Skip to main content
  • Micro report
  • Open access
  • Published:

Electrophysiological characterization of a Cav3.2 calcium channel missense variant associated with epilepsy and hearing loss


T-type calcium channelopathies encompass a group of human disorders either caused or exacerbated by mutations in the genes encoding different T-type calcium channels. Recently, a new heterozygous missense mutation in the CACNA1H gene that encodes the Cav3.2 T-type calcium channel was reported in a patient presenting with epilepsy and hearing loss—apparently the first CACNA1H mutation to be associated with a sensorineural hearing condition. This mutation leads to the substitution of an arginine at position 132 with a histidine (R132H) in the proximal extracellular end of the second transmembrane helix of Cav3.2. In this study, we report the electrophysiological characterization of this new variant using whole-cell patch clamp recordings in tsA-201 cells. Our data reveal minor gating alterations of the channel evidenced by a mild increase of the T-type current density and slower recovery from inactivation, as well as an enhanced sensitivity of the channel to external pH change. To what extend these biophysical changes and pH sensitivity alterations induced by the R132H mutation contribute to the observed pathogenicity remains an open question that will necessitate the analysis of additional CACNA1H variants associated with the same pathologies.

Mutations in the CACNA1H gene that encodes the Cav3.2 T-type calcium channel are risk factors for a number of human channelopathies including epilepsy [1], primary aldosteronism [2], autism spectrum disorder [3, 4], amyotrophic lateral sclerosis [5, 6], congenital amyotrophy [7], and trigeminal neuralgia [8, 9]. Recently, Algahtani and colleagues reported a new heterozygous missense mutation in a 50-year-old female patient with a clinical condition involving epilepsy and hearing loss which appears to be the first CACNA1H variant to be associated with sensorineural hearing alterations [10]. This mutation results in the substitution of an arginine at position 132 with a histidine (R132H) in the proximal extracellular end of the second transmembrane helix of Cav3.2 (Fig. 1a) and has not yet been reported in the gnomAD database ( Molecular simulation using the AlphaFold-generated model of the human Cav3.2 channel shows that replacement of the arginine 132 with a histidine leads to an additional hydrogen bond with methionine 119 of the first transmembrane helix (Fig. 1a) that has the potential to alter the gating of the channel. In addition, a histidine residue has a highly variable pKa value depending of its direct environment indicating that its charge may vary subtly as a function of external pH. To challenge this hypothesis, we assessed the functional impact of the R132H variant on the biophysical properties of Cav3.2 using patch-clamp recordings in tsA-201 cells bathed in 5 mM barium as the charge carrier (see Additional file 1). Both cells expressing Cav3.2 wild-type (WT) and R132H mutant channels displayed characteristic low-voltage activated T-type currents (Fig. 1a, b). A significant 40% (p = 0.0285) increase of the maximal macroscopic T-type conductance (Gmax) was observed in cells expressing the R132H channel variant (0.52 ± 0.06 nS/pF, n = 26) compared to cells expressing the WT channel (0.37 ± 0.03 nS/pF, n = 24) (Fig. 1d) without any alteration of the voltage dependence of activation (Fig. 1e) or steady-state inactivation (Fig. 1f). An additional significant (p = 0.0342) slowing of the time constant (τ) of recovery from inactivation was observed for R132H channels (467 ± 21 ms, n = 18) compared to WT channels (284 ± 34 ms, n = 10) (Fig. 1g) while fast activation and inactivation kinetics of the current remained unaltered (Fig. 1h).

Fig. 1
figure 1

Functional properties of the Cav3.2 R132H variant associated with epilepsy and hearing loss. a AlphaFold model of the human Cav3.2 channel showing the location of the R132H mutation (left panel). In this model, the arginine (R) 132 is located within the extracellular-exposed proximal end of the second transmembrane helix (S2) of Cav3.2 and forms two intra-helix hydrogen bonds with serine (S) 130 and leucine (L) 136 (left panel). Substitution of the R132 with a histidine (H) residue creates an additional hydrogen bond with methionine (M) 119 located within the first transmembrane helix (S1) of the channel. b Representative sets of whole-cell T-type current traces recorded in tsA-201 cells expressing Cav3.2 wild-type (WT) (black traces) and R132H variant (red traces). Currents were elicited by depolarizing steps to values ranging between − 90 mV and + 30 mV from a holding potential of − 100 mV. c Corresponding mean current/voltage (I/V) relationships. The continuous lines represent the fit of the I/V curves with the modified Boltzmann Eq. (1). d Corresponding mean maximal macroscopic conductance values (Gmax) obtained from the fit of the I/V curves. e Corresponding mean normalized voltage dependence of activation fitted (continuous lines) with the modified Boltzmann Eq. (2). The inset shows the mean half-activation potential values obtained from the fit of the conductance curves. f Mean normalized voltage dependence of steady-state inactivation fitted with the two-state Boltzmann Eq. (3). The inset represents the mean half-inactivation potential values obtained from the fit of the inactivation curves. g Mean normalized recovery from inactivation kinetic fitted with the single-exponential function (4). Inset shows the mean time constant values obtained from the fit of the recovery from inactivation curves. h Mean time constant of fast activation (diamond symbols) and inactivation (round symbols) kinetics of T-type currents as a function of the membrane potential. i Relative change in peak current amplitude in response to extracellular pH alkalinization (pHe 8.0, green symbols) and acidification (pHe 6.5, orange symbols) from physiological pHe 7.2 (top panels) as well as corresponding mean current change amplitude values (bottom panels). T-type currents were elicited by repetitive depolarizing steps to -20 mV from a holding potential of -100 mV. j, k Legend same as (i) but for T-type current activation and inactivation kinetics. Data are presented as mean ± S.E.M. and statistical analysis was performed using a two-tailed Student’s t test

Next, we aimed to assess the effect of extracellular pH (pHe) on the regulation of the channels. Indeed, histidine residues theoretically bear a partial charge at physiological pH, although this is largely influenced by the direct environment of the residue, and therefore act as [H+] sensor as a result of protonation. Protonation may in turn mediate modulatory effects on voltage-gated channels, including Cav3.2 [11]. Given that the R132H variant implicates the introduction of histidine within the extracellular end of the second transmembrane helix of Cav3.2, we assessed the effects of extracellular pH changes, alkalization (pHe 8.0) and acidification (pHe 6.5), on T-type currents. Consistent with previous results on T-type channels [11, 12], extracellular alkalization and acidification produced a significant increase and decrease of the T-type current, respectively, in both Cav3.2 WT- and R132H-expressing cells (Fig. 1i, top panels). However, these effects were emphasized on Cav3.2 R132H-mediated currents. For instance, alkalization-mediated increase of the T-type current was 82% higher (p = 0.0176) in cells expressing the R132H channel (24.0 ± 3.5% increase, n = 23) compared to cells expressing the WT channel (13.1 ± 1.2% increase, n = 16), whereas acidification-mediated decrease of the current was enhanced by 37% greater (p = 0.0087) (from − 30.9 ± 2.9% decrease in WT, n = 15, to − 42.5 ± 2.9% for R132H, n = 19) (Fig. 1i, bottom panels). In addition, extracellular alkalization produced an acceleration of the kinetics of current activation and inactivation, whereas acidification produced the exact opposite (Fig. 1j, k, top panels). However, these effects were proportionally similar between WT and R132H channels (Fig. 1j, k, bottom panels).

Previous studies in animal models have documented the importance of T-type channels in the functioning of the auditory system. For instance, Cav3.2 channels are highly expressed in mouse spiral ganglion neurons (SGN) where they are necessary for spatiotemporal auditory processing [13]. However, they also exhibit age-dependent increases in expression levels that are causally associated with SGN degeneration, whereas T-type channel blockers are protective against age-related SGN and hearing loss [14]. Here, we showed that the Cav3.2 R132H mutation causes mixed alterations of the channel as evident from an increase in current density (that can be attributed to an alteration of the single channel gating properties and/or an increased expression of Cav3.2 at the cell surface) consistent with a gain-of-channel function. There is also a slowing of the recovery from inactivation which is consistent with a loss-of-function of the channel. However, the extent to which this loss-of-gating will manifest under physiological conditions will largely depend on the firing properties of nerve cells expressing the mutant channel. In addition, we illustrate that the R132H mutation enhances the impact of pHe regulation of the channel. While the alterations may seem relatively mild, they have the merit to be observed and will require further experimentation to define their meaning in terms of pathogenicity. Clearly, there is evidence that alteration of pH homeostasis in response to primary metabolic disorders such as renal tubular acidosis is often accompanied with sensorineural hearing alterations [15]. In such context, altered pHe-dependent modulation of Cav3.2 by the R132H mutation may represent a risk factor for hearing loss. Likewise, there is evidence that brain pH levels are significantly increased in experimental animal models of epilepsy [16,17,18,19] and patients [20] and precipitates the development of seizures. Therefore, it is a possibility that alkalinization-meditated increase of Cav3.2 R132H currents may also exacerbate seizures. An interesting consideration is whether a primary epilepsy could be the initiator of subsequent hearing loss in the patient carrying the Cav3.2 R132H mutation.

In conclusion, it is premature to recommend classifying the Cav3.2 R132H mutation as disease-causing variant at this stage in the absence of a larger number of variants causing the same pathologies. Moreover, since our functional analysis was performed in a recombinant expression system, there remains the possibility that the R132H mutation may exhibit a more pronounced phenotype in a native neuronal environment, and additional analysis will help to fully comprehend to which extent this mutation alters Cav3.2 channel function in the context of auditory function and epilepsy.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its additional files.


Cav :

Voltage-gated calcium channel

G max :

Maximal macroscopic conductance


  1. Weiss N, Zamponi GW. Genetic T-type calcium channelopathies. J Med Genet. 2020;57(1):1–10.

    Article  PubMed  CAS  Google Scholar 

  2. Daniil G, Fernandes-Rosa FL, Chemin J, Blesneac I, Beltrand J, Polak M, et al. CACNA1H mutations are associated with different forms of primary aldosteronism. EBioMedicine. 2016;13:225–36.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Splawski I, Yoo DS, Stotz SC, Cherry A, Clapham DE, Keating MT. CACNA1H mutations in autism spectrum disorders. J Biol Chem. 2006;281(31):22085–91.

    Article  PubMed  CAS  Google Scholar 

  4. Viggiano M, D’Andrea T, Cameli C, Posar A, Visconti P, Scaduto MC, et al. Contribution of CACNA1H variants in autism spectrum disorder susceptibility. Front Psychiatry. 2022;13: 858238.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Rzhepetskyy Y, Lazniewska J, Blesneac I, Pamphlett R, Weiss N. CACNA1H missense mutations associated with amyotrophic lateral sclerosis alter Cav3.2 T-type calcium channel activity and reticular thalamic neuron firing. Channels (Austin). 2016;10(6):466–77.

    Article  PubMed  Google Scholar 

  6. Stringer RN, Jurkovicova-Tarabova B, Huang S, Haji-Ghassemi O, Idoux R, Liashenko A, et al. A rare CACNA1H variant associated with amyotrophic lateral sclerosis causes complete loss of Cav3.2 T-type channel activity. Mol Brain. 2020;13(1):33.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Carter MT, McMillan HJ, Tomin A, Weiss N. Compound heterozygous CACNA1H mutations associated with severe congenital amyotrophy. Channels (Austin). 2019;13(1):153–61.

    Article  PubMed  Google Scholar 

  8. Gambeta E, Gandini MA, Souza IA, Zamponi GW. CaV3.2 calcium channels contribute to trigeminal neuralgia. Pain. 2022;163:2315.

    Article  PubMed  CAS  Google Scholar 

  9. Mustafá ER, Gambeta E, Stringer RN, Souza IA, Zamponi GW, Weiss N. Electrophysiological and computational analysis of Cav3.2 channel variants associated with familial trigeminal neuralgia. Mol Brain. 2022;15(1):91.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Algahtani HA, Shirah BH, Samman A, Alhazmi A. Epilepsy and hearing loss in a patient with a rare heterozygous variant in the CACNA1H gene. J Epilepsy Res. 2022;12(1):33–5.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Delisle BP, Satin J. pH modification of human T-type calcium channel gating. Biophys J. 2000;78(4):1895–905.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Maksemous N, Blayney CD, Sutherland HG, Smith RA, Lea RA, Tran KN, et al. Investigation of CACNA1I Cav3.3 Dysfunction in Hemiplegic Migraine. Front Mol Neurosci. 2022;15:892820.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Lundt A, Seidel R, Soós J, Henseler C, Müller R, Bakki M, et al. Cav3.2 T-type calcium channels are physiologically mandatory for the auditory system. Neuroscience. 2019;409:81–100.

    Article  PubMed  CAS  Google Scholar 

  14. Lei D, Gao X, Perez P, Ohlemiller KK, Chen CC, Campbell KP, et al. Anti-epileptic drugs delay age-related loss of spiral ganglion neurons via T-type calcium channel. Hear Res. 2011;278(1–2):106–12.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Ay E, Gurses E, Aslan F, Gulhan B, Alniacik A, Duzova A et al. Hearing loss related to gene mutations in distal renal tubular acidosis. Audiol Neurootol. 2023;1–10.

  16. de Curtis M, Manfridi A, Biella G. Activity-dependent pH shifts and periodic recurrence of spontaneous interictal spikes in a model of focal epileptogenesis. J Neurosci. 1998;18:7543–51.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Schuchmann S, Schmitz D, Rivera C, Vanhatalo S, Salmen B, Mackie K, et al. Experimental febrile seizures are precipitated by a hyperthermia-induced respiratory alkalosis. Nat Med. 2006;12(7):817–23.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Helmy MM, Tolner EA, Vanhatalo S, Voipio J, Kaila K. Brain alkalosis causes birth asphyxia seizures, suggesting therapeutic strategy. Ann Neurol. 2011;69(3):493–500.

    Article  PubMed  Google Scholar 

  19. Lu D, Ji Y, Sundaram P, Traub RD, Guan Y, Zhou J, et al. Alkaline brain pH shift in rodent lithium-pilocarpine model of epilepsy with chronic seizures. Brain Res. 2021;1758: 147345.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Schuchmann S, Hauck S, Henning S, Grüters-Kieslich A, Vanhatalo S, Schmitz D, et al. Respiratory alkalosis in children with febrile seizures. Epilepsia. 2011;52(11):1949–55.

    Article  PubMed  Google Scholar 

Download references


Not applicable.


L.C. is supported by a Barrande fellowship (Campus France). 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.

Author information

Authors and Affiliations



RNS and LC performed the electrophysiology and analyzed the data. NW designed and supervised the study. NW wrote the manuscript. GWZ and MDW edited the manuscript. All authors critically revised the manuscript and contributed significantly to this work. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Norbert Weiss.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors have no competing interests to declare.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1.

Supplementary material and methods.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Stringer, R.N., Cmarko, L., Zamponi, G.W. et al. Electrophysiological characterization of a Cav3.2 calcium channel missense variant associated with epilepsy and hearing loss. Mol Brain 16, 68 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: