Electrophysiological and computational analysis of Cav3.2 channel variants associated with familial trigeminal neuralgia
Molecular Brain volume 15, Article number: 91 (2022)
Trigeminal neuralgia (TN) is a rare form of chronic neuropathic pain characterized by spontaneous or elicited paroxysms of electric shock-like or stabbing pain in a region of the face. While most cases occur in a sporadic manner and are accompanied by intracranial vascular compression of the trigeminal nerve root, alteration of ion channels has emerged as a potential exacerbating factor. Recently, whole exome sequencing analysis of familial TN patients identified 19 rare variants in the gene CACNA1H encoding for Cav3.2T-type calcium channels. An initial analysis of 4 of these variants pointed to a pathogenic role. In this study, we assessed the electrophysiological properties of 13 additional TN-associated Cav3.2 variants expressed in tsA-201 cells. Our data indicate that 6 out of the 13 variants analyzed display alteration of their gating properties as evidenced by a hyperpolarizing shift of their voltage dependence of activation and/or inactivation resulting in an enhanced window current supported by Cav3.2 channels. An additional variant enhanced the recovery from inactivation. Simulation of neuronal electrical membrane potential using a computational model of reticular thalamic neuron suggests that TN-associated Cav3.2 variants could enhance neuronal excitability. Altogether, the present study adds to the notion that ion channel polymorphisms could contribute to the etiology of some cases of TN and further support a role for Cav3.2 channels.
Trigeminal neuralgia (TN) also referred as “tic douloureux” is a rare form of chronic neuropathic pain syndrome originating from the trigeminal nerve that supplies sensation to the face. TN is characterized by recurrent and chronic paroxysms of electric shock-like or stabbing pain in the orofacial region (for reviews see [1, 2]). The pain usually lasts from a few seconds to a few minutes and may be so intense that it triggers involuntary wincing, hence the term tic. Most cases of TN are sporadic but familial forms exist and are likely to be underestimated . In both situations, the etiology of TN remains largely unknown and neurovascular compression of the trigeminal root nerve represents the primary theory for the underlying cause of the disease. However, the observation that many TN patients do not show any sign of neurovascular compression, and conversely that individuals with compression do not necessarily develop symptoms, suggested the existence of additional factors. Hence, an alteration of neuronal excitability resulting from abnormal functioning of ion channels has emerged as a potential underlying mechanism [4,5,6,7] and consistent with this notion, the sodium channel blockers carbamazepine and oxcarbazepine represent the first line therapy in TN . Moreover, alterations of the expression of several ion channels including sodium, calcium, and potassium channels have been reported in TN patients  as well as in preclinical rodent models [10,11,12,13,14,15,16,17]. In addition, rare polymorphisms in ion channel genes were identified in TN patients [18,19,20] suggesting the existence of predisposing genetic factors and gain-of-function mutations (GoF) were reported for Nav1.6 , Cav2.1 , TRPM7 [22, 23], and TRPM8 channels .
Recently, TN-associated polymorphisms in the gene CACNA1H encoding Cav3.2 calcium channels were reported . Cav3.2 channels belong to the subfamily of low-voltage-activated T-type channels and are widely expressed throughout the nervous system where they play an essential role in the control of neuronal excitability . Importantly, Cav3.2 is expressed in all structures of the trigeminal pathway including trigeminal ganglion sensory neurons [27, 28], the spinal trigeminal nucleus (SpV)  as well as several thalamic nuclei such as the ventroposterior nucleus (VPM)  that receives projections from the SpV. Hence, Cav3.2 channels may be of direct relevance for the transmission of trigeminal sensory information and a role for Cav3.2 in TN-like syndrome was reported in a preclinical rodent model .
In this study, we aimed to provide a comprehension analysis of TN-associated CACNA1H variants with regard to their impact on the functioning of Cav3.2 channels. Of the 19 variants reported , four had already been assessed for their impact on the biophysical properties of Cav3.2 channels and revealed a variant-dependent effect such that G563R and P566T produced a GoF of the channel, E286K caused a mild loss-of-function (LoF), and H526Y did not cause any alteration [17, 30]. We now report the functional characterization of 13 additional variants. Seven of these variants are located within cytoplasmic regions of Cav3.2 including the N-terminal region (P30L), the loop connecting domains II and III (Q1049H and P1120L), the loop connecting domains III and IV (P1605H), the linker connecting transmembrane segments S2-S3 of domain IV (R1674), and the C-terminal region (P2280H and E2291K). Four additional variants are mapped within important structural determinants of the channel including the transmembrane segment S1 of domain II (I799V and A802V), the end of the S4 voltage-sensor of domain IV (R1736C), and the fourth pore-forming loop (D1779Y). The two remaining variants are localized within the extracellular linkers connecting transmembrane segments S3-S4 of domain I (S187L) and S1-S2 of domain II (E819K) (Fig. 1). Electrophysiological analysis of recombinant TN-associated Cav3.2 variants in tsA-201 cells revealed a significant alteration in the gating properties of 7 out of the 13 variants analyzed. In addition, introduction of these variants in a computational model of reticular thalamic neuron (nRT) enhanced rebound burst firing of action potentials. Taken together, these data suggest that altered gating of TN-associated Cav3.2 variants may enhance neuronal excitability which could potentially contribute to the etiology of TN.
Materials and methods
Plasmid cDNA constructs and site-directed mutagenesis
The Cav3.2 variants were generated by site directed mutagenesis performed by GenScript using the wild-type human Cav3.2 (containing exon 26) in pcDNA3.1 (kindly provided by Dr. Terrance Snutch) as template. The fidelity of all constructs was confirmed by full-length sequencing of the coding region.
Cell culture and heterologous expression
Human embryonic kidney tsA-201 cells were grown in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (all media purchased from Invitrogen) and maintained under standard conditions at 37 °C in a humidified atmosphere containing 5% CO2. Heterologous expression was performed by transfecting cells with 5 μg of plasmid cDNAs encoding for Cav3.2 variants and empty pEGFP vector as transfection marker using the calcium/phosphate method.
Patch clamp electrophysiology
Patch clamp recordings of T-type currents in tsA-201 cells expressing Cav3.2 variants were performed 72 h after transfection in the whole-cell configuration at room temperature (22–24 °C) in a bath solution containing (in millimolar): 10 BaCl2, 125 CsCl, 1 MgCl2, 10 D-glucose, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4 with CsOH). Patch pipettes were filled with a solution containing (in millimolar): 110 CsCl, 3 Mg-ATP, 0.5 Na-GTP, 2.5 MgCl2, 5 D-glucose, 10 EGTA, and 10 HEPES (pH 7.4 with CsOH), and had a resistance of 2–4MΩ. The calculated liquid junction potential was about − 2.6 mV and therefore was corrected from the recordings. Recordings were performed using an Axopatch 200B amplifier (Axon Instruments) and acquisition and analysis were performed using pClamp 10 and Clampfit 10 softwares, respectively (Axon Instruments). The linear leak component of the current was corrected using a P/4 subtraction protocol and current traces were digitized at 10 kHz and filtered at 2 kHz.
The voltage dependence of activation of Cav3.2 channels was determined by measuring the peak of the T-type current in response to 140 ms depolarizing steps from − 80 mV to + 20 mV in 5 mV increments preceded by a 200 ms hyperpolarizing prepulse to − 110 mV from a holding membrane potential of − 100 mV. The current–voltage relationship (I/V) curve was fitted with the following modified Boltzmann Eq. (1):
with I(V) being the peak current amplitude at the command potential V, Gmax the maximum conductance, Vrev the reversal potential, V0.5 the half-activation potential, and k the slope factor. The voltage dependence of the whole-cell T-type channel conductance was calculated using the following modified Boltzmann Eq. (2):
with G(V) being the T-type channel conductance at the command potential V.
The voltage dependence of the steady-state inactivation of Cav3.2 channels was determined by measuring the peak T-type current amplitude in response to a 50 ms depolarizing step to -30 mV applied after a 1 s-long conditioning prepulse ranging from -110 mV to -15 mV in 5 mV increments. The current amplitude obtained during each test pulse was normalized to the maximal current amplitude and plotted as a function of the prepulse potential. The voltage dependence of the steady-state inactivation was fitted with the following two-state Boltzmann function (3):
with Imax corresponding to the maximal peak current amplitude and V0.5 to the half-inactivation voltage.
The recovery from inactivation was assessed using a double-pulse protocol preceded by a 50 ms-long hyperpolarizing prepulse to -110 mV from a holding potential of -100 mV. The cell membrane was depolarized for 2 s at -20 mV (inactivating prepulse) to ensure complete inactivation of the channel, and then to -20 mV for 150 ms (test pulse) after an increasing time period (interpulse) ranging between 1 ms and 8 s at -110 mV. The peak current from the test pulse was plotted as a ratio of the maximum prepulse current versus interpulse interval. The data were fitted with the following single-exponential function (4):
where τ is the time constant for channel recovery from inactivation.
Simulation of thalamic reticular neuron (nRT) firing was performed using the NEURON simulation environment (https://senselab.med.yale.edu/ModelDB/)  in the three-compartment model previously described . The electrophysiological properties of wild-type and TG-associated Cav3.2 variants obtained experimentally were modeled using Hodgkin-Huxley equations as previously described  and introduced into the model. To take into account the relative expression of Cav3.2 channels in nRT neurons (about 40% of Cav3.2 and 60% of Cav3.3 ) and the heterozygous nature of TN-associated Cav3.2 variants, only 20% of the T-type channel conductance described in the original model was altered with experimental values obtained for WT and TN-associated Cav3.2 variants. The simulation was performed at a holding potential set to -70 mV and the electrical membrane potential of the virtual soma was monitored in response to a 200ms-long hyperpolarizing and depolarizing current injection in order to assess rebound and tonic firing, respectively.
Average data are presented as mean ± S.E.M. for n measurements. Statistical analysis was performed using GraphPad Prism 8. A Kolmogorov–Smirnov normality test was performed and statistical significance was assessed using Kruskal–Wallis test with Dunn’s post-test. Datasets were considered significantly different for p ≤ 0.05.
Expression of TN-associated Ca v 3.2 variants
To assess the functional impact of TN-associated CACNA1H variants, tsA-201 cells were transiently transfected with plasmids encoding human Cav3.2 wild-type (WT) and TN-associated variants for electrophysiological analysis. Whole-cell patch clamp recordings in tsA-201 cells expressing wild-type (WT) and TN-associated Cav3.2 variants revealed that all variants were functionally expressed and generated a characteristic low-voltage-activated T-type current similar to WT channels (Fig. 2a–n, left panels). The maximal whole-cell macroscopic T-type channel conductance (Gmax) obtained from the fit of the current–voltage relationships (Fig. 2a–n, right panels) revealed no significant difference between cells expressing Cav3.2 variants compared to cells expressing the WT channel except for the R1674H variant were Gmax was reduced by 56% (p = 0.0185) (Fig. 2o and Table 1).
TN-associated CACNA1H variants alter the gating properties of Ca v 3.2 channels
Next, we aimed to assess the gating properties of TN-associated Cav3.2 variants. First, we analyzed the voltage dependence of activation of the channels. In 6 (S187L, A802V, E819K, P1120L, P2280H, and E2291K) out of the 13 variants analyzed, the mean half-activation potential of the T-type current was significantly shifted toward more hyperpolarized potentials by − 8.0 mV (E2291K, p = 0.0268) up to − 15.0 mV (P2280H, p < 0.0001) relative to WT channels which is consistent with a GoF of the channels (Fig. 3a–o and Table 1). In addition, a significant decrease of the activation slope factor (k) was observed for S187L, A802V, E819K, and P2280H variants suggesting an increased coupling between the channel voltage-sensor and the pore opening again consistent with a GoF which may be particularly relevant for voltage changes close to the resting membrane potential where first openings of the channel occur (Table 1). To gain additional insights into the electrophysiological properties of TN-associated Cav3.2 variants, we then assessed their voltage dependence of inactivation. A statistically significant hyperpolarizing shift of the voltage dependence of inactivation by -12.6 mV (p = 0.0015) relative to WT channels was observed for the P2280H variant and a similar trend albeit not statistically significant was observed for S187L (− 8.6 mV, p = 0.8716) and A802V variants (− 9.2 mV, p = 0.3699) whereas the remaining variants remained unaltered (Fig. 4a–o and Table 2). The alteration of the voltage dependence of inactivation is consistent with a LoF of the channel variants although the extent to which it may affect channel activity will largely depend on the resting membrane potential of cells, with a more pronounced effect in cells with a comparatively depolarized resting potential. In contrast, the kinetics of recovery from inactivation were accelerated by 2.7-fold for A802V (p = 0.0001) and by 3.5-fold for Q1049H variants (p = 0.0005) compared to WT channels, and a similar trend (albeit not statistically significant) was observed for several other variants indicative of a GoF (Fig. 5a–o and Table 2).
Ca v 3.2-dependent window current is altered by TN-associated CACNA1H variants
Because several TN-associated Cav3.2 variants showed alterations in the voltage dependence of activation and/or inactivation, we aimed to assess the impact on the T-type window current by visualizing the overlapping area between the activation and inactivation curves (Fig. 6a–g). In all TN-associated Cav3.2 variants for which the voltage dependence of activation and/or inactivation was altered, the window current was displaced toward more hyperpolarized potentials with the peak-voltage shifted by − 5 mV (P1120L) up-to − 12 mV (P2280H) (Fig. 6h). This effect was accompanied by an increased magnitude of the window current (except for the P2280H variant) ranging from 14% (E2291K) up-to 165% increase (P1120L) (Fig. 6i).
TN-associated Ca v 3.2 variants increase neuronal firing in a computational model of thalamic neurons
Given that Cav3.2 channels are highly expressed in thalamic neurons where they play an essential role in regulating neuronal excitability  and considering that the thalamus is a key relay station in the trigeminal sensory pathway , we aimed to simulate the functional consequence of TN-associated Cav3.2 variants on neuronal electrical activities using a computational model of reticular thalamic neuron (nRT). The simulation was performed with Cav3.2 variants for which an alteration of the voltage dependence of activation and/or inactivation was observed and the original model was altered in order to account for the relative contribution of Cav3.2 channels to the overall native T-type conductance and also to account for the heterozygous nature of TN-associated Cav3.2 variants (see Methods). Simulation of the neuronal membrane potential showed that hyperpolarizing current injections triggered rebound burst firing with WT as well as with TN-associated Cav3.2 variants (Fig. 7a–g). However, the minimum current necessary to trigger rebound firing (rheobase) was significantly less for TN-associated Cav3.2 variants (except A802V) compared to WT channels (Fig. 7h). Moreover, the firing frequency was increased (Fig. 7i). In contrast, when the firing was triggered with depolarizing current injections there was no major effect between WT and TN-associated Cav3.2 variants (Fig. 7j–r).
Polymorphisms in the CACNA1H gene have been reported in a number of human disorders  and GoF mutations in Cav3.2 are linked to primary aldosteronism (PA) [38,39,40] and idiopathic generalized epilepsy (IGE) . In contrast, LoF mutations were documented in autism spectrum disorders , neuromuscular disorders [43,44,45], and developmental and epileptic encephalopathy .
In this study, we report the functional characterization of 13 Cav3.2 missense variants identified in TN patients. Patch clamp recordings of T-type currents in tsA-201 cells expressing recombinant TN-associated Cav3.2 variants showed that all variants were functional with no significant alteration in their maximal macroscopic conductance except for the R1674H variant for which the conductance was reduced. This effect was not further investigated but may have been caused by a decreased trafficking of the channel to the plasma membrane and/or decreased stability. In contrast, of the 13 Cav3.2 variants analyzed, 6 variants (S187L, A802V, E819K, P1120L, P2280H, and E2291K) displayed alterations in their gating properties evidenced by a recurrent hyperpolarized shift of the voltage dependence of activation consistent with a GoF of the channels. An additional acceleration of the recovery from inactivation was also observed for A802V and Q1049H. Although these variants showed similar alterations in their gating properties, they did not segregate into a particular region of Cav3.2. Nonetheless, some of the channel molecular determinants containing TN-associated variants are known to contribute to the gating of Cav3.2. For instance, the II-III loop containing variant P1120L and the C-terminus containing variants P2280H and E2291K were previously reported to affect the voltage dependence of T-type channels [47,48,49]. Moreover, the GoF effect of TN-associated variants in the C-terminus of Cav3.2 is reminiscent of what was reported for several variants associated with (IGE) and PA [40, 50]. Importantly, when introduced into a computational model of nRT neuron, the 6 variants reduced the threshold for rebound burst firing implying an overall GoF effect. This is consistent with previous findings in various types of neurons showing that upregulation of T-type channel activity underlies reduced threshold for rebound burst firing [51,52,53,54,55]. While our modeling was performed in a computational model of nRT neurons, it may anticipate some of the possible effects of TN-associated Cav3.2 variants on the functioning of the trigeminal pathway for several reasons. First, although T-type dependent rebound burst firing has yet to be shown in trigeminal ganglion (TG) sensory neurons, it has been documented in dorsal root ganglion sensory neurons  and it is a possibility that it does also occur in TG neurons. Second, a low-threshold calcium conductance (presumably mediated by T-type channels) leading to calcium spikes and rebound burst firing has been reported in neurons of the brain stem trigeminal nuclei . Third, the trigeminal pathway gates through the thalamus in particular via the VPM where T-type channels contribute to rebound burst firing . And finally, alteration of thalamocortical rhythmic activities mediated by T-type channels has been implicated in the development of trigeminal pain . Hence, all of these aspects suggest that alteration of rebound burst firing caused by TN-associated Cav3.2 variants could potentially contribute to the sensitization of the trigeminal pathway. In addition, alteration of the channel gating properties resulted in a hyperpolarizing displacement of the voltage dependence of the window current which implies an increased passive influx of calcium around the resting membrane potential of cells. Considering that the voltage range of the window current is an important determinant of neuronal electrical activities and calcium oscillations , this may further contribute to enhance neuronal activity. These data are consistent with a previous report showing that re-expression of a GoF TN-associated Cav3.2 variant in cultured TG neuron increased neuronal excitability . The question then remains as to why TN patients harboring GoF Cav3.2 variants did not show signs of IGE or PA. It is a possibility that the gating alterations caused by these variants and affecting only the rebound burst firing in the absence of general alteration of the tonic firing is not enough to cause additional disease phenotypes.
In conclusion, our functional analysis of 13 Cav3.2 variants identified in TN patients revealed an overall GoF of the channel for 7 of these variants that could potentially contribute to the sensitization of the trigeminal pathway. Although these gating effects are reminiscent of what was previously reported for TN-associated variants in Nav1.6 , Cav2.1 , TRPM7 [22, 23], and TRPM8 channels , it is important to consider that our functional analysis in a heterologous expression system provides only a snapshot of the phenotype of a mutation. Hence, additional analysis of these variants in native conditions will be necessary to further validate these findings. Moreover, it is a possibility that the variants for which we did not observe any gating alteration will show different phenotypes in a more complex physiological environment. Finally, although most of the gating alterations were in general consistent with a GoF of the channel, it is important to consider that one variant was associated with a LoF suggesting that GoF phenotypes in ion channels may not represent a universal feature in TN. For instance, the expression level of SCN9A (Nav1.7) and SCN10A (Nav1.8) is reduced in gingival tissue of TN patients , as well as in preclinical models of TN  implying a LoF phenotype, although this mechanism may occur as a protective mechanism to normalize neuronal excitability. Nonetheless, it is striking to note that all patients exhibiting idiopathic TN with concomitant continuous pain (iTN-2) harbored GoF Cav3.2 variants. In contrast, all Cav3.2 variants identified in patients with congenital TN and concomitant pain (cTN-2) did not cause any alteration of the channel (Table 3). While additional studies are necessary to assess the exact role of Cav3.2 in the processing of trigeminal sensory information, our data add to the notion that rare CACNA1H variants may contribute to the etiology of TN. In that respect, the T-type channel blocker valproic acid was shown to be effective to mitigate pain in some TN patients  suggesting that other antiepileptic T-type channel blockers ethosuximide, zonizamide, and nimodipine  should also be considered especially in patients resistant to first line therapies.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information files.
Classical trigeminal neuralgia purely paroxysmal
Classical trigeminal neuralgia with concomitant continuous pain
Idiopathic generalized epilepsy
Idiopathic trigeminal neuralgia purely paroxysmal
Idiopathic trigeminal neuralgia with concomitant continuous pain
Reticular thalamic neuron
Spinal trigeminal nucleus
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R.N.S. holds a studentship from Charles University. E.G. holds a studentship from Alberta Innovates. G.W.Z. is supported by a grant from the Canadian Institutes of Health Research and holds a Canada Research Chair. N.W. is supported by a grant from the Czech Science Foundation (GACR #22-23242S) and the National Institute for Research of Metabolic and Cardiovascular Diseases (Program EXCELES #LX22NPO5104), funded by the European Union—Next Generation EU.
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Mustafá, E.R., Gambeta, E., Stringer, R.N. et al. Electrophysiological and computational analysis of Cav3.2 channel variants associated with familial trigeminal neuralgia. Mol Brain 15, 91 (2022). https://doi.org/10.1186/s13041-022-00978-9
- Trigeminal neuralgia
- Ion channel
- Calcium channel
- Cav3.2 channel