Structural Determinants of L-type Channel Activation in Segment IIS6 Revealed by a Retinal Disorder

Abstract

The mechanism of channel opening for voltage-gated calcium channels is poorly understood. The importance of a conserved isoleucine residue in the pore-lining segment IIS6 has recently been highlighted by functional analyses of a mutation (I745T) in the CaV1.4 channel causing severe visual impairment (Hemara-Wahanui, A., Berjukow, S., Hope, C. I., Dearden, P. K., Wu, S. B., Wilson-Wheeler, J., Sharp, D. M., Lundon-Treweek, P., Clover, G. M., Hoda, J. C., Striessnig, J., Marksteiner, R., Hering, S., and Maw, M. A. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7553–7558). In the present study we analyzed the influence of amino acids in segment IIS6 on gating of the CaV1.2 channel. Substitution of Ile-781, the CaV1.2 residue corresponding to Ile-745 in CaV1.4, by residues of different hydrophobicity, size and polarity shifted channel activation in the hyperpolarizing direction (I781P > I781T > I781N > I781A > I781L). As I781P caused the most dramatic shift (-37 mV), substitution with this amino acid was used to probe the role of other residues in IIS6 in the process of channel activation. Mutations revealed a high correlation between the midpoint voltages of activation and inactivation. A unique kinetic phenotype was observed for residues 779–782 (LAIA) located in the lower third of segment IIS6; a shift in the voltage dependence of activation was accompanied by a deceleration of activation at hyperpolarized potentials, a deceleration of deactivation at all potentials (I781P and I781T), and decreased inactivation. These findings indicate that Ile-781 substitutions both destabilize the closed conformation and stabilize the open conformation of CaV1.2. Moreover there may be a flexible center of helix bending at positions 779–782 of CaV1.2. These four residues are completely conserved in high voltage-activated calcium channels suggesting that these channels may share a common mechanism of gating. The mechanism of channel opening for voltage-gated calcium channels is poorly understood. The importance of a conserved isoleucine residue in the pore-lining segment IIS6 has recently been highlighted by functional analyses of a mutation (I745T) in the CaV1.4 channel causing severe visual impairment (Hemara-Wahanui, A., Berjukow, S., Hope, C. I., Dearden, P. K., Wu, S. B., Wilson-Wheeler, J., Sharp, D. M., Lundon-Treweek, P., Clover, G. M., Hoda, J. C., Striessnig, J., Marksteiner, R., Hering, S., and Maw, M. A. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7553–7558). In the present study we analyzed the influence of amino acids in segment IIS6 on gating of the CaV1.2 channel. Substitution of Ile-781, the CaV1.2 residue corresponding to Ile-745 in CaV1.4, by residues of different hydrophobicity, size and polarity shifted channel activation in the hyperpolarizing direction (I781P > I781T > I781N > I781A > I781L). As I781P caused the most dramatic shift (-37 mV), substitution with this amino acid was used to probe the role of other residues in IIS6 in the process of channel activation. Mutations revealed a high correlation between the midpoint voltages of activation and inactivation. A unique kinetic phenotype was observed for residues 779–782 (LAIA) located in the lower third of segment IIS6; a shift in the voltage dependence of activation was accompanied by a deceleration of activation at hyperpolarized potentials, a deceleration of deactivation at all potentials (I781P and I781T), and decreased inactivation. These findings indicate that Ile-781 substitutions both destabilize the closed conformation and stabilize the open conformation of CaV1.2. Moreover there may be a flexible center of helix bending at positions 779–782 of CaV1.2. These four residues are completely conserved in high voltage-activated calcium channels suggesting that these channels may share a common mechanism of gating. The entry of Ca2+ through voltage-gated Ca2+ channels has direct effects on muscle contraction, release of hormones and neurotransmitters, hearing, vision, gene expression, and other important physiological functions (2.Catterall W.A. Striessnig J. Snutch T.P. Perez-Reyes E. Pharmacol. Rev. 2003; 55: 579-581Crossref PubMed Scopus (208) Google Scholar). The pore-forming α1-subunits of voltage-gated Ca2+ channels are composed of four homologous domains formed by six transmembrane segments (S1–S6) that are linked together on a single polypeptide (3.Catterall W.A. Annu. Rev. Cell Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1950) Google Scholar). A membrane depolarization initiates channel openings (activation) and closures (inactivation). These events can be considered a multistep process consisting of a conformational change in the voltage sensor, a transmission of the signal to the pore region, the opening of the pore, and channel closure due to inactivation. The voltage-sensing machinery is formed by multiple charged amino acids located in segment S4 and adjacent structures of each domain (4.Yifrach O. MacKinnon R. Cell. 2002; 111: 231-239Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). A large number of amino acids involved in Ca2+ channel inactivation have been identified and several molecular mechanisms for this process have been proposed (for reviews see Refs. 5.Hering S. Berjukow S. Sokolov S. Marksteiner R. Weiss R.G. Kraus R. Timin E.N. J. Physiol. 2000; 528: 237-249Crossref PubMed Scopus (117) Google Scholar, 6.Stotz S.C. Zamponi G.W. Trends Neurosci. 2001; 24: 176-181Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 7.Shi C. Soldatov N.M. J. Biol. Chem. 2002; 277: 6813-6821Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The molecular mechanism of the voltage-dependent pore opening of Ca2+ channels, however, is less studied and largely unknown. The first attempt to localize the structural elements in Ca2+ channel α1-subunits that are involved in channel activation was made by Tanabe et al. (8.Tanabe T. Adams B.A. Numa S. Beam K.G. Nature. 1991; 352: 800-803Crossref PubMed Scopus (107) Google Scholar) who constructed chimeric channels in which sequence stretches of a slow activating (“skeletal muscle-like”) CaV1.1 α1-subunit were replaced by sequences from a fast activating (“cardiac-like”) CaV1.2 α1-subunit. The chimeras activated slowly if repeat I of the CaV1.2 α1-subunit was replaced by the CaV1.1 α1-sequence. In a later study, replacement of domains I, II, and III of the low voltage and fast activating CaV3.1 α1-subunit with the corresponding domains of the high voltage-activated CaV1.2 α1-subunit resulted in a high voltage-activated channel (9.Li J. Stevens L. Klugbauer N. Wray D. J. Biol. Chem. 2004; 279: 26858-26867Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). An important role of domains I and III but not II and IV on midpoint voltage and time constants of activation was reported by Garcia et al. (10.Garcia J. Nakai J. Imoto K. Beam K.G. Biophys. J. 1997; 72: 2515-2523Abstract Full Text PDF PubMed Scopus (54) Google Scholar) who mutated the arginines in the S4 segments of all four domains of a chimeric channel to neutral or negative amino acids. The removal of prolines that are conserved in segments IS4 and IIIS4 of voltage-gated Ca2+ channels resulted in shortening of channel open time, whereas introduction of extra prolines to corresponding positions of IIS4 and IVS4 lengthened the channel open time (11.Yamaguchi H. Muth J.N. Varadi M. Schwartz A. Varadi G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1357-1362Crossref PubMed Scopus (25) Google Scholar). Our present study was initiated by the recent finding that a novel retinal disorder is caused by a point mutation (I745T) in segment IIS6 of the CaV1.4 α1-subunit that shifts the voltage dependence of CaV1.4 channel activation by approximately -30 mV (1.Hemara-Wahanui A. Berjukow S. Hope C.I. Dearden P.K. Wu S.B. Wilson-Wheeler J. Sharp D.M. Lundon-Treweek P. Clover G.M. Hoda J.C. Striessnig J. Marksteiner R. Hering S. Maw M.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7553-7558Crossref PubMed Scopus (118) Google Scholar, 12.Hope C.I. Sharp D.M. Hemara-Wahanui A. Sissingh J.I. Lundon P. Mitchell E.A. Maw M.A. Clover G.M. Clin. Exp. Ophthalmol. 2005; 33: 129-136Crossref PubMed Scopus (69) Google Scholar). As CaV1.4 channels express only at low density in mammalian cell lines (13.Koschak A. Reimer D. Walter D. Hoda J.C. Heinzle T. Grabner M. Striessnig J. J. Neurosci. 2003; 23: 6041-6049Crossref PubMed Google Scholar) we have decided to study the functional roles of this residue and neighboring residues in segment IIS6 by introducing and characterizing mutations in the homologous CaV1.2 channel. Our findings demonstrate that residue Ile-781 and three neighboring residues (Leu-779, Ala-780, and Ala-782) play a key role in gating of the CaV1.2 channel. Mutagenesis—The CaV1.2 α1-subunit coding sequence (GenBank™ X15539) in-frame 3′ to the coding region of a modified green fluorescent protein (GFP) 4The abbreviations used are: GFP, green fluorescent protein; WT, wild-type; NaChBac, bacterial sodium channel; Bay K8644, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)pyridine-3-carboxylic acid methyl ester. was kindly donated by Dr. M. Grabner (14.Grabner M. Dirksen R.T. Beam K.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1903-1908Crossref PubMed Scopus (136) Google Scholar). For electrophysiological studies we used the plasmid lacking the GFP tag. Substitutions in segment IIS6 of the CaV1.2 α1-subunit were introduced by the “gene SOEing” technique (15.Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2639) Google Scholar). In particular, an isoleucine to threonine mutation was introduced in position 781, and further substitutions were made at this position to proline, leucine, alanine, asparagine, glutamine, and arginine. The mutated fragments were cloned into a BamHI-AflII-cassette (nucleotides 1265 and 2689, numbering according to the CaV1.2 α1-subunit coding sequence). This cassette was also used in creation of C769P, G770P, N771P, Y772P, I773P, L774P, L775P, N776P, V777P, F778P, L779P, A780P, A782P, and V783P. Mutations that did not lead to functional channels were recloned into the GFP-tagged vector (14.Grabner M. Dirksen R.T. Beam K.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1903-1908Crossref PubMed Scopus (136) Google Scholar) to study membrane targeting. All constructs were checked by restriction site mapping and sequencing. Cell Culture and Transient Transfection—Human embryonic kidney tsA-201 cells were grown at 5% CO2 and 37 °C to 80% confluence in Dulbecco's modified Eagle's/F-12 medium supplemented with 10% (v/v) fetal calf serum and 100 units/ml penicillin/streptomycin. Cells were split via trypsin EDTA and plated on 35-mm Petri dishes (Falcon) at 30–50% confluence ∼16 h before transfection. Subsequently tsA-201 cells were co-transfected with cDNAs encoding wild-type or mutant CaV1.2 α1-subunits with auxiliary β1a (16.Ruth P. Rohrkasten A. Biel M. Bosse E. Regulla S. Meyer H.E. Flockerzi V. Hofmann F. Science. 1989; 245: 1115-1118Crossref PubMed Scopus (257) Google Scholar) or β2a (17.Perez-Reyes E. Castellano A. Kim H.S. Bertrand P. Baggstrom E. Lacerda A.E. Wei X.Y. Birnbaumer L. J. Biol. Chem. 1992; 267: 1792-1797Abstract Full Text PDF PubMed Google Scholar) as well as α2-δ1-subunits (18.Ellis S.B. Williams M.E. Ways N.R. Brenner R. Sharp A.H. Leung A.T. Campbell K.P. McKenna E. Koch W.J. Hui A. et al.Science. 1988; 241: 1661-1664Crossref PubMed Scopus (442) Google Scholar). The transfection of tsA-201 cells was performed using the FuGENE 6 transfection reagent (Roche Applied Science) following standard protocols. Ionic Current Recordings and Data Acquisition—Barium currents (IBa) through voltage-gated Ca2+ channels were recorded at 22–25 °C using the patch clamp technique (19.Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pflugers Arch. Eur. J. Physiol. PubMed Scopus Google Scholar) by of an patch clamp h transfection. The and to with with of were made from and with and to with All were using a by of a and on and were at and at for the inactivation currents were at and at and currents were at and at currents were using of currents by a mV hyperpolarizing or by of an and voltage were The was used for and was for and The voltage dependence of activation was from that were according to the following modified I is is membrane I is is membrane is voltage for and is The time of activation was to a is at time A is the is a time and is The voltage dependence of inactivation was using a multistep to for A. Berjukow S. Hope C.I. Dearden P.K. Wu S.B. Wilson-Wheeler J. Sharp D.M. Lundon-Treweek P. Clover G.M. Hoda J.C. Striessnig J. Marksteiner R. Hering S. Maw M.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7553-7558Crossref PubMed Scopus (118) Google Scholar). The sequence was from a of were according to a is membrane is midpoint is the and is the of Data are as was with the were h transfection. Data are for tsA-201 cells from three were with a using a The membrane was with were using an for the GFP-tagged CaV1.2 α1-subunits and a for I781T CaV1.2 to Ile-745 in segment IIS6 of the CaV1.4 α1-subunit to residue Ile-781 in the CaV1.2 α1-subunit The effects of I781T substitution on CaV1.2 channel activation were wild-type or mutant CaV1.2 α1-subunits together with auxiliary β1a and α2-δ1-subunits in tsA-201 of currents and the corresponding and voltage of channel activation and inactivation are For both wild-type and I781T channels, the at mV that the I781T mutation not The midpoint voltage of activation from mV in the wild-type channel to mV in the I781T channel I781T also shifted the voltage dependence of inactivation. The midpoint voltage of the inactivation from mV in wild-type channels to mV in I781T channels and channels were by whereas I781T channels were by a at the of -30 substitutions shift the voltage of CaV1.2 channel activation and inactivation. voltage of activation of wild-type I781P I781T I781A and I781N voltage of inactivation for wild-type I781P and I781T lines to correlation between potentials of activation and inactivation of the Ile-781 a correlation of influence of IIS6 pore mutations on voltage-dependent gating of CaV1.2 and of the activation and of channel inactivation a to the potentials of the of of 6 in a The of CaV1.2 by Ile-781 in and demonstrate that Ile-781 a role in CaV1.2 gating. into the structural at this we replaced Ile-781 by residues of different polarity and of Ile-781 by leucine, alanine, asparagine, and caused a shift in channel activation from approximately mV for to approximately mV for I781P The mutations also shifted the channel inactivation by from approximately mV to approximately -30 mV and the in the voltage of channel activation and inactivation in This is from the midpoint voltages of the activation are the midpoint voltages of the inactivation the with a revealed a correlation of Ile-781 Substitutions at and voltage of the activation time constants for the Ile-781 substitution were also -30 mV and activation of the wild-type and mutant channels with a time between and between and voltages negative to the of wild-type CaV1.2 activation mutant channels activation channel activation was observed in wild-type and whereas the four mutant channels activated at slow for time activation of I781P at mV was In other the the voltage dependence of channel activation was shifted the slowly the channels activated at hyperpolarized voltages into the of the open channel we also analyzed the voltage dependence of deactivation for I781P and the that the effects on channel activation. The mutant channels slowly the wild-type channel at all potentials with deactivation of I781P that of I781T Substitution of kinetic phenotype to that observed for the I781T substitution slow activation at hyperpolarized and was reported for a bacterial sodium channel a conserved in the third of was by V. T. W.A. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). an role of in the corresponding position of segment IIS6 of CaV1.2 we mutated this amino acid to however, effects on the from on the voltage dependence of channel activation and inactivation adjacent to was to shift the voltage dependence of channel activation and inactivation and to slow the time of inactivation did however, slow channel activation at hyperpolarized voltages between and at all and channel deactivation was not of the effects of substitution on the voltage dependence of CaV1.2 gating. voltage dependence of activation of wild-type and mutant lines to correlation between potentials of activation and inactivation is a correlation of of kinetic phenotype in was not in the correlation of substitution of Ile-781 caused the most dramatic in channel activation the of I781P on CaV1.2 channel gating we all amino acids between and by prolines and Substitution of the residues Ile-781 and and of shifted the voltage dependence of activation and also channel activation at hyperpolarized voltages channel inactivation was for and not for and a were observed for This mutation shifted the voltage dependence of channel activation but not inactivation and this mutation the of channel activation at all voltages substitutions C769P, I773P, and caused or shifts in the voltage dependence of activation for of constructs L774P, L775P, V777P, and did not the of observed for these be of an impairment of membrane we the of GFP-tagged (14.Grabner M. Dirksen R.T. Beam K.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1903-1908Crossref PubMed Scopus (136) Google Scholar) by and mutant GFP-tagged CaV1.2 α1-subunits were co-transfected with β1a and α2-δ1-subunits in tsA-201 and the membrane was by with with U. B. K. Striessnig J. J. Physiol. 1999; PubMed Scopus Google Scholar, T. M. H. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google the wild-type CaV1.2 α1-subunit at the membrane with also from cells the channels are that all mutant were to the with also These findings demonstrate that the of observed for these be to a of the mutant GFP-tagged CaV1.2 α1-subunits to the Bay and of Ile-781 shift of voltage dependence of activation negative voltages is a of Bay was to if the shift in activation gating caused by the mutation I781T potentials corresponding to the of the voltage mV for and -30 mV for 100 of in and in the I781T A and The voltage of channel activation were shifted to mV in I781T and mV in is well that the gating of high voltage-activated Ca2+ channels (for reviews see Refs. L. N. R. E. D. J. E. J. 1998; PubMed Scopus Google Scholar, J. Campbell K.P. 2003; PubMed Scopus Google Scholar, S. Trends Pharmacol. Sci. 2002; 23: Full Text Full Text PDF PubMed Scopus Google Scholar). the observed in the voltage dependence of channel activation with we and I781P CaV1.2 α1-subunits with or as well as the and analyzed the activation For each of these channels, of the a hyperpolarizing shift in the voltage dependence of channel activation and channel inactivation with with the These findings that these Ile-781 not In this study we demonstrate a role of a of amino acids (Leu-779, Ala-780, Ile-781, Ala-782) in the pore-forming segment IIS6 in activation and inactivation of the CaV1.2 channel. This finding is to a recently a mutation of the corresponding isoleucine to threonine in CaV1.4 (I745T) a severe retinal disorder C.I. Sharp D.M. Hemara-Wahanui A. Sissingh J.I. Lundon P. Mitchell E.A. Maw M.A. Clover G.M. Clin. Exp. Ophthalmol. 2005; 33: 129-136Crossref PubMed Scopus (69) Google Scholar). As for the CaV1.4 mutant (1.Hemara-Wahanui A. Berjukow S. Hope C.I. Dearden P.K. Wu S.B. Wilson-Wheeler J. Sharp D.M. Lundon-Treweek P. Clover G.M. Hoda J.C. Striessnig J. Marksteiner R. Hering S. Maw M.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7553-7558Crossref PubMed Scopus (118) Google I781T resulted in an shift in the voltage dependence of activation and inactivation of CaV1.2 suggesting a common mechanism of gating in both channel analyses revealed that the I781T mutation also caused a negative shift in the voltage dependence of inactivation activation at hyperpolarized voltages and deactivation at all potentials These in gating of CaV1.2 to the open and can be by a of the closed an of the to the open see a of the open a deceleration of the to the closed see V. T. W.A. 2004; Full Text Full Text PDF PubMed Scopus Google or Ile-781 Substitutions the Ile-781 substitutions that functional channels caused in channel gating to this finding by a by a of channel A mechanism for of the closed be a in in position with neighboring residues in the This is in with the lower and shifts in the activation of threonine with and of Ile-781 with neighboring residues in the region to of the closed conformation in wild-type in however, not the is less threonine J. J. Biol. PubMed Scopus Google a shift As from isoleucine by the position of only methyl and the we that Ile-781 of a region in the closed channel caused the on channel activation. however, to the wild-type isoleucine to threonine and the with neighboring amino acids in the closed by bending of the J.N. J. Biol. 2002; PubMed Scopus Google Scholar) and may an open pore for of the deceleration of deactivation observed for I781P and I781T is a of a of the open channel A of the open is also by the of channel inactivation that was observed for the of Ile-781 a in the open channel in a from the open to the These findings indicate that a point mutation in segment can be to slow channel that closure of voltage-gated Ca2+ channels the of all four pore-forming elements into the of the not the the pore may open for a of the of I781P we the residues between and by prolines acids causing shifts in the voltage dependence of channel activation and inactivation were in a the channel (I781P > L779P, A782P, The the voltage dependence of channel activation was the these channels activated at hyperpolarized potentials and decreased whereas inactivation was for and suggesting that all three residues to both activation and inactivation gating to different In the of caused a shift of the activation and but did not shift the inactivation and did not the of channel activation at hyperpolarized I781P and the voltage dependence of channel deactivation was in this mutant these findings that mutation not stabilize the open but is to destabilize the closed The correlation between shifts in the activation and inactivation observed for all with the of an finding and A channel activation in of a voltage-sensing a mechanism of the pore opening and inactivation events this to the did not currents These that substitutions in Ca2+ channel segments may the of the were recently made for substitutions in the of segment T. W.A. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus (54) Google Scholar). however, that other substitutions in these positions functional Ca2+ channels with gating Bay and of deactivation and a shift of activation as observed for the mutation I781T and other mutations is of mutation I781T a further shift of channel activation the of the These that Bay and mutation I781T the channel via and structural studies that the of channel activation and inactivation by by a direct of the of the pore-forming segment S. Trends Pharmacol. Sci. 2002; 23: Full Text Full Text PDF PubMed Scopus Google Scholar, F. Nature. 2004; PubMed Scopus Google Scholar). The I781P and I781T the shift in the voltage dependence of channel activation and inactivation that wild-type are with β2a β1a These findings that of segment and the effects of residue on pore are by of Ile-781 and for of Ca2+ of the helix channel revealed that a conserved at position is for bending the pore-lining helix to channel opening A. J. M. MacKinnon R. Nature. 2002; PubMed Scopus Google Scholar). studies have that the corresponding in the bacterial voltage-gated sodium channel a V. T. W.A. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). This is on shifts the activation to hyperpolarized and this shift is accompanied by a deceleration of deactivation the corresponding in CaV1.2 to did not effects on channel gating of a gating at residue is not The kinetic phenotype of Ile-781 and adjacent residues (Leu-779, Ala-780, and Ala-782) observed in the present study however, to the for V. T. W.A. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Our that helix bending is to to the channel in CaV1.2. 779–782 are conserved in high voltage-activated Ca2+ channels, and is to that this sequence (LAIA) in a common mechanism of gating. Dr. for in The of was