Calcium‐sensing receptor regulates Kv7 channels via Gi/o protein signalling and modulates excitability of human induced pluripotent stem cell‐derived nociceptive‐like neurons

Neuropathic pain, a debilitating condition with unmet medical needs, can be characterised as hyperexcitability of nociceptive neurons caused by dysfunction of ion channels. Voltage‐gated potassium channels type 7 (Kv7), responsible for maintaining neuronal resting membrane potential and thus excitability, reside under tight control of G protein‐coupled receptors (GPCRs). Calcium‐sensing receptor (CaSR) is a GPCR that regulates the activity of numerous ion channels, but whether CaSR can control Kv7 channel function has been unexplored until now.


| INTRODUCTION
Neuropathic pain, defined as a pain caused by a lesion or disease affecting the somatosensory system, exerts significant impacts on individuals' quality of life (Shueb et al., 2015) and causes a serious socio-economic burden (Schaefer et al., 2014).The pathophysiological mechanisms of neuropathic pain involve hyperexcitability of nociceptive neurons associated with dysfunction of neuronal ion channels, such as voltage-gated sodium (Na V ), voltage-gated potassium (K V ), and voltage-gated calcium (Ca V ) channels (Li et al., 2014;Siqueira et al., 2009;Takeda et al., 2011).Carbamazepine, a Na V channel blocker and gabapentinoids, Ca V channel blockers, have been used for neuropathic pain management.However, these medications are not always effective in producing a satisfactory pain reduction (Haviv et al., 2014), suggesting that direct targeting of specific ion channels may not be an optimal solution.Upstream regulatory mechanisms of key ion channels, via G protein-coupled receptors (GPCRs), may be implicated in the pathophysiology of neuronal hyperresponsiveness, suggesting that GPCRs might provide a more appropriate therapeutic avenue for neuropathic pain management (Pan et al., 2008).
Negative allosteric modulators (NAMs) of CaSR, more commonly known as calcilytics, have demonstrated promising therapeutic uses in certain neurological diseases, for example, traumatic brain injury and Alzheimer's disease (Armato et al., 2013;Xue et al., 2017), whereas positive allosteric modulators (PAMs) or calcimimetics were suggested for neuroblastoma therapy (Rodríguez-Hernández et al., 2016).The role of CaSR in inflammation (Iamartino & Brandi, 2022) also suggests that CaSR might be associated with neuropathic pain, because inflammatory processes are relevant in the pathophysiology of neuropathic pain (Ellis & Bennett, 2013).
What is already known?
• Neuronal Kv7 channels regulate excitability, and their dysfunction is associated with neuropathic pain.
• CaSR can modulate the activity of neuronal ion channels and thus excitability.
What does this study add?
• Activation of CaSR inhibits Kv7 channels via G i/o protein signalling.
• Calcilytic drugs can rescue hiPSC-derived nociceptive-like neurons from hyperexcitability induced by an algogenic cocktail.

What is the clinical significance?
• CaSR may be a therapeutic target for Kv7 channelassociated hyperexcitability disorders, such as neuropathic pain.
Based on the evidence that neuronal hyperexcitability is associated with dysfunctions of CaSR (Kapoor et al., 2008) and neuronal Kv7.2/7.3 channels (Chokvithaya et al., 2023;Schroeder et al., 1998), it is conceivable that a crosslink between CaSR and Kv7.2/7.3 channels exists and contributes to the regulation of neuronal excitability.
This study aimed to establish the molecular pathway linking CaSR and Kv7.2/7.3 channels, and to explore the potential of calcilytics in suppressing neuronal hyperexcitability, using a human induced pluripotent stem cell (hiPSC)-derived nociceptive-like neuronal model.

| Materials
The following compounds and antibodies were used in this study:  10% foetal bovine serum (Life Technologies), 1% L-glutamine (Life Technologies) and 1% penicillin/streptomycin (Life Technologies), and maintained in a humidified incubator at 37 C with 5% CO 2 /95% O 2 .The culture medium was replaced every 2 to 3 days and confluency was assessed with a Leica DM IL inverted microscope (Leitz, Wetzlar, Germany).The cells were passaged using 0.25% trypsin-ethylenediamine tetra acetic acid (EDTA; Life Technologies) upon reaching 80% confluency.

| Isolation and culture of primary DRG neurons
Animal procedures were approved by the Animal Welfare and Ethical Review Body of Newcastle University.Animal studies are reported in compliance with the ARRIVE guidelines (Percie du Sert et al., 2020) and with the recommendations made by the British Journal of Pharmacology (Lilley et al., 2020).Naïve 6-month-old male CB57BL/6 mice (n = 12 mice, Comparative Biology Centre, Newcastle University, UK) were housed in 500-cm 2 polycarbonate cages under environmentally controlled conditions (20-24 C, 12-h light-dark cycle, 45%-65% humidities) and with free access to food and water.
The mice were killed by cervical dislocation and their dorsal root ganglia (DRG) extracted from all spinal levels under a microscope.
The collected DRG were enzymatically digested using collagenase Experiments were carried out three to 14 days after plating.

| Immunocytochemistry
Cells (CHO-Kv7.2/7.3,HEK-CaSR, or hiPSC-derived neurons) were plated on 13-mm diameter circular glass coverslips, fixed with 4% paraformaldehyde (Fisher Scientific) for 15 min and washed with phosphate buffer solution (PBS) three times.The fixed cells were blocked and permeabilised with 1% bovine serum albumin (Merck) and 0.1% Triton™ X-100 (Merck) in PBS for 1 h at room temperature.The coverslips were incubated with the primary antibody diluted in the blocking/permeabilisation solution, at a concentration as indicated above, and kept at 4 C overnight.The next day, the coverslips were washed with PBS three times followed by an application of the secondary antibody for 1 h at room temperature.
Afterwards, the coverslips were washed with PBS three times before being mounted using ProLong™ Diamond Antifade Mountant with 4 0 ,6-diamidino-2-phenylindole (DAPI; Life Technologies).Images were obtained with Olympus BX61 fluorescence microscope or Zeiss LSM800 laser confocal scanning microscope and processed using cellSens imaging software (Olympus, Tokyo, Japan).For the determination of the spontaneous activity, cells were held with a current of 0 pA for 1 min to allow for observation of any spontaneous action potentials (sAP) being generated.The cells were categorised into three groups based on the sAP type: quiet, attempting sAP and complete sAP (Figure 6a) (Telezhkin et al., 2016).The ability of the cells to generate induced action potentials (iAPs) was determined using a 1-s current step injection protocol.First, the cells were artificially hyperpolarised and held between À80 and À90 mV.Afterwards, the current step was incrementally increased by 10 pA per sweep up to a maximum of 180 pA; 30 ms was allowed between steps for recovery.

| Electrophysiology
Classification of the iAP type was done at the threshold level when the first iAP was produced, and the cells were categorised into three groups: complete single iAP, attempting train of iAP and complete train of iAP (Figure 6c) (Telezhkin et al., 2016).The cells, producing immature iAPs that did not reach 0 mV, were excluded from analyses.
Spike analysis of the first iAP was performed on cells that produced at least a complete single iAP.Using Clampfit 10.2, several characteristics of an iAP were determined as shown in Figure 7a.Some characteristics can be measured directly, including overshoot, afterhyperpolarisation and spike height.To determine the half-height width, the voltage at 50% of the spike height was first measured, and the time difference between which this voltage was reached during depolarisation and repolarisation was calculated.The depolarisation rate and repolarisation rate were defined as the maximum rising slope and maximum declining slope, respectively, which were measured from the first differentiation of voltage with respect to time.The threshold was determined at the peak of the third differentiation of voltage with respect to time during the depolarisation phase of an AP (Henze & Buzsáki, 2001;Telezhkin et al., 2016).were incubated at 37 C with 5% CO 2 /95% O 2 .The culture medium in each chamber was replaced the next day.For the control group, KCNQ2/3 cDNA was omitted.
To investigate the effect of a calcilytic on excitability, 1 μM NPS-2143 was applied both alone and in combination with the algogenic cocktail to separate cultures of hiPSC-derived nociceptive-like neurons for 3 to 4 days.Analyses of the spontaneous activity, iAP type, characteristics of the first iAP and spike frequency were performed.

| Data and statistical analyses
All data are presented as mean ± standard error of the mean (SEM).Data were analysed and visualised using Clampfit 10.2 (Molecular Devices) and GraphPad Prism 9 (GraphPad Software, San Diego, USA).
Following characterisation of the CaSR-Kv7 channel crosslink in several cellular models, our next series of experiments explored the possibility of targeting such crosslink for controlling neuronal hyperexcitability using the hiPSC-derived hyperexcitability model due to its scalability and being a human-derived model.3.5 | An algogenic cocktail enhances induced excitability of hiPSC-derived nociceptive-like neurons without affecting spontaneous activity Functional assessment with patch-clamp electrophysiology demonstrated a spectrum of spontaneous and induced excitability in the hiPSC-derived nociceptive-like neurons (Figure 6a-d).Following 3 to 4 days of incubation with an algogenic cocktail, most of the hiPSCderived nociceptive-like neurons showed no sAP, which was similar to the controls (chi-squared [6]=3.7,P=0.72; Figure 6).The proportion of the algogenic cocktail-treated neurons with a complete train of iAPs, however, was more than twice relative to the control (24.5% vs. 11.9%) (chi-squared [6]=11.8,P=0.068; Figure 6d).In addition, spike analysis of the first iAP was performed in the hiPSC-derived nociceptive-like neurons (Figure 7a-e).The total spike height of the algogenic cocktail-treated neurons was significantly increased by 15.7 ± 5.7 mV, compared to the controls (Figure 7c).

| NPS-2143 rescues hiPSC-derived nociceptive-like neurons from hyperexcitability induced by the algogenic cocktail
Like the algogenic cocktail, 1-μM NPS-2143 did not impose a significant effect on the spontaneous excitability of hiPSC-derived nociceptive-like neurons (Figure 6b).When NPS-2143 was applied simultaneously with the algogenic cocktail, a similar distribution of the neurons in each induced excitability classification to the controls was observed (Figure 6d).
Spike analysis of the first iAP showed that NPS-2143 enhanced certain characteristics associated with the repolarisationhyperpolarisation phase of APs, including afterhyperpolarisation, that in the presence of algogenic cocktail + NPS-2134 and NPS-2134 alone, demonstrated significant difference from the control (P=0.02 and P=0.04, respectively; Figure 7b), repolarisation rate significantly decreased in the presence of NP-S2134 (P=0.003; Figure 7d) and half-height width ssignificantly decreased in the presence of algogenic cocktail + NP-S2134 and NP-S2134 alone (P=0.016 and P=0.001, respectively; Figure 7e).The effect of the algogenic cocktail in enhancing the total spike height was sgnificant (P=0.021),whereas that effect was not apparent in the presence of NPS-2143 (Figure 7c).No significant difference in the threshold, overshoot and depolarisation rate was noted between the four groups.
3.7 | Algogenic cocktail alters the relationship between the injected current levels and spike frequency in hiPSC-derived nociceptive-like neurons The relationship between the injected current level and iAP spike frequency (the number of iAPs generated per second) of the hiPSCderived nociceptive-like neurons exhibiting a complete train of iAPs was analysed in four different conditions (Figure 8a-f).Two-way ANOVA revealed significant effects of both the injected current level and condition on the spike frequency.In the controls, increasing the current amplitude correlated to a certain extent with an increase in the spike frequency, with two peaks observed at 30 pA and 140 pA (Figure 8a).In the algogenic cocktail-treated neurons, the spike frequency reached its peak at 50 pA then continuously declined with no secondary peak observed, so the mean spike frequency was significantly reduced compared to the Control (Figure 8b); a similar trajectory was found when the cells were treated in the algogenic cocktail plus NPS-2143 (Figure 8c).The NPS-2143 group showed a similar current-spike frequency relationship to the control, but the two peaks were slightly shifted to 50 and 170 pA, respectively (Figure 8d).Post hoc analysis with Dunnett's multiple comparison tests revealed the algogenic cocktail significantly reduced the mean iAP spike frequency (mean difference: -2.0 ± 0.6 Hz; P=0.002), which was rescued by cotreatment with NPS-2143 (Figure 8f).

| DISCUSSION
The significance of this study is two-fold: i) the discovery of the CaSR-Kv7.2/7.3 channel crosslink and ii) the therapeutic potential of targeting CaSR for neuronal hyperexcitability management.] o in our study, which was to be expected and consistent with a previous report (Leong et al., 2021).The EC 50 value for [Ca 2+ ] o could be underestimated as we did not test [Ca 2+ ] o at concentrations higher Effects of an algogenic cocktail and NPS-2143 on the spontaneous and induced excitability of hiPSC-derived nociceptive-like neurons.(a) hiPSC-derived nociceptive-like neurons were classified as having complete sAP (i) when at least one AP with its peak above 0 mV was observed.Attempting sAP (ii) was defined when an abrupt change in the RMP that looked like an AP occurred but did not reach 0 mV.When no AP was present during a 1-min period, the neurons were classified as quiet (iii).(b) Graph shows the percentage of hiPSC-derived nociceptivelike neurons at each spontaneous excitability level in four conditions: control (Ct; n = 125 cells from nine cultures), the algogenic cocktail (AC; n = 62 cells from three cultures), the algogenic cocktail plus 1-μM NPS-2143 (AC + N; n = 56 cells from four cultures) and 1-μM NPS-2143 (N; n = 38 cells from three cultures).(c) hiPSC-derived nociceptive-like neurons were classified as having a complete train of iAPs when more than one iAP overshot above 0 mV (i).When multiple iAPs were observed but only one iAP overshot above 0 mV, the cells were classified as having an attempting train of iAPs (ii).Cells with a single iAP overshooting above 0 mV were defined as having a complete single iAP (iii).(d) Graph shows the percentage of hiPSC-derived nociceptive-like neurons at each induced excitability level in four conditions: control (Ct; n = 29 cells from seven cultures), the algogenic cocktail (AC; n = 19 cells from three cultures), the algogenic cocktail plus 1-μM NPS-2143 (AC + N; n = 19 cells from four cultures) and 1-μM NPS-2143 (N; n = 16 cells from three cultures).Statistical analyses were performed using the chi-squared test.
than 10 mM.Since inhibition of neuronal Kv7 channel activity leads to augmented excitability (Liu et al., 2010;Peng et al., 2017), our findings fit into the paradigm that CaSR activation enhances neuronal excitability (Vyleta & Smith, 2011) and it follows that CaSR inhibition with calcilytics may suppress neuronal hyperexcitability.
The lack of retigabine's consistent effect on I m and RMP in our hiPSC-derived model may suggest that a tonic activity of the CaSR suppresses the function of Kv7 channels.This notion is supported by a previous study, which showed that neurons isolated from CaSR-positive mice had more depolarised RMP and firing rates than those from CaSRdeficient mice (Martiszus et al., 2021), further supporting our theory that unlocking CaSR-mediated inhibition of Kv7 channels can be a strategy to control hyperexcitability.An alternative explanation is that our hiPSC-derived model may express a different Kv7 channel isoform that is less sensitive to retigabine (Schenzer et al., 2005).This possibility may explain the inconsistency concerning the effect of retigabine on I m of our hiPSC-derived model and DRG neurons.The inconsistency in I m modulation by high [Ca 2+ ] o implies that a higher concentration may be required for the hiPSC-derived model.
Our findings are in accordance with previous studies (Chang et al., 1998;Di Mise et al., 2018) in demonstrating that NPS-R568 reduced intracellular cAMP levels, confirming the coupling of CaSR to ] o , however, did not have a significant effect in our study, which could be explained by the preferential coupling of CaSR to G q/11 protein when CaSR is stimulated by [Ca 2+ ] o , compared to NPS-R568 (Cook et al., 2015;Nemeth et al., 1998).G q/11 protein triggers inositol triphosphate (IP 3 )-induced [Ca 2+ ] i mobilisation, which can activate G i/o protein-insensitive soluble AC and subsequently increases cAMP levels (Tresguerres et al., 2011), attenuating the overall response.Given our current understanding that cAMP signalling occurs in discrete intracellular microdomains (Tresguerres et al., 2011), the G i/o protein-mediated cAMP reduction near the cell membrane can be masked by an increase in cAMP levels within the cytoplasmic microdomain when measured by ELISAs.the G q/11 protein pathway (Cook et al., 2015) and PKA being compartmentalised in a similar fashion to cAMP (Tresguerres et al., 2011).
A previous study has shown that I m is potentiated by cAMPdependent PKA phosphorylation at Ser 52 of the Kv7.2 channel subunit (Schroeder et al., 1998), suggesting that a reduction in cAMP levels, PKA activity and serine phosphorylation would inhibit the G i/o protein-transmembrane AC-cAMP-PKA signalling pathway mediates the CaSR-Kv7.2/7.3 channel crosslink (Figure 9).

| Modulation of neuronal excitability via the CaSR-Kv7 channel crosslink
Unlike mouse DRG neurons (Figure S2), our hiPSC-derived nociceptive-like neurons demonstrated a variable iAP firing rate, with most generating a single iAP.Such a characteristic aligns with that of human sensory neurons (Davidson et al., 2014), highlighting interspecies differences in iAP firing.
Our data demonstrated that an algogenic cocktail induced hyperexcitability.Firstly, a higher percentage of the neurons with a complete train of iAPs was observed following algogenic cocktail treatment, possibly via Kv7.2/7.3 channel activity suppression (Passmore et al., 2003).However, the compliance of these neurons to generate iAPs also was reduced as a steady decrease in the iAP spike frequency at high injected current levels was found.A previous study showed that a more hyperpolarised Na V current availability was associated with lower AP firing rates in hiPSC-derived neurons (Telezhkin et al., 2016).Hence, the algogenic cocktail likely induced a hyperpolarising shift of the Na V current availability window, contributing to the observed reduction in the iAP spike frequency.Secondly, the total iAP spike height increased following algogenic cocktail treatment, which is similar to what was previously reported in hiPSC-derived nociceptive neurons of persistent pain patients and might suggest that Na V 1.7 and Na V 1.8 currents were enhanced (Meents et al., 2019;Renganathan et al., 2001).
The well-established interplay between CaSR and proinflammatory cytokines (Iamartino & Brandi, 2022) and the significance of the inflammatory process in neuropathic pain (Ellis & Bennett, 2013;Li et al., 2023)  Although NPS-2143 inhibited the algogenic cocktail in driving the hiPSC-derived nociceptive-like neurons to develop a complete iAP train characteristic, it appeared to synergise with the algogenic cocktail in increasing the iAP spike frequency at lower current injection of the neurons that presumably already expressed a train of iAPs.Blocking of Ca 2+ -activated K + channels enhanced iAP firing in rodent DRG neurons (Pagadala et al., 2013;Tsantoulas & McMahon, 2014;Zhang et al., 2003).Based on existing evidence that CaSR activation up-regulates Ca 2+ -activated K + channel function (Vassilev et al., 1997), one might speculate that NPS-2143 would inhibit Ca 2+ -activated K + channels and augment iAP firing.A previous study demonstrated that most human DRG neurons produced a single iAP (Davidson et al., 2014).Therefore, when it comes to modulation of collective excitability, the effect of NPS-2143 in preventing algogenic cocktail-induced transformation of the neurons with a single iAP towards multiple iAPs may be greater than its effect in enhancing iAP firing of the cells already at the high-end of the excitability spectrum.The effect of the calcilytic on neuronal excitability, thus, is likely to be multimodal with several knowledge gaps worth pursuing in the future.
Considering the calcilytic's potential in regulating Kv7 channels and neuronal excitability, a calcilytic might help address shortcomings of direct Kv7 channel modulators.Unlike Kv7 channel openers such as flupirtine, calcilytics appeared to be safer and more tolerable (Fitzpatrick et al., 2012;Halse et al., 2014) and could be repurposed for neuropathic pain management.A combined use of the calcilytic and Kv7 channel opener is worth investigating and might reduce the required dosage of the Kv7 channel opener, thus limiting its adverse effects.

| Limitations
First, the effect of [Ca 2+ ] o could be due to its off-target actions.This limitation was circumvented by using NPS-R568 to supplement our findings, and the same effect was observed.Secondly, ELISAs only gave information about overall changes within the cell but no information regarding localisation of the effects.This warrants future investigations into the effect of CaSR activation on spatially distinct cAMP levels and PKA activity.The hiPSC-derived nociceptive neuron-like model provides a promising platform to explore novel therapeutics for pain but is limited by how well the model can replicate in vivo human nociception.Our model still lacked the full complexity of in vivo models, such as the interaction with glia, which is crucial to neuropathic pain development (Zhang et al., 2017) and the complete assemblage of algogenic mediators released in vivo (Ellis & Bennett, 2013).
Our findings spearhead CaSR as a potential therapeutic target for Kv7 channel-associated hyperexcitability disorders such as neuropathic pain, which is worth further investigation.

The
RMP was recorded with the gap free protocol (current = 0 pA) using an Axopatch 200A amplifier and Digidata 1440 A/D interface (Axon Instruments, Forster City, USA).The macroscopic transmembrane I m was recorded using a voltage-stepped deactivation protocol, by holding the cells at a potential of À20 mV and stepped down to À60 mV for 4 s.All recordings were filtered at 2 kHz and digitised at 5 kHz.Clampex 10.2 (Molecular Devices, Sunnyvale, USA) was used for data acquisition.Quantification of I m was performed during the voltage activation step.
Detection of intracellular cAMP levels and PKA activity CHO-Kv7.2/7.3 were plated in 12-well plates at a density of 5 Â 10 5 cells per well and incubated at 37 C with 5% CO 2 /95% O 2 .The following day, wells were divided into groups and received appropriate media (Figure5g,h).The following commercial kits were used to determine intracellular cAMP levels and PKA activity, respectively, according to the manufacturers' instructions: Enzo ® Life Sciences Direct cAMP enzyme-linked immunosorbent assay (ELISA) kit (ADI-900-066, Fisher Scientific) and Invitrogen™ PKA Colorimetric Activity kit (EIAPKA, Life Technologies).The intracellular cAMP levels and PKA activity were normalised to total protein concentrations, which were determined using the Pierce™ BCA Protein Assay Kit (Life Technologies) for samples prepared for cAMP detection, and the Pierce™ 660-nm Protein Assay Reagent (Life Technologies) for samples prepared for PKA activity detection according to the manufacturers' instructions.Each assay was performed in duplicate.2.7 | Phosphoserine-KCNQ2 PLA CHO-Kv7.2/7.3 were plated on 13-mm diameter circular glass coverslips fitted in 24-well plates at a density of 2 Â 10 4 cells per coverslip and incubated at 37 C with 5% CO 2 /95% O 2 .The following day, the cells were treated with either 5-mM [Ca 2+ ] o or 10-μM NPS-R568 for 4 h, fixed with 4% paraformaldehyde for 15 min, and stored in 100% methanol at À20 C. A commercial Duolink ® proximity ligation assay (PLA) system (Merck) was used according to the manufacturer's protocol.The following antibodies were used: rabbit polyclonal anti-KCNQ2 (ab22897, 1:200) and mouse monoclonal anti-phosphoserine conjugated to Duolink ® PLA MINUS probe (DUO87012, 1:50).An Olympus BX61 fluorescence microscope was used to capture three random areas per coverslip; the density of PLA signals (red fluorescent puncta/cell) were counted using ImageJ software.2.8 | Transient transfection of HEK-CaSR with KCNQ2/3 cDNA HEK-CaSR were plated on specialised silicone chambers as mentioned above and incubated at 37 C with 5% CO 2 /95% O 2 overnight.Transfection was performed the following day.In an Eppendorf tube, 2 μL of human KCNQ2/3 cDNA plasmid (0.9 gÁL À1 ) and 1 μL of pmax green fluorescent protein vector (0.5 gÁL À1 ) (Lonza, Cologne, Germany) were diluted in 125 μL of Opti-MEM medium (Life Technologies).In a separate tube, 5 μL of Lipofectamine™ 2000 transfection reagent (Life Technologies) was diluted in 125 μL of Opti-MEM ® medium and incubated at room temperature for 5 min.The diluted cDNA and diluted Lipofectamine™ 2000 transfection reagent were gently mixed then incubated at room temperature for a further 20 min.Approximately 35 μL of the mixture was added to each chamber and all chambers

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4. 1 |
Functional and molecular crosslink between CaSR and Kv7.2/7.3 channels Our experiments on several cellular models demonstrated that CaSR activation by [Ca 2+ ] o and NPS-R568 reduced I m and caused depolarisation, whereas inhibition by NPS-2143 increased I m .In the absence of Kv7.2/7.3 channel expression, or when the channels were blocked by XE991, CaSR-mediated depolarisation was nearly absent.Collectively, our findings suggest that CaSR activation causes depolarisation via Kv7.2/7.3 channel function inhibition.The allosteric modulators, NPS-R568 and NPS-2143, induced slower responses than high [Ca 2+

F I G R E 7
Effects of the algogenic cocktail and NPS-2143 on the characteristics of the first iAP of hiPSC-derived nociceptive-like neurons.(a) Exemplar trace illustrates the seven characteristics of an iAP investigated: threshold, overshoot, afterhyperpolarisation, total spike height, depolarisation rate, repolarisation rate and half-height width.(b-e) Graphs compare afterhyperpolarisation (, total spike height, repolarisation rate and half-height width of the first iAP between four conditions: control (Ct; n = 29 cells from seven cultures), the algogenic cocktail (AC; n = 19 cells from three cultures), the algogenic cocktail plus 1-μM NPS-2143 (AC + N; n = 19 cells from four cultures) and 1-μM NPS-2143 (N; n = 16 cells from three cultures).Data are shown as means ± SEM.Statistical analyses were performed using one-way ANOVA with Dunnett's multiple comparison test to the control (b and c) and the Kruskal-Wallis test with Dunn's multiple comparison test to the control (d and e).Where P is not provided, no significant difference was observed.Since PKA function depends on cAMP levels (Søberg & Skålhegg, 2018), CaSR signalling may interfere with PKA activity.In our study, the forskolin-stimulated PKA activity was attenuated by NPS-R568 and to a lesser extent by high [Ca 2+ ] o .Our findings thus confirm the association between CaSR activation and G i/o protein-transmembrane AC-cAMP-PKA signalling.The smaller effect of [Ca 2+ ] o may pertain to the biased signalling of [Ca 2+ ] o towards

Kv7. 2
/7.3 channel activity.In the present study, depolarisation and I m reduction induced by high [Ca 2+ ] o and NPS-R568 were abated in PTX-treated CHO-Kv7.2/7.3.Furthermore, CaSR-mediated depolarisation was mitigated by 8-Br-cAMP.The PLA showed that CaSR activation significantly reduced phosphorylated serine signals associated with the Kv7.2 channel subunit.Altogether, our results suggest that F I G U R E 8 Effects of the algogenic cocktail and NPS-2143 on the iAP spike frequency of hiPSC-derived nociceptivelike neurons.(a-d) Graphs show the iAP spike frequency at each injected current level of hiPSC-derived nociceptive-like neurons expressing a complete train of iAPs in each condition.(e) Exemplar traces illustrate trains of induced action potentials in HD33n1 hiPSC-derived nociceptive-like neurons recorded in the current-clamp mode (upper panel) and the 1-s current injection protocol used for recording (lower panel).(f) Graphs compare the mean iAP spike frequency between four conditions: control (Ct; n = 8 cells from five cultures), the algogenic cocktail (AC; n = 13 cells from three cultures), the algogenic cocktail plus 1-μM NPS-2143 (AC + N; n = 5 cells from four cultures) and 1-μM NPS-2143 (N; n = 6 cells from three cultures).Data are shown as means±SEM.Statistical analysis was performed using two-way ANOVA with Dunnett's multiple comparison test to the control.Where P is not provided, no significant difference was observed.
EC 50 is the half maximal depolarisation response and H is Hill slope.
Statistical tests (chi-squared test, one-sample t test, Wilcoxon signed rank test, paired t test, unpaired t test, Mann-Whitney U test, oneway analysis of variance (ANOVA), two-way ANOVA and Kruskal-Wallis test as stated in text and figure legends) were selected based on normality of the data and carried out in GraphPad Prism 9; statistical significance differences were recognised at P < 0.05 throughout the study.2.11 | Nomenclature of targets and ligands 3 | RESULTS 3.1 | Activation of CaSR attenuates I m and causes depolarisation through neuronal Kv7.2/7.3 channelsThe functional crosslink between the CaSR and Kv7.2/7.3 channels was investigated using several cellular models: CHO-Kv7.2/7.3,HEK-CaSR, mouse DRG neurons and hiPSC-derived