Characterization of the Gating Brake in the I-II Loop of Cav3.2 T-type Ca2+ Channels

Abstract

Mutations in the I-II loop of Cav3.2 channels were discovered in patients with childhood absence epilepsy. All of these mutations increased the surface expression of the channel, whereas some mutations, and in particular C456S, altered the biophysical properties of channels. Deletions around C456S were found to produce channels that opened at even more negative potentials than control, suggesting the presence of a gating brake that normally prevents channel opening. The goal of the present study was to identify the minimal sequence of this brake and to provide insights into its structure. A peptide fragment of the I-II loop was purified from bacteria, and its structure was analyzed by circular dichroism. These results indicated that the peptide had a high α-helical content, as predicted from secondary structure algorithms. Based on homology modeling, we hypothesized that the proximal region of the I-II loop may form a helix-loop-helix structure. This model was tested by mutagenesis followed by electrophysiological measurement of channel gating. Mutations that disrupted the helices, or the loop region, had profound effects on channel gating, shifting both steady state activation and inactivation curves, as well as accelerating channel kinetics. Mutations designed to preserve the helical structure had more modest effects. Taken together, these studies showed that any mutations in the brake, including C456S, disrupted the structural integrity of the brake and its function to maintain these low voltage-activated channels closed at resting membrane potentials. Mutations in the I-II loop of Cav3.2 channels were discovered in patients with childhood absence epilepsy. All of these mutations increased the surface expression of the channel, whereas some mutations, and in particular C456S, altered the biophysical properties of channels. Deletions around C456S were found to produce channels that opened at even more negative potentials than control, suggesting the presence of a gating brake that normally prevents channel opening. The goal of the present study was to identify the minimal sequence of this brake and to provide insights into its structure. A peptide fragment of the I-II loop was purified from bacteria, and its structure was analyzed by circular dichroism. These results indicated that the peptide had a high α-helical content, as predicted from secondary structure algorithms. Based on homology modeling, we hypothesized that the proximal region of the I-II loop may form a helix-loop-helix structure. This model was tested by mutagenesis followed by electrophysiological measurement of channel gating. Mutations that disrupted the helices, or the loop region, had profound effects on channel gating, shifting both steady state activation and inactivation curves, as well as accelerating channel kinetics. Mutations designed to preserve the helical structure had more modest effects. Taken together, these studies showed that any mutations in the brake, including C456S, disrupted the structural integrity of the brake and its function to maintain these low voltage-activated channels closed at resting membrane potentials. Voltage-gated calcium (Cav) channels regulate calcium influx in response to membrane depolarization. Their main role is to couple the electrical activity of cells with intracellular processes such as contraction, secretion, neurotransmission, and gene expression in many different cell types (1Catterall W.A. Perez-Reyes E. Snutch T.P. Striessnig J. Pharmacol. Rev. 2005; 57: 411-425Crossref PubMed Scopus (983) Google Scholar). Cav channels have been divided in two subfamilies, comprising high voltage-activated and low voltage-activated or T-type channels (2Bean B.P. Annu. Rev. Physiol. 1989; 51: 367-384Crossref PubMed Scopus (844) Google Scholar, 3Matteson D.R. Armstrong C.M. J. Gen. Physiol. 1986; 87: 161-182Crossref PubMed Scopus (195) Google Scholar, 4Carbone E. Lux H.D. Nature. 1984; 310: 501-502Crossref PubMed Scopus (589) Google Scholar). These proteins are composed of four or five distinct subunits that are encoded by multiple genes. The main subunit, α1, contains four homologous repeats (I–IV), each composed of six transmembrane segments (S1–S6). Each repeat shows significant homology to voltage-gated K+ channels, such as Shaker (5Jan L.Y. Jan Y.N. Nature. 1990; 345: 672Crossref PubMed Scopus (199) Google Scholar), and mutagenesis studies have established the conservation of function. For example, mutations in the pore loop between S5 and S6 affect channel permeation (6Cataldi M. Perez-Reyes E. Tsien R.W. J. Biol. Chem. 2002; 277: 45969-45976Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 7Talavera K. Janssens A. Klugbauer N. Droogmans G. Nilius B. J. Gen. Physiol. 2003; 121: 529-540Crossref PubMed Scopus (41) Google Scholar). Similarly, mutations in the S4 voltage sensors of T-channels affect channel gating as in K+ channels (8Lam A.D. Chikina M.D. McNulty M.M. Glaaser I.W. Hanck D.A. Pfluegers Arch. Eur. J. Physiol. 2005; 451: 349-361Crossref PubMed Scopus (17) Google Scholar). Therefore crystal structures of K+ channels (9Long S.B. Campbell E.B. Mackinnon R. Science. 2005; 309: 897-903Crossref PubMed Scopus (1846) Google Scholar), and models developed for Na+ voltage-gated channels (10Yarov-Yarovoy V. Baker D. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7292-7297Crossref PubMed Scopus (200) Google Scholar) are likely to provide insights into T-channel gating. In these models a change in membrane potential triggers an outward movement of the S4 voltage sensors, a concomitant movement of the S4-S5 linker, and a widening of the pore walls formed by S6 segments (10Yarov-Yarovoy V. Baker D. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7292-7297Crossref PubMed Scopus (200) Google Scholar, 11Long S.B. Campbell E.B. Mackinnon R. Science. 2005; 309: 903-908Crossref PubMed Scopus (815) Google Scholar, 12Tombola F. Pathak M.M. Isacoff E.Y. Annu. Rev. Cell Dev. Biol. 2006; 22: 23-52Crossref PubMed Scopus (244) Google Scholar). In addition, S6 segments also play a similar role in T-channel inactivation as observed in high voltage-activated channels. For example, mutations in IIIS6 affect inactivation from the open state in Cav3.1 much as they did in Cav2.2 channels (13Marksteiner R. Schurr P. Berjukow S. Margreiter E. Perez-Reyes E. Hering S. J. Physiol. (Lond.). 2001; 537: 27-34Crossref Scopus (44) Google Scholar). Mutations in Cav1.2 discovered in Timothy syndrome patients dramatically slow inactivation of Cav1.2 channels (14Splawski I. Timothy K.W. Sharpe L.M. Decher N. Kumar P. Bloise R. Napolitano C. Schwartz P.J. Joseph R.M. Condouris K. Tager-Flusberg H. Priori S.G. Sanguinetti M.C. Keating M.T. Cell. 2004; 119: 19-31Abstract Full Text Full Text PDF PubMed Scopus (1205) Google Scholar), and similarly located mutations in Cav3.1 produce a similar effect (15Arias J.M. Murbartián J. Vitko I. Lee J.H. Perez-Reyes E. FEBS Lett. 2005; 579: 3907-3912Crossref PubMed Scopus (47) Google Scholar). Despite these advances, little is known about why T-channels activate at lower voltages than other voltage-gated channels. Toward this end, a recent study (15Arias J.M. Murbartián J. Vitko I. Lee J.H. Perez-Reyes E. FEBS Lett. 2005; 579: 3907-3912Crossref PubMed Scopus (47) Google Scholar) reported that the transfer of the I-II loop of a high to a low voltage-activated channel created a chimera that gated at even lower voltages than the low voltage-activated wild type (WT). 4The abbreviations used are:WTwild typePPGpoly-proline-glycinePApolyalanineMBPmaltose-binding protein. A similar result was reported in a functional study of single nucleotide polymorphisms present in childhood absence epilepsy patients (16Chen Y.C. Lu J.J. Pan H. Zhang Y.H. Wu H.S. Xu K.M. Liu X.Y. Jiang Y.W. Bao X.H. Yao Z.J. Ding K.Y. Lo W.H.Y. Qiang B.Q. Chan P. Shen Y. Wu X.R. Ann. Neurol. 2003; 54: 239-243Crossref PubMed Scopus (318) Google Scholar), where it was observed that the mutation C456S in Cav3.2 channels (located in the proximal I-II loop) shifted the voltage dependence of activation to more negative potentials (17Vitko I. Chen Y. Arias J.M. Shen Y. Wu X.R. Perez-Reyes E. J. Neurosci. 2005; 25: 4844-4855Crossref PubMed Scopus (158) Google Scholar). This observation was followed up with a set of deletions exploring the role of the I-II loop in Cav3.2 channels (18Vitko I. Bidaud I. Arias J.M. Mezghrani A. Lory P. Perez-Reyes E. J. Neurosci. 2007; 27: 322-330Crossref PubMed Scopus (95) Google Scholar). This study revealed that the I-II loop has two separable roles: one to regulate surface expression and another to modulate the biophysical properties of Cav3.2 channels. The deletions performed within the amino-terminal region of this loop modified the voltage dependence of these channels, allowing them to open at even more negative potentials. Such results suggested that the proximal region of this loop normally functions as a “brake” that regulates channel opening. The goal of the present study was to establish the identity of the brake and to make inferences about its structure. For this purpose we used a combination of molecular biological and electrophysiological techniques to test the hypothesis that the region forms a helix-loop-helix motif. To localize the distal end of the brake, we designed a series of deletion mutants. To test the helical structures we made a series of mutations that either stabilized (polyalanine) or disrupted the putative helices (polyglycine and proline). To test the importance of the loop region, we attempted to straighten it by either replacement with an α-helix or by deletion, to of these mutations and deletions revealed a similar of each of the to the activity of Cav3.2 channels. that the gating brake has a helix-loop-helix structure within the and that the structural integrity of the brake is in T-channels closed at the resting membrane wild type protein. mutagenesis was performed and The was a fragment of into reported a mutation was into this fragment to a (18Vitko I. Bidaud I. Arias J.M. Mezghrani A. Lory P. Perez-Reyes E. J. Neurosci. 2007; 27: 322-330Crossref PubMed Scopus (95) Google Scholar), of the by the fragment into All of the were by and the sequence of the fragment was of cells were in modified with and The cells were with each Cav3.2 cells were for electrophysiological cell were at an with a The was to a a and The were at and at cell were the and to with The the and to with The were made from was of the these the was and cell were to the by a Cell The cells where the were series was in a voltage of than and inactivation were to at each voltage were used to the voltage dependence of activation for each cell the where is the is the is the of and is the of the The voltage dependence of steady state inactivation was to potentials followed by a test to to channel The each test was to that observed the potential was and the from each cell was with the was also at each test potential some of the deletion shifted the voltage dependence of activation to more negative potentials where the for is To of was to cell All of the results are as the in the were analyzed of followed by multiple test channels or multiple test fragment within the proximal region of the I-II loop of Cav3.2 was into the expression The was performed of the to a protein. this was into were by and single were in in the presence of the cell an of at expression was by the of for at The cells were with and at The was a and the was with the was from with and purified by on a The were a peptide in the presence of results were in the presence of The were with an model were at of and The were by the To is structural on the I-II loop of T-type calcium channels. we used secondary structure homology modeling, and mutagenesis techniques to insights into the structure of the Cav3.2 gating of the a that secondary structure C. G. Google Scholar), it was found that the proximal I-II loop form a helix-loop-helix structure. showed that the α-helix from to and that the one from to of Cav3.2 channels The also predicted that of the are in α-helical are in and are in the predicted a similar structure K. A. D. 2003; PubMed Scopus Google Scholar) and a for structure contains a helix-loop-helix structure the the structure of for a model where the helices are in an and the membrane The presence of α-helical in this brake region was by circular dichroism. A peptide to of the I-II loop of the Cav3.2 channel was purified in E. of the peptide a negative between and the presence of an α-helical structure. The secondary structure was from the between and the The structures in this intracellular were α-helix and whereas was These results are with the made by the and for the the of the a study we showed that deletions of the proximal I-II loop gating, whereas deletions in the distal loop surface expression (18Vitko I. Bidaud I. Arias J.M. Mezghrani A. Lory P. Perez-Reyes E. J. Neurosci. 2007; 27: 322-330Crossref PubMed Scopus (95) Google Scholar). To the end of the brake region, we a series of deletions that from the The gating properties of these channels were and steady state inactivation the are in These were analyzed to the at each voltage and with to the of activation and The were with a form of the that also and the potential In this the of activation was from for each cell and To the in the we also the for each cell to the observed and present the in These results that deletions and had a profound effect on the voltage dependence of activation and whereas had significant A of T-channel is that they with the A.D. Tsien R.W. PubMed Scopus Google Scholar). Therefore mutations that the voltage dependence of activation produce a similar in the voltage dependence of and this was observed with the and channels and These mutations also increased the observed results of this by the increased where these channels open to we the and it to cell as by and increased the distal of the brake of cell from cells and Cav3.2 channels. The were by from to from a potential of The to are in type to the in the of the The to both of for and deletion mutants. modified to the The in the from to the from each of to test and voltage dependence of the of activation and inactivation kinetics. from two of the were as a function of membrane The used to and are in of deletions on steady state of at to of voltages between and in cells and Cav3.2 channels. steady state inactivation the test was the potential was was by the at by and as a function of the The to the The results from the of to each cell are reported in properties of the I-II loop at of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of in a state inactivation was followed by a test to to channel The deletion of this the shifting the of the by and In the other deletions did have a significant effect on this of closed state These results that the gating brake around observed with any deletions proximal to this end have effects on channel gating. These results also that the brake a role in channel activation than in inactivation from closed the of the of each to or secondary structure has been and a is that whereas and them J.M. J. Biol. PubMed Scopus Google Scholar, Science. 1990; PubMed Scopus Google Scholar). In a we used circular to the predicted in the structure of a peptide six of an α-helix are with or (15Arias J.M. Murbartián J. Vitko I. Lee J.H. Perez-Reyes E. FEBS Lett. 2005; 579: 3907-3912Crossref PubMed Scopus (47) Google Scholar). In this study we the region of each with either six and or repeats and both and mutations shifted the voltage dependence of activation and inactivation to a similar indicated that these mutations disrupted a between in and either or in Therefore we a set of that and for was similar to with in the a in the and modest effects on kinetics. In the mutation shifted the and the and activation and inactivation and Similarly, the in gating in in the and inactivation whereas the mutation in both the and and both activation and inactivation the brake region from as a the of the brake with to the To test this hypothesis we one two or into the putative The were six distal to a Cav3.2 that these was M. I. and E. All disrupted channel gating, shifting the shifting the and channel and the voltage dependence of activation for was shifted to a than the whereas the inactivation of showed voltage the of the structure that the loop to may composed of two by and hypothesized that the loop to the of the To test this we performed two types of one to straighten the by it with α-helix and another by the loop region, on the the sequence in it was channels. The was also in and both were in In four the of activation were shifted to more negative potentials to and these mutations negative in the of steady state of inactivation with the the observed with the activation and inactivation were at test potentials and and were in the voltage that these were to in gating and to in activation gating. of the together, the results the structure of the brake region is to channel gating and provide for the hypothesis that this region may form a helix-loop-helix structure. To into the effects of the mutations, we models a of the region also of the mutants. The model of the sequence shows a distal to followed by a helix-loop-helix structure. The model also the presence of a between in and either or in of this may why activity more than To test this we made two in and the in The voltage dependence of activation and inactivation of channels were shifted from to the as whereas were the of any in and play in the function of the gating The goal of this study was to the of the T-type calcium channel on the intracellular loop repeats This loop is of the mutations found in childhood absence epilepsy patients are to it (16Chen Y.C. Lu J.J. Pan H. Zhang Y.H. Wu H.S. Xu K.M. Liu X.Y. Jiang Y.W. Bao X.H. Yao Z.J. Ding K.Y. Lo W.H.Y. Qiang B.Q. Chan P. Shen Y. Wu X.R. Ann. Neurol. 2003; 54: 239-243Crossref PubMed Scopus (318) Google Scholar). In we have that these I-II loop mutations are of both the gating and surface expression of Cav3.2 channels, a for role in epilepsy (17Vitko I. Chen Y. Arias J.M. Shen Y. Wu X.R. Perez-Reyes E. J. Neurosci. 2005; 25: 4844-4855Crossref PubMed Scopus (158) Google Scholar, I. Bidaud I. Arias J.M. Mezghrani A. Lory P. Perez-Reyes E. J. Neurosci. 2007; 27: 322-330Crossref PubMed Scopus (95) Google Scholar). These studies also showed that deletions in the proximal I-II loop gating, whereas deletions of the distal loop had effect on gating to surface expression of the The present study was on the proximal I-II of its to the voltage dependence of activation and we a gating The hypothesis tested in this study is that the gating brake is formed by a helix-loop-helix structure. The of the I-II a of structure and proteins of known structure that are likely to similar to the sequence K. A. D. 2003; PubMed Scopus Google Scholar). This of the proximal Cav3.2 I-II loop to the that the proximal I-II loop form a helix-loop-helix structure similar to that found in a test of this we purified a fragment of the I-II loop from E. and its structure circular a for structure Sci. PubMed Scopus Google Scholar). The circular indicated that the proximal I-II loop contains a high α-helical content, as of purified were for these results establish that the proximal loop has a high helical and provide for the structure Mutations to the types of mutations were used to test the we used deletion to the distal end of the gating we tested for the of α-helical by six with and to the and as a we the with to the we tested for the of the loop by either it to an α-helix or by it to we also tested the of to the of the of the and the of the helix-loop-helix with to the In a study we found that deletion of a similar effect on gating as and that deletions made did gating (18Vitko I. Bidaud I. Arias J.M. Mezghrani A. Lory P. Perez-Reyes E. J. Neurosci. 2007; 27: 322-330Crossref PubMed Scopus (95) Google Scholar). To the distal we a series of the distal end at of from a profound in the voltage dependence of both activation and inactivation and kinetics. effects were in the study with and (18Vitko I. Bidaud I. Arias J.M. Mezghrani A. Lory P. Perez-Reyes E. J. Neurosci. 2007; 27: 322-330Crossref PubMed Scopus (95) Google Scholar), that the deletion disrupted the function of the gating the proximal an had a more modest effect on the and significant effect on the both activation and inactivation of were to channels. In mutations that shifted activation also shifted the voltage dependence of this was the for some mutations open state with little or effect on the voltage dependence of the proximal more in channels that gated Taken these results that the in play an role in the function of the we tested the effect of either these or them to The mutation a of channel gating, shifting the voltage dependence of activation and and accelerating kinetics. In the mutation had a effect on channel gating, shifting the effect on the of the or on channel kinetics. A is that the putative that the gating brake this region and that this region a role in its function. The of Mutations in the hypothesis for these studies was that replacement of six of an α-helix with such as and the helices and the function of the gating brake, whereas replacement with such as preserve structure and function J.M. J. Biol. PubMed Scopus Google Scholar, Science. 1990; PubMed Scopus Google Scholar). the of the putative and six with either a repeat or the in the voltage dependence of activation and inactivation and kinetics. The effects were observed with the the also disrupted gating. The structural model of these a for these replacement of with in an between and In to channels, the in this putative are also in the the mutations two these the mutation had little or whereas the to function and The of Mutations in the studies of the loop region were by the presence of two putative one to in an with to replacement of either region with six channels with modest effects on gating. In mutation of both or deletion of the loop region in gating. The studies well with the replacement of either was to the helix-loop-helix structure of by the of the suggested that more and that of and a more region, allowing between the of that the loop was and that was to The gating of this is similar to the we that function of the gating brake was The model also that the epilepsy C456S, is at a in the brake, the of and hypothesis is that the are in an in and that the form with of the channel of the brake In either the results that the loop region is for the function of the brake and that of its structure to functional effects on channel gating. of into of the test of the α-helical structure of the region was to or into the of the hypothesis was that each change the of the of the by and the these in the voltage dependence of activation and inactivation and kinetics. The gating properties of and were different from channels different from each other with the of where channels more These results the hypothesis that the brake region is with to the of the to in of the mutations than wild type channels, even for cell as by cell of this for by the for in the were at more negative potentials and from the To for this we the and to cell and the observed this and showed a significant of likely for this are that these mutations to in the of channel in the surface expression of channels. In study we found for deletion of to increased with change in surface in whereas deletion of to both surface expression and (18Vitko I. Bidaud I. Arias J.M. Mezghrani A. Lory P. Perez-Reyes E. J. Neurosci. 2007; 27: 322-330Crossref PubMed Scopus (95) Google Scholar). studies are to the of Cav3.2 channels, the I-II loop is in this In results that the brake is within the of the I-II loop of Cav3.2 channels, and that the structural integrity of this region is for the low voltage activation of this T-type of this structure to channels that open at more negative at more negative and kinetics. inactivation of T-channels is to activation K. Nilius B. Pfluegers Arch. Eur. J. Physiol. 2006; PubMed Scopus Google Scholar), and this may the concomitant in steady state activation and inactivation observed in this results that the function of this region is as a brake that prevents channel of T-channel gating that of the channels open of movement (8Lam A.D. Chikina M.D. McNulty M.M. Glaaser I.W. Hanck D.A. Pfluegers Arch. Eur. J. Physiol. 2005; 451: 349-361Crossref PubMed Scopus (17) Google Scholar, Klugbauer N. F. FEBS Lett. 2002; PubMed Scopus Google Scholar). This that T-channels open minimal movement of voltage sensors, and studies that this why T-channel are (8Lam A.D. Chikina M.D. McNulty M.M. Glaaser I.W. Hanck D.A. Pfluegers Arch. Eur. J. Physiol. 2005; 451: 349-361Crossref PubMed Scopus (17) Google Scholar, K. Nilius B. Pfluegers Arch. Eur. J. Physiol. 2006; PubMed Scopus Google Scholar, A. Perez-Reyes E. J. Gen. Physiol. 2001; PubMed Scopus Google Scholar). that the brake region the closed the role of one of the S4-S5 to the pore closed at resting membrane potentials. of the brake channels to open at more negative membrane potentials and open of the brake region is much channels than suggesting in the role of the brake in the low voltage gating activation of T-type channels. with