistochemistry in the mouse distal colon
  7. Electrophysiological identification of IK channels
  8. Discussion
  9. General overview
  10. Biochemical characterization
  11. Subcellular localization
  12. Pharmacological characterization
  13. Correlation of the AHP with IK channel immunoreactivity
  14. Dogiel type I neurones
  15. Re-evaluation of AHPs in other neurones
  16. Potential clinical uses of IK channel blockers
  17. Acknowledgements
  18. References

Expression of IK channel mRNA and protein in the mouse enteric nervous system was examined by RT–PCR and western blotting respectively. For RT–PCR, total RNA was extracted from EM–MP of the mouse distal colon. PCR amplicons were designed to overlap multiple introns in the IK channel gene to rule out the possibility that products were derived from genomic DNA. A typical RT–PCR experiment is shown in Fig. 1(a). The target sequence in this case (250 bp; nucleotides 849–1099 numbered from initiation of coding region) was amplified (left lane) using the PCR primers, forward 5′-CAACAAGGCGGAGAAACACG-3′ and reverse 5′-GCATCTTGGAGATGTCCAC-3′, based on the mIK1 cDNA sequence (Vandorpe et al. 1998; Wang et al. 2003). No PCR product was obtained when either reverse transcriptase (RT) (middle lane) or RNA (right lane) was omitted from the cDNA synthesis step, indicating that the PCR product was amplified from reverse transcribed RNA. Sequencing of the PCR product confirmed its identity as partial IK sequence. IK channel protein expression in mouse EM–MP was examined by western blotting. Figure 1(b) shows analysis of 30 µg EM–MP lysate separated by 10% SDS–PAGE and probed with three different sets of anti-IK channel antisera. A prominent band of 49 kDa was observed for each antiserum, which corresponds to the predicted size of a single IK channel subunit.

image

Figure 1. IK channel expression in the mouse distal colon myenteric plexus. (a) Expression of IK channel mRNA determined by RT–PCR. Total RNA was extracted from mouse distal colon EM–MP and reverse transcribed to form cDNA. The gel shows the results of PCR from reverse transcribed RNA (left lane), from RNA that was not reverse transcribed (middle lane), and from reactions in which the template was omitted (right lane). PCR was performed using IK channel-specific primers described in Results. DNA size was calculated against a ØX174 DNA-HaeIII digest standard. (b) IK channel protein expression in mouse enteric lysate determined by western blotting. Protein (30 µg) extracted from mouse distal colon EM–MP was resolved by 10% SDS–PAGE and transferred to polyvinylidene difluoride membranes for western analysis. Blots were probed with either rabbit (38/6) or sheep (559/3) antisera raised against a 15-amino acid N-terminal sequence of the rodent IK channel (Furness et al. 2003). Blots were also probed with the M20 antibody raised against the N-terminus of the human IK channel sequence (Boettger et al. 2002). Protein size markers are shown on left. Results are typical of at least three different preparations.

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The predicted amino acid sequence of the IK channel in mouse myenteric plexus was determined from the sequence of 5–18 overlapping RT–PCR products. The consensus full-length cDNA contained a 1275-bp open reading frame, which encodes a 425-amino acid protein that is identical to the IK channel expressed in other mouse tissues (Vandorpe et al. 1998). The mouse enteric IK channel shares 88% and 98% homology to the human and rat amino acid sequences respectively, and contains the six transmembrane domains and canonical K+ channel selectivity filter, GYG. Nine amino acids differ between the mouse and the previously published rat sequence (GenBank accession number AF190458) (Neylon et al. 1999): four on exofacial loops, two within the buried C-helix, and three close to the C-terminus. Residues T248 and V273, which are thought to be critical for clotrimazole binding (Hamilton et al. 2003), are present within the pore of the mouse sequence.

IK channel immunohistochemistry in the mouse distal colon

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression and characterization of IK channel by RT–PCR and western blotting
  6. IK channel immunohistochemistry in the mouse distal colon
  7. Electrophysiological identification of IK channels
  8. Discussion
  9. General overview
  10. Biochemical characterization
  11. Subcellular localization
  12. Pharmacological characterization
  13. Correlation of the AHP with IK channel immunoreactivity
  14. Dogiel type I neurones
  15. Re-evaluation of AHPs in other neurones
  16. Potential clinical uses of IK channel blockers
  17. Acknowledgements
  18. References

To determine which cells in the enteric nervous system express the IK channel, we performed immunohistochemistry using the rabbit anti-IK (38/6) and M20 antisera. EM–MP from the mouse distal colon was dissected and fixed for antibody labelling. IK channel immunoreactivity was found in a subset of neurones in the myenteric plexus with negligible binding in surrounding muscle.

The proportion of nerve cells in the longitudinal muscle myenteric plexus of the mouse distal colon exhibiting IK channel immunoreactivity to the IK38/6 antibody was 21.25% (n = 642), estimated by counting neurones in preparations that were double-stained using antiserum to the general neuronal marker, the Hu RNA-binding protein.

Neurones of three morphologies, Dogiel type II, Dogiel type I and stellate neurones (Fig. 2), were IK channel immunoreactive. The proportions of Dogiel type II neurones, identified by morphology and calretinin immunoreactivity, that were IK channel-immunoreactive were counted in six preparations from three mice. A high proportion of Dogiel type II neurones (97.7%; n = 130) expressed IK channel immunoreactivity. A similar number of Dogiel type I neurones expressed IK channel immunoreactivity but these represented only 20.9% (n = 173) of the total Dogiel type I population. Large stellate neurones were also IK channel immunoreactive but these neurones were very rare (only eight were found) and were not immunoreactive for calretinin. IK channel immunoreactivity was found at the cell surface and in the cytoplasm, but not in the cell nucleus.

image

Figure 2. IK channel immunoreactivity in neurones of the mouse distal colon. Confocal images from wholemount preparations show examples of cell bodies in myenteric ganglia. Immunoreactivity was found in neurones of three different morphologies. (a) Oval-shaped Dogiel type II cell with strong IK channel immunoreactivity in the cytoplasm and at the cell surface. A minority of these cells show expression in the initial segment of the process (arrow) and, although these cells are often multipolar, immunoreactivity was rarely found in more than one process. (b) Dogiel type I cell with lamellar dendrites (arrow). Immunoreactivity was strong at the surface of the soma but not in the axons. Immunoreactive non-varicose axons can be seen traversing the ganglia. (c) Large stellate neurones showing immunoreactivity in broad branching dendrites. (d, e) Identification of neurones by double-staining with IK channel and calretinin antisera. IK channel-immunoreactive Dogiel type II cells are positive for calretinin (arrow) whereas IK channel-immunoreactive Dogiel type I cells are negative (asterisks). Neurones that are calretinin-immunoreactive but not IK-channel immunoreactive are indicated by arrowheads. All preparations were stained with IK38/6 antisera to label IK channels. Scale bars represent 25 µm.

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The Dogiel type II cells were oval in profile, and the majority showed immunoreactivity in the soma, but not in the processes. In a minority of cases, one clearly immunoreactive process that generally had immunoreactivity only in about the first 50 µm was observed (Fig. 2a). In some cases the IK channel immunoreactivity of the process was more prominent than that of the cell body, as has been reported for Dogiel type II neurones in the rat enteric nervous system (Furness et al. 2003), but more often the processes were weakly immunoreactive, or not revealed at all. Despite the fact that these neurones are generally multipolar, as demonstrated when the cells are filled with biocytin through a recording electrode (Nurgali et al. 2004), it was rare to detect immunoreactivity in more than one process. In rare cases, processes could be followed out of the ganglia, and in some instances these entered nerve strands that ran through the circular muscle, towards the mucosa. The numerous terminals of calretinin neurones in the ganglia were not IK channel immunoreactive (Fig. 2).

The Dogiel type I neurones that were IK channel immunoreactive varied considerably in size, from about 10–20 µm in diameter. These neurones often had their cell bodies and lamellar dendrites in different planes, as has been reported in guinea pig (Pompolo and Furness 1990). Focus at the level of the cell body showed strong staining at the cell membrane (Fig. 2b). At the level of the dendrites, the immunoreactive processes were revealed. The axons of Dogiel type I neurones were not immunoreactive.

Large stellate neurones with IK channel immunoreactivity (Fig. 2c) were rare. These had immunoreactivity in broad branching dendrites, but not in their axons. Immunoreactive non-varicose axons, and some immunoreactive axons with undulating profiles, ran through the ganglia but, as mentioned above, there was no immunoreactivity of the varicose axons in the ganglia, or of the axons that innervate the longitudinal and circular muscle.

Electrophysiological identification of IK channels

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression and characterization of IK channel by RT–PCR and western blotting
  6. IK channel immunohistochemistry in the mouse distal colon
  7. Electrophysiological identification of IK channels
  8. Discussion
  9. General overview
  10. Biochemical characterization
  11. Subcellular localization
  12. Pharmacological characterization
  13. Correlation of the AHP with IK channel immunoreactivity
  14. Dogiel type I neurones
  15. Re-evaluation of AHPs in other neurones
  16. Potential clinical uses of IK channel blockers
  17. Acknowledgements
  18. References

About 45% of neurones expressing IK channel immunoreactivity were Dogiel type II neurones. These neurones are also known as AH neurones because of the slow AHP that follows the action potential. To investigate whether IK channels are responsible for the AHP in the mouse Dogiel type II neurones, we examined the effect of the IK channel blocker clotrimazole. Intracellular recordings were made from 20 neurones with AH type electrophysiology. All these neurones had large-amplitude action potentials (81.9 ±2.4 mV) with a hump on the repolarizing phase. Average cell capacitance was 24.9 ± 2.5 pF. The presence of a hump on the falling phase was confirmed by analysing the first time derivative (dV/dt) of the action potential. These neurones fired a single action potential in response to a 500-ms depolarizing current pulse, at just greater than threshold strength, injected through the recording electrode. The threshold current for action potential firing was reached at 0.10 ± 0.01 nA. Slow AHPs followed single or multiple soma action potentials in these neurones. In some neurones, the amplitudes of slow AHPs were too small (< 3 mV) for accurate measurement of their amplitude and duration to be made.

Slow AHPs (average amplitude before drug −6.9 ± 2.0 mV; width at half-amplitude 3.5 ± 1.4 s) were completely inhibited by clotrimazole (5–9 µm) within 20–30 min (Figs 3a and b) in all AH neurones tested (n =4). At this concentration clotrimazole did not affect action potential amplitude (81.9 ± 2.4 mV) or duration (1.7 ±0.2 ms width at half-amplitude). There was no significant effect of clotrimazole on input resistance (200 ± 28 MOhm) or resting membrane potential (− 60.8 ± 2.1 mV). The inhibitory effects of clotrimazole on the slow AHP were fully reversed after 20–30 min of washout with normal physiological saline.

image

Figure 3. Effect of IK channel antagonist clotrimazole on slow AHP in morphologically identified myenteric neurones. (a) i. Action potentials followed by a slow AHP elicited by a train of three intracellular current pulses (0.2 nA, 10 ms duration, 10-ms intervals). ii. Slow AHP was inhibited by clotrimazole (9 µm). (b) i. Slow AHP elicited by three-pulse stimulation in another neurone. ii. The AHP, which was slightly smaller in amplitude, was blocked by a lower concentration of clotrimazole (5 µm). (c) Camera lucida drawing of the neurone from which the recordings shown in (b) were taken after it had been filled through the recording electrode with biocytin, which was subsequently converted to a permanent diaminobenzidine deposit. This is a Dogiel type II neurone with multiple processes (full extent not shown). Scale bar 50 µm.

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Neurones in which the AHP was blocked by clotrimazole were filled with biocytin and their morphology was determined. All these AH neurones had Dogiel type II morphology with round or oval cell bodies and multiple axon-like circumferentially orientated processes (Fig. 3c).

General overview

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression and characterization of IK channel by RT–PCR and western blotting
  6. IK channel immunohistochemistry in the mouse distal colon
  7. Electrophysiological identification of IK channels
  8. Discussion
  9. General overview
  10. Biochemical characterization
  11. Subcellular localization
  12. Pharmacological characterization
  13. Correlation of the AHP with IK channel immunoreactivity
  14. Dogiel type I neurones
  15. Re-evaluation of AHPs in other neurones
  16. Potential clinical uses of IK channel blockers
  17. Acknowledgements
  18. References

Initial reports indicating only limited expression of IK channel mRNA in brain, the lack of electrophysiological evidence for IK channels in neurones, and its abundant expression in non-excitable cells, have led to the consensus that IK channels do not play a role in neurones (Sah and Faber 2002). Nonetheless, several examples exist in the literature that describe Ca2+-dependent potassium currents in neurones that cannot be explained by conventional K+ channels thought to exist in these cells. In particular, the Ca2+-activated long-lasting AHP that follows action potentials in a subset of enteric neurones has biophysical and pharmacological characteristics that indicate it is not caused by BK or SK channel openings (Vogalis et al. 2002b). Electrophysiological evidence has implicated IK-like channels in the long-lasting AHP, but only in guinea pig intestine where the protein has not been identified owing to the lack of suitable antibodies. In the present study, we demonstrate that IK channels are expressed on the same myenteric neurones in the mouse that exhibit a clotrimazole-sensitive, long-lasting AHP. Analysis of RNA isolated from the myenteric plexus revealed an IK channel mRNA transcript that was identical to the IK channel expressed in other mouse tissues. Taken together, these data provide evidence that AHP currents in myenteric neurones are mediated by IK channels.

Biochemical characterization

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression and characterization of IK channel by RT–PCR and western blotting
  6. IK channel immunohistochemistry in the mouse distal colon
  7. Electrophysiological identification of IK channels
  8. Discussion
  9. General overview
  10. Biochemical characterization
  11. Subcellular localization
  12. Pharmacological characterization
  13. Correlation of the AHP with IK channel immunoreactivity
  14. Dogiel type I neurones
  15. Re-evaluation of AHPs in other neurones
  16. Potential clinical uses of IK channel blockers
  17. Acknowledgements
  18. References

IK channels were detected in the EM–MP by western blotting. The apparent size of the IK channel protein (49 Da) in western blots is close to that predicted by translation of the open reading frame of the mIK1 gene (47 784 Da). A slightly smaller protein (40–45 kDa) was found in western blots of extracts of mouse colon (Joiner et al. 2003) and the rat ileum myenteric plexus (Furness et al. 2003), and an even smaller polypeptide is produced by in vitro translation of the mIK1 cRNA by rabbit reticulocyte lysate (Vandorpe et al. 1998). The reason for these differences in molecular weight is not known, although it does not appear to be due to differences in glycosylation state of the protein (Vandorpe et al. 1998). It is possible that the IK channel subunits can be degraded during extraction and experimental manipulation or subjected to post-translational modification.

Subcellular localization

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression and characterization of IK channel by RT–PCR and western blotting
  6. IK channel immunohistochemistry in the mouse distal colon
  7. Electrophysiological identification of IK channels
  8. Discussion
  9. General overview
  10. Biochemical characterization
  11. Subcellular localization
  12. Pharmacological characterization
  13. Correlation of the AHP with IK channel immunoreactivity
  14. Dogiel type I neurones
  15. Re-evaluation of AHPs in other neurones
  16. Potential clinical uses of IK channel blockers
  17. Acknowledgements
  18. References

Immunoreactivity was found predominantly in the soma in mouse enteric neurones, and rarely in the processes. This differs from our finding in rat in which immunoreactivity was localized at the initial segment of the process. These data may point to a difference in trafficking of the IK channel between species, or indicate that action potentials are modulated by the AHP at the soma in mouse neurones and also in the processes in rat neurones. Synaptic inputs to these neurones in the guinea pig are mainly on the soma, but they also occur on the initial segments (Pompolo and Furness 1988), suggesting that excitability is controlled in both regions. However, the location of synapses on mouse IPANs has not been investigated.

Pharmacological characterization

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression and characterization of IK channel by RT–PCR and western blotting
  6. IK channel immunohistochemistry in the mouse distal colon
  7. Electrophysiological identification of IK channels
  8. Discussion
  9. General overview
  10. Biochemical characterization
  11. Subcellular localization
  12. Pharmacological characterization
  13. Correlation of the AHP with IK channel immunoreactivity
  14. Dogiel type I neurones
  15. Re-evaluation of AHPs in other neurones
  16. Potential clinical uses of IK channel blockers
  17. Acknowledgements
  18. References

Clotrimazole concentrations of 2–10 µm were sufficient to block the slow AHP in the mouse distal colon myenteric neurones. Clotrimazole is a selective blocker of IK channels at nanomolar (Alvarez et al. 1992; Ishii et al. 1997; Wang et al. 2003) and micromolar (Dunn 1998; Jensen et al. 1999; Joiner et al. 2003) concentrations. Clotrimazole blocks the whole-cell current underlying the slow AHP in cultured hippocampal pyramidal neurones with an IC50 of 1–2 µm (Shah et al. 2001) and blocks IK channel openings in guinea pig myenteric neurones in excised patches (Vogalis et al. 2002a). Clotrimazole can also inhibit BK channels in ferret portal vein vascular smooth muscle cells (Rittenhouse et al. 1997) and Ca2+-independent voltage-gated K+ channels in carotid body cells (Hatton and Peers 1996). However, in our experiments clotrimazole did not affect the shape of the action potential implying that there was no effect of clotrimazole on BK channels. These results suggest that clotrimazole inhibits the AHP by directly blocking IK channels. It is noteworthy that the IK channel subunit mRNA we isolated from mouse myenteric plexus contains the essential amino acids (T248 and V273) required for clotrimazole binding.

Correlation of the AHP with IK channel immunoreactivity

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression and characterization of IK channel by RT–PCR and western blotting
  6. IK channel immunohistochemistry in the mouse distal colon
  7. Electrophysiological identification of IK channels
  8. Discussion
  9. General overview
  10. Biochemical characterization
  11. Subcellular localization
  12. Pharmacological characterization
  13. Correlation of the AHP with IK channel immunoreactivity
  14. Dogiel type I neurones
  15. Re-evaluation of AHPs in other neurones
  16. Potential clinical uses of IK channel blockers
  17. Acknowledgements
  18. References

Clotrimazole has been shown previously to block the channels underlying the AHP in the guinea pig duodenum (Vogalis et al. 2002b). Available antisera, raised against the N-terminus of the mouse and rat IK sequence, are not successful in guinea pig tissues, and so no correlation with demonstration of functional IK channel expression has been possible. Conversely, we have described IK channel immunoreactivity predominantly on Dogiel type II neurones in the rat gastrointestinal tract, although no electrophysiological recordings were made owing to the excessive connective tissue in this species. AHPs occur in Dogiel type II neurones in the mouse distal colon (Nurgali et al. 2004), and these cells are morphologically similar to those in the guinea pig small and large intestine (Iyer et al. 1988; Lomax et al. 1999). Thus, the present study shows for the first time that the clotrimazole-sensitive AHP is correlated with expression of IK channel immunoreactivity on Dogiel type II type neurones.

Dogiel type I neurones

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression and characterization of IK channel by RT–PCR and western blotting
  6. IK channel immunohistochemistry in the mouse distal colon
  7. Electrophysiological identification of IK channels
  8. Discussion
  9. General overview
  10. Biochemical characterization
  11. Subcellular localization
  12. Pharmacological characterization
  13. Correlation of the AHP with IK channel immunoreactivity
  14. Dogiel type I neurones
  15. Re-evaluation of AHPs in other neurones
  16. Potential clinical uses of IK channel blockers
  17. Acknowledgements
  18. References

IK channel immunoreactivity was also expressed on neurones with Dogiel type I morphology, and this accounts for about half of the total number of IK channel-immunoreactive neurones in the mouse myenteric plexus. The majority of Dogiel type I neurones in this tissue have S type electrophysiology and do not express a slow AHP after action potentials; only 4.3% of S neurones exhibit AH type electrophysiology (Nurgali et al. 2004). As about 20% of Dogiel type I neurones express IK channel immunoreactivity but only a small proportion (4.3%) exhibit slow AHPs, the functional role of IK channels on Dogiel type I neurones remains to be determined. Dogiel type I cells exhibit action potentials that are completely blocked by tetrodotoxin, indicating that there is little or no inward Ca2+ current (Bornstein et al. 1994). The lack of a transient Ca2+ increase associated with the action potential would explain the absence of an AHP in neurones that express IK channels. In these neurones IK channel activity may be modulated by Ca2+ fluctuations caused in other ways, for example by the actions of neurotransmitters and hormones.

Re-evaluation of AHPs in other neurones

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression and characterization of IK channel by RT–PCR and western blotting
  6. IK channel immunohistochemistry in the mouse distal colon
  7. Electrophysiological identification of IK channels
  8. Discussion
  9. General overview
  10. Biochemical characterization
  11. Subcellular localization
  12. Pharmacological characterization
  13. Correlation of the AHP with IK channel immunoreactivity
  14. Dogiel type I neurones
  15. Re-evaluation of AHPs in other neurones
  16. Potential clinical uses of IK channel blockers
  17. Acknowledgements
  18. References

Because IK channels have been implicated in the slow AHP in intrinsic primary afferent neurones of the enteric nervous system, it may be timely to re-evaluate the possible role of IK channels in other neuronal systems, including the CNS. The finding that IK channel mRNA can be detected in brain by RT–PCR (Vandorpe et al. 1998), but not by northern analysis (Ishii et al. 1997), suggests that IK channels may be expressed only in specialized regions of the CNS. One of the regions in which it may play a role is the hippocampus, the part of the CNS involved in spatial learning and memory. Neurones in this region undergo large changes in firing properties, associated with pronounced synaptic plasticity. The slow AHP observed in hippocampal pyramidal neurones (Storm 1990; Sah 1996; Shah et al. 2001) is modulated by neurotransmitters acting through kinase–phosphatase reactions (Lancaster and Adams 1986; Pedarzani et al. 1998; Krause and Pedarzani 2000), a property consistent with an involvement of IK channels. Shah and co-workers reported pharmacological evidence for IK channels mediating the slow AHP component (Shah et al. 2001). It is therefore possible that IK channels are preferentially expressed in such specialized regions because of their unique sensitivity to modulation by second messenger signalling pathways. In contrast to SK channels, which do not appear to be modulated by protein kinases (Xia et al. 1998), IK channels contain multiple phosphorylation consensus sites (Neylon et al. 1999; Gerlach et al. 2000; Jensen et al. 2001; von Hahn et al. 2001; Wulf and Schwab 2002) and phosphorylation leads to changes in the firing properties of the neurones (Kawai et al. 2003; Vogalis et al. 2003). Thus, our finding of a unique role of IK channels in a specialized class of enteric neurones highlights the importance of a detailed examination of their expression in the CNS and other neural systems.

Potential clinical uses of IK channel blockers

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression and characterization of IK channel by RT–PCR and western blotting
  6. IK channel immunohistochemistry in the mouse distal colon
  7. Electrophysiological identification of IK channels
  8. Discussion
  9. General overview
  10. Biochemical characterization
  11. Subcellular localization
  12. Pharmacological characterization
  13. Correlation of the AHP with IK channel immunoreactivity
  14. Dogiel type I neurones
  15. Re-evaluation of AHPs in other neurones
  16. Potential clinical uses of IK channel blockers
  17. Acknowledgements
  18. References

Clotrimazole is used currently as an antimycotic agent, however, it is in clinical trial for the treatment of sickle cell disease, diarrhoea and rheumatoid arthritis (Jensen et al. 2001; Wulff et al. 2003). Its clinical use is limited by its side-effect of blocking cytochrome P450 enzymes, and related compounds without that activity have been developed. ICA-17043 is in clinical trials for the treatment of sickle cell anaemia (Stocker et al. 2003). In view of the recent discovery of the importance of IK channels in proliferative diseases, it is likely that channel blockers will have more widespread use. For example, the chemically related TRAM-34 has recently been noted to prevent vascular restenosis in a rat model (Köhler et al. 2003), and it and related compounds hold therapeutic promise as potent immunosuppressants (Wulff et al. 2000). In future, IK channel blockers are likely to be tried as anticancer therapy following recent findings of their inhibition of cancer cell proliferation (Ouadid-Ahidouch et al. 2004).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Expression and characterization of IK channel by RT–PCR and western blotting
  6. IK channel immunohistochemistry in the mouse distal colon
  7. Electrophysiological identification of IK channels
  8. Discussion
  9. General overview
  10. Biochemical characterization
  11. Subcellular localization
  12. Pharmacological characterization
  13. Correlation of the AHP with IK channel immunoreactivity
  14. Dogiel type I neurones
  15. Re-evaluation of AHPs in other neurones
  16. Potential clinical uses of IK channel blockers
  17. Acknowledgements
  18. References
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