Gating Effects of Mutations in the Cav3.2 T-type Calcium Channel Associated with Childhood Absence Epilepsy

Childhood absence epilepsy (CAE) is a type of generalized epilepsy observed in 2–10% of epileptic children. In a recent study by Chen et al. (Chen, Y., Lu, J., Pan, H., Zhang, Y., Wu, H., Xu, K., Liu, X., Jiang, Y., Bao, X., Yao, Z., Ding, K., Lo, W. H., Qiang, B., Chan, P., Shen, Y., and Wu, X. (2003) Ann. Neurol. 54, 239–243) 12 missense mutations were identified in the CACNA1H (Cav3.2) gene in 14 of 118 patients with CAE but not in 230 control individuals. We have functionally characterized five of these mutations (F161L, E282K, C456S, V831M, and D1463N) using rat Cav3.2 and whole-cell patch clamp recordings in transfected HEK293 cells. Two of the mutations, F161L and E282K, mediated an ∼10-mV hyperpolarizing shift in the half-activation potential. Mutation V831M caused a ∼50% slowing of inactivation relative to control and shifted half-inactivation potential ∼10 mV toward more depolarized potentials. Mean time to peak was significantly increased by mutation V831M but was unchanged for all others. No resolvable changes in the parameters of the IV relation or current kinetics were observed with the remaining mutations. The findings suggest that several of the Cav3.2 mutants allow for greater calcium influx during physiological activation and in the case of F161L and E282K can result in channel openings at more hyperpolarized (close to resting) potentials. This may underlie the propensity for seizures in patients with CAE. Childhood absence epilepsy (CAE) is a type of generalized epilepsy observed in 2–10% of epileptic children. In a recent study by Chen et al. (Chen, Y., Lu, J., Pan, H., Zhang, Y., Wu, H., Xu, K., Liu, X., Jiang, Y., Bao, X., Yao, Z., Ding, K., Lo, W. H., Qiang, B., Chan, P., Shen, Y., and Wu, X. (2003) Ann. Neurol. 54, 239–243) 12 missense mutations were identified in the CACNA1H (Cav3.2) gene in 14 of 118 patients with CAE but not in 230 control individuals. We have functionally characterized five of these mutations (F161L, E282K, C456S, V831M, and D1463N) using rat Cav3.2 and whole-cell patch clamp recordings in transfected HEK293 cells. Two of the mutations, F161L and E282K, mediated an ∼10-mV hyperpolarizing shift in the half-activation potential. Mutation V831M caused a ∼50% slowing of inactivation relative to control and shifted half-inactivation potential ∼10 mV toward more depolarized potentials. Mean time to peak was significantly increased by mutation V831M but was unchanged for all others. No resolvable changes in the parameters of the IV relation or current kinetics were observed with the remaining mutations. The findings suggest that several of the Cav3.2 mutants allow for greater calcium influx during physiological activation and in the case of F161L and E282K can result in channel openings at more hyperpolarized (close to resting) potentials. This may underlie the propensity for seizures in patients with CAE. Generalized epileptic disorders involve both brain hemispheres and are characterized by abnormal synchronous electrical (electroencephalographic) activity, recorded bilaterally at seizure onset (1Adams R.D. Victor M. Ropper A.H. Principles of Neurology.Sixth Ed. McGraw-Hill Health Professions Division, New York1998Google Scholar). Childhood absence epilepsy (CAE) 1The abbreviations used are: CAE, childhood absence epilepsy; SWD, spike-and-wave discharge; ANOVA, analysis of variance. is a type of idiopathic generalized epilepsy and is typified by sudden brief impairment of consciousness followed by ∼3-Hz spike- and-wave discharges (SWDs) over both brain hemispheres (2Goetz C.G. Pappert E.J. Textbook of Clinical Neurology. W. B. Saunders, Philadelphia1999Google Scholar). A typical absence seizure is without convulsions and there are no reported neuropathological changes associated with this disorder (3Crunelli V. Leresche N. Nat. Rev. Neurosci. 2002; 3: 371-382Crossref PubMed Scopus (500) Google Scholar). Spike-wave discharges in absence epilepsy involve interactions between cortical and thalamic structures (4Avoli M. Rogawski M.A. Avanzini G. Epilepsia. 2001; 42: 445-457Crossref PubMed Scopus (82) Google Scholar). The classical view of SWD-based seizures, including absence epilepsy, implicates the thalamus as the site of seizure generation (5Jasper H.H. Droogleever-Fortuyn J. Res. Publ. Assoc. Nerv. Ment. Dis. 1947; 26: 272-298Google Scholar, 6Pollen D.A. Perot P. Reid K.H. Electroencephalogr. Clin. Neurophysiol. 1963; 15: 1017-1028Abstract Full Text PDF PubMed Scopus (64) Google Scholar). Recently, an increasing body of evidence suggests that spike-wave seizures are initiated in the neocortex and then rapidly progress to involve thalamic structures (7Steriade M. Contreras D. J. Neurophysiol. 1998; 80: 1439-1455Crossref PubMed Scopus (276) Google Scholar, 8Steriade M. Contreras D. J. Neurosci. 1995; 15: 623-642Crossref PubMed Google Scholar, 9Meeren H.K. Pijn J.P. Van Luijtelaar E.L. Coenen A.M. Lopes da Silva F.H. J. Neurosci. 2002; 22: 1480-1495Crossref PubMed Google Scholar). The thalamus and cortex then engage in complex interplay that underlies SWD generation and is dependent on the activation of low voltage-activated (T-type) calcium channels (4Avoli M. Rogawski M.A. Avanzini G. Epilepsia. 2001; 42: 445-457Crossref PubMed Scopus (82) Google Scholar). Indeed, reticular thalamic neurons are endowed with large T-type currents that mediate bursting behavior associated with SWDs. The critical role of T-type channels in SWD epilepsies is also supported by treatment of absence seizures using ethosuximide, an inhibitor of T-type Ca2+ currents (10Coulter D.A. Huguenard J.R. Prince D.A. Ann. Neurol. 1989; 25: 582-593Crossref PubMed Scopus (403) Google Scholar, 11Kostyuk P.G. Molokanova E.A. Pronchuk N.F. Savchenko A.N. Verkhratsky A.N. 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Snutch T.P. J. Biol. Chem. 2001; 276: 3999-4011Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 23Chemin J. Monteil A. Bourinet E. Nargeot J. Lory P. Biophys. J. 2001; 80: 1238-1250Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). It was recently shown that Cav3.1 knock-out mice display reduced burst mode firing activity, and that the Cav3.1-deficient thalamus is specifically resilient to SWD generation (24Kim D. Song I. Keum S. Lee T. Jeong M.J. Kim S.S. McEnery M.W. Shin H.S. Neuron. 2001; 31: 35-45Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). In a complimentary study, although P/Q-type Ca2+ channel knock-out mice experienced absence seizures with 4–5 Hz SWDs, an additional knock-out of T-type channels rescued the animals such that SWDs were abolished (25Song, I., Kim, D., Jun, K., and Shin, H. S. (2001) Annual Meeting of the Society for Neuroscience, November 10–15, San Diego, CA (abstracts)Google Scholar). In addition, a functional study has demonstrated that different splice isoforms of human Cav3.1 channels, when exogenously expressed, can display radically different electrophysiological properties, resulting in differential ability to maintain T-type channel activity during stimulation with rapid spike trains (23Chemin J. Monteil A. Bourinet E. Nargeot J. Lory P. Biophys. J. 2001; 80: 1238-1250Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). A recent study by Chen et al. (26Chen Y. Lu J. Pan H. Zhang Y. Wu H. Xu K. Liu X. Jiang Y. Bao X. Yao Z. Ding K. Lo W.H. Qiang B. Chan P. Shen Y. Wu X. Ann. Neurol. 2003; 54: 239-243Crossref PubMed Scopus (321) Google Scholar) identified 12 mutations in the CACNA1H (Cav3.2) gene in 14 of 118 children with CAE, whereas the mutations were not found in 230 control subjects. We have generated five of these mutations in the rat Cav3.2 T-type channel homologue and functionally characterized them in HEK293 cells. We find that three of the missense mutations mediate significant gain of function effects on T-type channel activity. Given that T-type channels are expressed in both thalamus and neocortex (27Talley E.M. Cribbs L.L. Lee J.-H. Daud A. Perez-Reyes E. Bayliss D.A. J. Neurosci. 1999; 19: 1895-1911Crossref PubMed Google Scholar), and their association with SWD generation, this increase in activity may perhaps play a role in altering seizure threshold in patients with CAE. Site-directed Mutagenesis—Site-directed mutagenesis of the rat Cav3.2 calcium channel α1 subunit in pCDNA-3 (22McRory J.E. Santi C.M. Hamming K.S. Mezeyova J. Sutton K.G. Baillie D.L. Stea A. Snutch T.P. J. Biol. Chem. 2001; 276: 3999-4011Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) was carried out using the Quick change mutagenesis kit (Stratagene) following the manufacturer’s instructions. For each of the five mutations, the entire Cav3.2-pCDNA-3 plasmid was used as the mutagenesis template, and then the entire coding sequence of the channel was sequenced to rule out the presence of errors associated with the mutagenesis (DNA Sequencing Facility, University of Calgary) before transfection into HEK293 (tsA-201) cells for electrophysiological characterization. Cell Culture and Transient Transfection—Tissue culture and transfection of tsA-201 cells was described by us previously in detail (20Beedle A.M. Hamid J. Zamponi G.W. J. Membr. Biol. 2002; 187: 225-238Crossref PubMed Scopus (90) Google Scholar). Briefly, HEK cells were grown to 85% confluence at 37 °C (5% CO2) in Dulbecco’s modified Eagle’s medium (+10% fetal bovine serum, 200 units/ml penicillin, and 0.2 mg/ml streptomycin, Invitrogen). Cells were dissociated with trypsin (0.25%)-EDTA before and plated on glass coverslips. Mutant and wild type Cav3.2 channel α1 subunits (8 μg) and green fluorescent protein marker (1 μg) DNA were transfected into cells by the calcium phosphate method. Cells were transferred to 28 °C 24 h after transfection, and recordings were conducted 2 days later. Electrophysiology and Data Analysis—Prior to recordings, cells were transferred into an external bath solution of 5 mm barium (in mm: 5 BaCl2, 1 MgCl2, 10 HEPES, 40 tetraethylammonium chloride (TEA-Cl), 10 glucose, 88 CsCl, pH 7.2 adjusted with TEA-OH). Borosilicate glass pipettes were pulled and polished to 2–4-MΩ resistance and filled with internal solution (in mm: 108 CsCH3SO4, 4 MgCl2, 9 EGTA, 9 HEPES, pH 7.2 adjusted with Cs-OH). Data were acquired at room temperature using an Axopatch 200B amplifier and pClamp 9.0 software (Axon Instruments), low pass-filtered at 1 kHz, and digitized at 10 kHz. Series resistance was compensated to 80%. Data analysis and offline leak subtraction was carried out in Clamp-fit 9.0 (Axon Instruments), and all curves were fitted using Origin analysis software (OriginLab). Current-voltage (IV) plots were fitted using the Boltzmann equation, I=(V-Erev)×G×(1/(1+exp(-(V-V0.5a)/S))) where Erev is the reversal potential, G is the maximum slope conductance, V0.5a is the half-maximal activation potential, and S is the slope factor. Individual inactivation curves were fitted with the Boltzmann equation, I(normalized)=X+(1-X)/(1+exp(-z×(V0.5i-V)/25.6)) where I(normalized) is the fraction of available channels, X is the non-inactivating fraction of current, z is the slope factor, and V0.5i and V are the half-inactivating potential. Time constants for inactivation, τinact, were obtained from monoexponetial fits to the raw current data. Time constants for recovery from inactivation, τr, were obtained by monexoponetial fits to the time course of recovery from inactivation. These data were obtained by applying an inactivating conditioning pulse followed by a variable recovery period preceding the test pulse. All averaged data are plotted as mean ± S.E., and numbers in parentheses reflect the number of cells. Statistical analysis was carried out using one way analysis of variance, where p < 0.05 was considered as significant. We used site-directed mutagenesis to introduce five of the recently identified CAE-associated missense mutations (26Chen Y. Lu J. Pan H. Zhang Y. Wu H. Xu K. Liu X. Jiang Y. Bao X. Yao Z. Ding K. Lo W.H. Qiang B. Chan P. Shen Y. Wu X. Ann. Neurol. 2003; 54: 239-243Crossref PubMed Scopus (321) Google Scholar) into the rat Cav3.2 sequence (22McRory J.E. Santi C.M. Hamming K.S. Mezeyova J. Sutton K.G. Baillie D.L. Stea A. Snutch T.P. J. Biol. Chem. 2001; 276: 3999-4011Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). As shown in Fig. 1A, these mutations are distributed throughout the Cav3.2 channel protein, including the domain IS2-S3 linker (F161L), the domain I S5-S6 region (E282K), the domain I-II linker (C465S), the domain II S2 segment (V831M), and the domain III S5-S6 region (D1463N) and include two substitutions of negatively charged amino acids. Each of the mutant channels expressed well in HEK cells, had current densities similar to those obtained with the wild type channels (not shown), and produced typical current waveforms expected from T-type calcium channels (Fig. 1B). Two of the mutations, F161L and E282K, resulted in statistically significant hyperpolarizing shifts in the half-activation potential of the channel by ∼10 mV (Fig. 1, C and D), without affecting the reversal potential. Hence, mutations F161L and E282K, due to a shift in their activation potentials, can open in response to smaller membrane voltage fluctuations and therefore allow for greater Ca2+ influx as compared with wild type. In neurons, this behavior could result in enhanced bursting activity and might facilitate intracellular changes associated with elevated Ca2+i during epochs of intense neuronal activity. The voltage dependence of activation of the remaining mutants did not differ significantly from that observed with the wild type channel (Fig. 1D). The time course of activation was significantly slowed in the V831M mutant at potentials more positive than –30 mV (Fig. 1E), suggesting that this channel might conduct less inward current during brief membrane depolarizations. However, the V831M mutant also exhibited significantly altered inactivation characteristics. At moderate depolarizations (i.e. –20 mV), the time constant for inactivation was significantly slowed in the V831M mutant (Fig. 2A), whereas the remaining mutants behaved roughly similar to wild type channels. In addition, the position of the steady state inactivation curve of the V831M mutant was shifted toward more depolarized potentials by ∼10 mV, whereas that of the other mutants did not differ significantly from that of the wild type channel (Fig. 2B). This suggests that V831M channels, although slower to activate, are more readily available for opening and when conducting remain open for longer durations relative to wild type. The observation that V831M activation was slowed only at depolarized potentials might suggest that in this mutant, one or more of the gating transitions during channel opening become rate-limiting at positive voltages but not at more negative potentials at which activation is generally slower. Recovery from inactivation was not significantly affected by any of the mutations (Fig. 2C). Taken together, three of the five mutants examined exhibited statistically significant altered gating behavior, whereas mutant channels C456S and D1463N were indistinguishable from wild type channels. The findings with C456S and D1463N are consistent with a number of reports of calcium channel mutations in various that not result in effects on channel For several mutations in the calcium channel to not the properties of expressed channels J.E. Hamid J. E. R. Hamming K.S. Chen M. Beedle A.M. Zamponi G.W. Snutch T.P. J. Neurosci. 24 (in PubMed Scopus Google Scholar). mutations associated with mediate only effects on channel inactivation da Silva J.M. Clin. 2002; PubMed Scopus Google Scholar). several of the mutations in P/Q-type calcium channels that are found in patients with not to channel function K. M. J. 2003; Scopus Google Scholar). However, a of effects on channel not to of functional in a neuronal For the of the is that interactions with could result in altered channel function for the C456S is that could altered in this the mutation to has as a site (26Chen Y. Lu J. Pan H. Zhang Y. Wu H. Xu K. Liu X. Jiang Y. Bao X. Yao Z. Ding K. Lo W.H. Qiang B. Chan P. Shen Y. Wu X. Ann. Neurol. 2003; 54: 239-243Crossref PubMed Scopus (321) Google Scholar). is that the effects of mutations are only observed in of the splice isoforms of Cav3.2 calcium channels J. Monteil A. Perez-Reyes E. Bourinet E. Nargeot J. Lory P. J. 2002; Scopus Google Scholar). In the obtained with the F161L and the E282K mutants are more with the epileptic of patients this A shift in the voltage dependence of activation to that are to membrane potentials of neurons result in increased calcium influx and of neuronal increased spike and discharges in cells that the F161L or E282K mutations. any slowing of inactivation and shifts of the half-inactivation potential toward more depolarized potentials such as in the V831M mutant result in increased of the channel for opening and increased channel activity. It is to that used the rat Cav3.2 calcium channel as the mutagenesis template, in the mutations were into that are sequence rat and human Cav3.2 calcium channels. Hence, is that the observed effects for rat Cav3.2 calcium channels. the observed effects of three of the five mutations examined are consistent with the of CAE. there have only reports of on T-type calcium channel et al. M. K. N. J. G. F. B. J. 2001; Scopus Google Scholar) the C of the Cav3.1 calcium channel in inactivation, on current of inactivation of voltage-activated calcium channels S.C. Zamponi G.W. J. Scopus Google Scholar), and on mutagenesis in Cav3.1 channels R. P. S. E. Perez-Reyes E. S. J. 2001; Scopus Google Scholar), the are also to to the inactivation the only CAE-associated mutation that affected channel inactivation is to a region that had not previously in the inactivation of any type of calcium the domain The of mutant channel E282K on activation are one might have expected a of an negatively for a to voltage than suggest that the effects of this mutation not from an with the voltage of the channel but may perhaps mediated to the activation gating In data the of mutations with functional on the gating behavior of T-type calcium channels. At in a of mutations reported in children with CAE, the altered channel gating that from the presence of these mutations is consistent with the The of these mutations in a of but not in a number of control suggests that their presence is which is supported by their functional effects in the of CAE may well by interactions with other such as other channels and intracellular all of which are of a of activity in the epileptic Cav3.2 has in several (27Talley E.M. Cribbs L.L. Lee J.-H. Daud A. Perez-Reyes E. Bayliss D.A. J. Neurosci. 1999; 19: 1895-1911Crossref PubMed Google Scholar), the of Cav3.2 protein has not It to as to the physiological effects of Cav3.2 channel mutations are to altered of spike-wave in the neocortex (7Steriade M. Contreras D. J. Neurophysiol. 1998; 80: 1439-1455Crossref PubMed Scopus (276) Google Scholar, 8Steriade M. Contreras D. J. Neurosci. 1995; 15: 623-642Crossref PubMed Google Scholar, 9Meeren H.K. Pijn J.P. Van Luijtelaar E.L. Coenen A.M. Lopes da Silva F.H. J. Neurosci. 2002; 22: 1480-1495Crossref PubMed Google Scholar). We for the wild type Cav3.2