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• W1991438270 abstract "Voltage dependence and kinetics of CaV1.2 activation are affected by structural changes in pore-lining S6 segments of the α1-subunit. Significant effects are induced by either proline or threonine substitutions in the lower third of segment IIS6 (“bundle crossing region”), where S6 segments are likely to seal the channel in the closed conformation (Hohaus, A., Beyl, S., Kudrnac, M., Berjukow, S., Timin, E. N., Marksteiner, R., Maw, M. A., and Hering, S. (2005) J. Biol. Chem. 280, 38471–38477). Here we report that S435P in IS6 results in a large shift of the activation curve (-25.9 ± 1.2 mV) and slower current kinetics. Threonine substitutions at positions Leu-429 and Leu-434 induced a similar kinetic phenotype with shifted activation curves (L429T by -6.6 ± 1.2 and L434T by -12.1 ± 1.7 mV). Inactivation curves of all mutants were shifted to comparable extents as the activation curves. Interdependence of IS6 and IIS6 mutations was analyzed by means of mutant cycle analysis. Double mutations in segments IS6 and IIS6 induce either additive (L429T/I781T, -34.1 ± 1.4 mV; L434T/I781T, -40.4 ± 1.3 mV; L429T/L779T, -12.6 ± 1.3 mV; and L434T/L779T, -22.4 ± 1.3 mV) or nonadditive shifts of the activation curves along the voltage axis (S435P/I781T, -33.8 ± 1.4 mV). Mutant cycle analysis revealed energetic coupling between residues Ser-435 and Ile-781, whereas other paired mutations in segments IS6 and IIS6 had independent effects on activation gating. Voltage dependence and kinetics of CaV1.2 activation are affected by structural changes in pore-lining S6 segments of the α1-subunit. Significant effects are induced by either proline or threonine substitutions in the lower third of segment IIS6 (“bundle crossing region”), where S6 segments are likely to seal the channel in the closed conformation (Hohaus, A., Beyl, S., Kudrnac, M., Berjukow, S., Timin, E. N., Marksteiner, R., Maw, M. A., and Hering, S. (2005) J. Biol. Chem. 280, 38471–38477). Here we report that S435P in IS6 results in a large shift of the activation curve (-25.9 ± 1.2 mV) and slower current kinetics. Threonine substitutions at positions Leu-429 and Leu-434 induced a similar kinetic phenotype with shifted activation curves (L429T by -6.6 ± 1.2 and L434T by -12.1 ± 1.7 mV). Inactivation curves of all mutants were shifted to comparable extents as the activation curves. Interdependence of IS6 and IIS6 mutations was analyzed by means of mutant cycle analysis. Double mutations in segments IS6 and IIS6 induce either additive (L429T/I781T, -34.1 ± 1.4 mV; L434T/I781T, -40.4 ± 1.3 mV; L429T/L779T, -12.6 ± 1.3 mV; and L434T/L779T, -22.4 ± 1.3 mV) or nonadditive shifts of the activation curves along the voltage axis (S435P/I781T, -33.8 ± 1.4 mV). Mutant cycle analysis revealed energetic coupling between residues Ser-435 and Ile-781, whereas other paired mutations in segments IS6 and IIS6 had independent effects on activation gating. Ca2+ current through CaV1.2 channels initiates muscle contraction, release of hormones and neurotransmitters, and affects physiological processes such as vision, hearing, and gene expression (1Catterall W.A. Striessnig J. Snutch T.P. Perez-Reyes E. Pharmacol. Rev. 2003; 55: 579-581Crossref PubMed Scopus (202) Google Scholar). Their pore-forming α1-subunit is composed of four homologous domains formed by six transmembrane segments (S1–S6) (2Catterall W.A. Annu. Rev. Cell Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1902) Google Scholar). The signal of the voltage-sensing machinery, consisting of multiple charged amino acids (located in segments S4 and adjacent structures of each domain), is transmitted to the pore region (3Jiang Y. Ruta V. Chen J. Lee A. MacKinnon R. Nature. 2003; 423: 42-48Crossref PubMed Scopus (708) Google Scholar). Conformational changes in pore lining S6 and adjacent segments finally lead to pore openings (activation) and closures (inactivation). Our understanding of how CaV1.2 channels open and close is largely based on extrapolations of structural information from potassium channels. The crystal structures of the closed conformation of two bacterial potassium channels (KcsA and MlotiK) (4Zhou Y. Morais-Cabral J.H. Kaufman A. MacKinnon R. Nature. 2001; 414: 43-48Crossref PubMed Scopus (1681) Google Scholar, 5Clayton G.M. Altieri S. Heginbotham L. Unger V.M. Morais-Cabral J.H. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 1511-1515Crossref PubMed Scopus (140) Google Scholar) show a gate located at the intracellular channel mouth formed by tightly packed S6 helices. The crystal structure of the open conformation of Kv1.2 (6Long S.B. Campbell E.B. MacKinnon R. Science. 2005; 309: 897-903Crossref PubMed Scopus (1811) Google Scholar, 7Long S.B. Tao X. Campbell E.B. MacKinnon R. Nature. 2007; 450: 376-382Crossref PubMed Scopus (1157) Google Scholar) revealed a bent S6 with the highly conserved PXP motif apparently acting as a hinge (see 8Webster S.M. Del Camino D. Dekker J.P. Yellen G. Nature. 2004; 428: 864-868Crossref PubMed Scopus (180) Google Scholar). The activation mechanism proposed for MthK channels involves helix bending at a highly conserved glycine at position 83 (see Ref. 9Jiang Y. Lee A. Chen J. Cadene M. Chait B.T. MacKinnon R. Nature. 2002; 417: 515-522Crossref PubMed Scopus (1196) Google Scholar, “glycine gating hinge” hypothesis). Compared with potassium channels, the pore of CaV is asymmetric, and none of the four S6 segments has a putative helix-bending PXP motif. Furthermore, the conserved glycine (corresponding to position 83 in MthK, see Ref. 10Jiang Y. Lee A. Chen J. Cadene M. Chait B.T. MacKinnon R. Nature. 2002; 417: 523-526Crossref PubMed Scopus (1065) Google Scholar) is only present in segments IS6 and IIS6 (for review see Ref. 11Hering S. Beyl S. Stary A. Kudrnac M. Hohaus A. Guy H.R. Timin E. Channels. 2008; 2: 61-69Crossref PubMed Scopus (12) Google Scholar). We have shown that substituting proline for this glycine in IIS6 of CaV1.2 does not significantly affect gating (12Hohaus A. Beyl S. Kudrnac M. Berjukow S. Timin E.N. Marksteiner R. Maw M.A. Hering S. J. Biol. Chem. 2005; 280: 38471-38477Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Zhen et al. (13Zhen X.G. Xie C. Fitzmaurice A. Schoonover C.E. Orenstein E.T. Yang J. J. Gen. Physiol. 2005; 126: 193-204Crossref PubMed Scopus (40) Google Scholar) investigated the pore lining S6 segments of CaV2.1 using the substituted cysteine accessibility method. The accessibility of cysteines was changed by opening and closing the channel, consistent with the gate being on the intracellular side. The general picture of a channel gate close to the inner channel mouth of CaV1.2 was recently supported by pharmacological studies (14Beyl S. Timin E.N. Hohaus A. Stary A. Kudrnac M. Guy R.H. Hering S. J. Biol. Chem. 2007; 282: 3864-3870Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Substitution of hydrophilic residues in the lower third of segment IIS6 of CaV1.2 (LAIA motif, 779–784, see Ref. 12Hohaus A. Beyl S. Kudrnac M. Berjukow S. Timin E.N. Marksteiner R. Maw M.A. Hering S. J. Biol. Chem. 2005; 280: 38471-38477Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) induces pronounced changes in channel gating as follows: a shift in the voltage dependence of activation accompanied by a slowing of the activation kinetics near the footstep of the m∞(V) curve and a slowing of deactivation at all potentials. Interestingly, these changes in channel gating resemble the effects of proline substitution of Gly-219 in the bacterial sodium channel from Bacillus halodurans (“Gly-219 gating hinge,” see Ref. 15Zhao Y. Yarov-Yarovoy V. Scheuer T. Catterall W.A. Neuron. 2004; 41: 859-865Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The strongest shifts of the activation curve reported so far were observed for proline substitutions (12Hohaus A. Beyl S. Kudrnac M. Berjukow S. Timin E.N. Marksteiner R. Maw M.A. Hering S. J. Biol. Chem. 2005; 280: 38471-38477Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). As prolines in an α-helix cause a rigid kink with an angle of about 26° (16Cordes F.S. Bright J.N. Sansom M.S. J. Mol. Biol. 2002; 323: 951-960Crossref PubMed Scopus (298) Google Scholar), we hypothesized that these mutants were causing a kink in helix IIS6 similar to a bend that would normally occur flexibly during the activation process (12Hohaus A. Beyl S. Kudrnac M. Berjukow S. Timin E.N. Marksteiner R. Maw M.A. Hering S. J. Biol. Chem. 2005; 280: 38471-38477Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Here we extend our previous study by systematically substituting residues in segment IS6 of CaV1.2 by proline or the small and polar threonine. Several functional IS6 mutants with shifted activation and inactivation characteristics were identified (S435P, L429T, and L434T), and the interdependence of IS6 and IIS6 mutations was analyzed. Mutant cycle analysis revealed both mutually independent and energetically coupled contributions of IS6 and IIS6 residues on activation gating. Mutagenesis—The CaV1.2 α1-subunit coding sequence (GenBank™ accession number X15539) in-frame 3′ to the coding region of a modified green fluorescent protein (GFP) 3The abbreviation used is: GFP, green fluorescent protein. was kindly donated by Dr. M. Grabner (17Grabner M. Dirksen R.T. Beam K.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1903-1908Crossref PubMed Scopus (134) Google Scholar). Substitutions in segment IS6 of the CaV1.2 α1-subunit were introduced by the “gene SOEing” technique (18Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2614) Google Scholar). For electrophysiological studies, we used the plasmid lacking the GFP tag. Proline mutations were introduced in segment IS6 in positions G422P to F424P and N428P to S439P. The mutated fragments were cloned into an SaII-AgeI cassette. The mutated fragment of L779T was cloned into an AgeI-AfIII cassette. The AgeI restriction site was introduced as a silent mutation. This cassette was used to create G422P, S423P, F424P, N428P, L429T/L429P, V430P/V430T, L431P/L431T, G432P/G432T, V433P/V433T, L434T, S435P, E437P, F438P, and S439P. The mutated fragment of L779T was cloned into a AgeI-AfIII cassette. This cassette was used to create L779T, L429T/L779T, L429T/I781T, L434T/L779T, L434T/I781T by two double mutants S435P/I781P and S435P/I781T. Mutants that did not conduct barium currents were recloned into the GFP-tagged vector (17Grabner M. Dirksen R.T. Beam K.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1903-1908Crossref PubMed Scopus (134) Google Scholar) to analyze whether the channels were expressed in the membrane. 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 (19Ruth P. Rohrkasten A. Biel M. Bosse E. Regulla S. Meyer H.E. Flockerzi V. Hofmann F. Science. 1989; 245: 1115-1118Crossref PubMed Scopus (253) Google Scholar) as well as α2-δ1 subunits (20Ellis 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. Schwartz A. Harpold M.M. Science. 1988; 241: 1661-1664Crossref PubMed Scopus (435) Google Scholar). The transfection of tsA-201 cells was performed using the FuGENE 6 transfection reagent (Roche Applied Science) following standard protocols. tsA-201 cells were used until passage number 15. No variation channel gating related to different cell passage numbers was observed. 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 (21Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pfluegers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15065) Google Scholar) by means of an Axopatch 200A patch clamp amplifier (Axon Instruments, Foster City, CA) 36–48 h after transfection. The extracellular bath solution contained (in mm) the following: BaCl2 5, MgCl2 1, HEPES 10, choline-Cl 140, titrated to pH 7.4 with methanesulfonic acid. Patch pipettes with resistances of 1 to 4 megohms were made from borosilicate glass (Clark Electromedical Instruments, UK) and filled with pipette solution containing (in mm) the following: CsCl 145, MgCl2 3, HEPES 10, EGTA 10, titrated to pH 7.25 with CsOH. All data were digitized using a DIGIDATA 1200 interface (Axon Instruments, Foster City), smoothed by means of a four-pole Bessel filter and saved to a disk. 100-ms current traces were sampled at 10 kHz and filtered at 5 kHz; for the steady-state inactivation protocol, currents were sampled at 1 kHz and filtered at 0.5 kHz; and tail currents were sampled at 50 kHz and filtered at 10 kHz. Leak currents were subtracted digitally using average values of scaled leakage currents elicited by a 10-mV hyperpolarizing pulse or electronically by means of an Axopatch 200 amplifier (Axon Instruments). Series resistance and offset voltage were routinely compensated. The pClamp software package (version 7.0, Axon Instruments) was used for data acquisition and preliminary analysis. Microcal Origin 5.0 was used for analysis and curve fitting. To analyze the voltage dependence of activation, we estimated the fractions of open (activated) channels at different potentials at the current peak when channel opening reached a saturating level, and inactivation was still small. Current-voltage (I-V) curves were fitted according to the following modified Boltzmann equation: I = Gmax(V - Vrev)/(1 + exp((V0.5,act - V)/kact)), where Vrev is extrapolated reversal potential; V is membrane potential; I is peak current; Gmax is maximum membrane conductance; V0.5, act is voltage for half-maximal activation; and kact is slope factor. The voltage dependence of activation was determined from I-V curves and fitted to m∞ = 1/(1 + exp((V0.5,act - V)/kact)). The time course of current activation was fitted to a monoexponential function as follows: I(t) = A(exp(-t/τ)) + C, where I(t) is current at time t; A is the amplitude coefficient; τ is time constant; and C is steady state current. The voltage dependence of IBa inactivation (inactivation curve) was measured using a multistep protocol to account for run down (22Hemara-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 (113) Google Scholar). The pulse sequence was applied every 40 s from a holding potential of -100 mV. Inactivation curves were drawn according to a Boltzmann equation as follows: IBa, inact = Iss + (1 - Iss)/(1 + exp((V - V0.5,inact)/k)), where V is membrane potential; V0.5, inact is midpoint voltage; k is slope factor; and Iss is fraction of noninactivating current. Data are given as mean ± S.E. Statistical significance was assessed with analysis of variance and post hoc test. Student's unpaired t test was used to analyze significance of differences from 0 (ΔΔG). Confocal Imaging—Images were obtained ∼30 h after transfection. The images were acquired with a Zeiss Axiovert 200 M microscope equipped with an LSM 510 laser scanning module, using a 63× (1.4 NA) oil immersion objective. Fluorescence from GFP-tagged CaV1.2 α1-subunits was excited at 488 nm using an argon laser, and emitted light was recorded with a 505–530 nm bandpass filter. The plasma membrane was stained with 2 μm FM4-64 (an amphiphilic styryl dye, Molecular Probes). The dye was detected with a helium-neon laser (excitation, 543 nm) in combination with a 650 nm long pass filter. The instrument was operated in the multitracking mode to minimize channel cross-talk. Pinholes were adjusted to obtain optical slices of 1-μm thickness for each channel. Homology Modeling—Models of the pore-forming α1-subunit of the closed CaV1.2 channel (accession number P15381) are based on the crystal structures of MlotiK (5Clayton G.M. Altieri S. Heginbotham L. Unger V.M. Morais-Cabral J.H. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 1511-1515Crossref PubMed Scopus (140) Google Scholar) and KcsA (4Zhou Y. Morais-Cabral J.H. Kaufman A. MacKinnon R. Nature. 2001; 414: 43-48Crossref PubMed Scopus (1681) Google Scholar). Sequence similarities between CaV1.2 S6 segments and MlotiK/KcsA M2 helices are in the range of 22–40%. The alignment used is the same one as suggested previously (Fig. 1A) (14Beyl S. Timin E.N. Hohaus A. Stary A. Kudrnac M. Guy R.H. Hering S. J. Biol. Chem. 2007; 282: 3864-3870Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Models were built with the Modeler software (23Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10289) Google Scholar) using the same modeling criteria as described previously (14Beyl S. Timin E.N. Hohaus A. Stary A. Kudrnac M. Guy R.H. Hering S. J. Biol. Chem. 2007; 282: 3864-3870Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 24Stary A. Shafrir Y. Hering S. Wolschann P. Guy H.R. Channels. 2008; 2: 210-215Crossref PubMed Scopus (21) Google Scholar). Briefly, the four CaV1.2 domains were arranged in a clockwise manner in agreement with previous studies (24Stary A. Shafrir Y. Hering S. Wolschann P. Guy H.R. Channels. 2008; 2: 210-215Crossref PubMed Scopus (21) Google Scholar, 25Shafrir Y. Durell S.R. Guy H.R. Biophys. J. 2008; 95: 3650-3662Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 26Huber I. Wappl E. Herzog A. Mitterdorfer J. Glossmann H. Langer T. Striessnig J. Biochem. J. 2000; 347: 829-836Crossref PubMed Scopus (68) Google Scholar, 27Zhorov B.S. Folkman E.V. Ananthanarayanan V.S. Biochem. Biophys. 2001; 393: 22-41Crossref PubMed Scopus (61) Google Scholar, 28Lipkind G.M. Fozzard H.A. Mol. Pharmacol. 2003; 63: 499-511Crossref PubMed Scopus (63) Google Scholar, 29Dudley Jr., S.C. Chang N. Hall J. Lipkind G. Fozzard H.A. French R.J. Gen. Physiol. 2000; 116: 679-690Crossref PubMed Scopus (89) Google Scholar). Large extra- and intracellular loops have been omitted from the model, due to lack of suitable structure templates. The selectivity filter of CaV1.2 is based on homology model of the bacterial sodium channel from B. halodurans (25Shafrir Y. Durell S.R. Guy H.R. Biophys. J. 2008; 95: 3650-3662Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The model quality has been analyzed using conventional validation programs. Proline Scan of IS6 Reveals Large Shift of Activation and Inactivation Curves for Mutant S435P—Amino acids of segment IS6 (most residues between positions 422 and 439) of the CaV1.2 α1-subunit were substituted by prolines and barium (Ba2+) currents measured with patch clamp after co-expression of the α1-subunits with auxiliary β1a and α2-δ1 subunits in tsA-201 cells. Only 1 out of the tested 14 proline mutants (S435P) conducted ionic currents. A family of inward Ba2+ currents and the corresponding current-voltage (I-V) curve of mutant S435P and the voltage dependences of steady-state inactivation are shown in Fig. 1. The current reversed at about 50 mV, indicating that the mutation did not affect ion selectivity (Fig. 1B, similar observations were made for mutants L429T and L434T, data not shown). The midpoint voltage of steady state activation changed from -9.9 ± 1.1 mV in the wild-type channel to -35.8 ± 0.6 mV in S435P mutant (Fig. 1). The mutation also shifted the midpoint voltage of the inactivation curve from -38.7 ± 1.0 mV in wild-type channels to -65.7 ± 0.7 mV (Fig. 1C) and substantially slowed channel inactivation. Wild-type channels were inactivated by 65 ± 4%, whereas mutation S435P was inactivated by 5 ± 4% during a 300-ms pulse at the peak of the I-V curve (see Table 1). In Fig. 1, E and G, the corresponding changes in current activation and deactivation kinetics are illustrated. The activation time constant of S435P ranged between 10.4 ms (-50 mV) and 3.1 ms (10 mV) (Fig. 1E), and at -30 mV the mutant displayed a 1.5-fold slower activation time course than wild type.TABLE 1Influence of pore mutations on voltage-dependent gating of CaV1.2 Midpoints and slope factors of the activation curves, midpoints of the inactivation curves, and amount of channel inactivation during a 300-ms pulse (r300) to the peak potentials of the I-V curve are shown. Numbers of experiments are indicated in parentheses.MutantV0.5, actkactV0.5, inactkinactr300mVmVmVmV%Wild-type–9.9 ± 1.1 (8)6.3 ± 0.7–38.7 ± 1.0 (3)8.1 ± 0.965 ± 4IS6 segmentL429T–16.5 ± 0.6 (5)7.5 ± 0.6–42.5 ± 1.1 (3)7.9 ± 0.969 ± 6L434T–21.9 ± 1.3 (5)6.9 ± 0.9–51.1 ± 1.1 (3)8.6 ± 1.063 ± 5S435T–11.3 ± 0.7 (5)6.1 ± 0.6–40.1 ± 0.9 (3)7.7 ± 0.864 ± 6S435A–15.9 ± 0.6 (6)6.9 ± 0.5–44.1 ± 0.9 (4)7.8 ± 0.970 ± 5S435P–35.8 ± 0.6 (6)8.8 ± 0.6–65.7 ± 0.7 (4)7.3 ± 0.75 ± 4aData are statistically significantly different from wild type (p < 0.05). Data for wild type, I781T, and I781P are from Hohaus et al. (12). Mutations G422P, S423P, F424P, N428P, L429P, V430T/V430P, L431T/L431P, G432T/G432P, V433T/V433P, L434P, E437P, F438P, and S439P were not functionalIIS6 segmentL779T–18.6 ± 0.7 (5)6.4 ± 0.6–50.5 ± 1.2 (3)7.1 ± 0.612 ± 5aData are statistically significantly different from wild type (p < 0.05). Data for wild type, I781T, and I781P are from Hohaus et al. (12). Mutations G422P, S423P, F424P, N428P, L429P, V430T/V430P, L431T/L431P, G432T/G432P, V433T/V433P, L434P, E437P, F438P, and S439P were not functionalI781T–37.7 ± 1.2 (7)7.2 ± 1.0–57.8 ± 0.7 (3)8.9 ± 0.547 ± 3aData are statistically significantly different from wild type (p < 0.05). Data for wild type, I781T, and I781P are from Hohaus et al. (12). Mutations G422P, S423P, F424P, N428P, L429P, V430T/V430P, L431T/L431P, G432T/G432P, V433T/V433P, L434P, E437P, F438P, and S439P were not functionalI781P–47.2 ± 1.1 (8)6.3 ± 0.6–68.7 ± 0.8 (5)5.8 ± 0.553 ± 6Double mutantsL429T/I781T–44.0 ± 1.0 (7)8.3 ± 0.9–61.2 ± 1.0 (4)8.7 ± 0.941 ± 5aData are statistically significantly different from wild type (p < 0.05). Data for wild type, I781T, and I781P are from Hohaus et al. (12). Mutations G422P, S423P, F424P, N428P, L429P, V430T/V430P, L431T/L431P, G432T/G432P, V433T/V433P, L434P, E437P, F438P, and S439P were not functionalL429T/L779T–22.5 ± 0.8 (5)6.4 ± 0.7–49.6 ± 1.3 (3)8.0 ± 0.712 ± 6aData are statistically significantly different from wild type (p < 0.05). Data for wild type, I781T, and I781P are from Hohaus et al. (12). Mutations G422P, S423P, F424P, N428P, L429P, V430T/V430P, L431T/L431P, G432T/G432P, V433T/V433P, L434P, E437P, F438P, and S439P were not functionalL434T/I781T–50.3 ± 0.8 (5)5.4 ± 0.8–70.0 ± 1.6 (3)8.5 ± 1.215 ± 4aData are statistically significantly different from wild type (p < 0.05). Data for wild type, I781T, and I781P are from Hohaus et al. (12). Mutations G422P, S423P, F424P, N428P, L429P, V430T/V430P, L431T/L431P, G432T/G432P, V433T/V433P, L434P, E437P, F438P, and S439P were not functionalL434T/L779T–32.3 ± 0.8 (7)7.1 ± 0.7–58.2 ± 0.8 (5)8.2 ± 0.952 ± 5S435A/I781T–35.3 ± 0.9 (6)8.7 ± 0.7–59.1 ± 0.9 (4)8.1 ± 0.848 ± 4aData are statistically significantly different from wild type (p < 0.05). Data for wild type, I781T, and I781P are from Hohaus et al. (12). Mutations G422P, S423P, F424P, N428P, L429P, V430T/V430P, L431T/L431P, G432T/G432P, V433T/V433P, L434P, E437P, F438P, and S439P were not functionalS435P/I781T–43.7 ± 0.9 (5)9.1 ± 0.9–64.1 ± 1.1 (3)8.6 ± 1.044 ± 5aData are statistically significantly different from wild type (p < 0.05). Data for wild type, I781T, and I781P are from Hohaus et al. (12). Mutations G422P, S423P, F424P, N428P, L429P, V430T/V430P, L431T/L431P, G432T/G432P, V433T/V433P, L434P, E437P, F438P, and S439P were not functionalS435P/I781PNo currenta Data are statistically significantly different from wild type (p < 0.05). Data for wild type, I781T, and I781P are from Hohaus et al. (12Hohaus A. Beyl S. Kudrnac M. Berjukow S. Timin E.N. Marksteiner R. Maw M.A. Hering S. J. Biol. Chem. 2005; 280: 38471-38477Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Mutations G422P, S423P, F424P, N428P, L429P, V430T/V430P, L431T/L431P, G432T/G432P, V433T/V433P, L434P, E437P, F438P, and S439P were not functional Open table in a new tab Membrane expression of “nonconducting” constructs was examined by analyzing the subcellular distribution of GFP-tagged mutants by confocal microscopy. Wild-type and mutant GFP-tagged CaV1.2 α1-subunits were co-transfected with β1a and α2-δ1 subunits in tsA-201 cells, and the plasma membrane was visualized by staining with FM4-64. Consistent with our previous report (12Hohaus A. Beyl S. Kudrnac M. Berjukow S. Timin E.N. Marksteiner R. Maw M.A. Hering S. J. Biol. Chem. 2005; 280: 38471-38477Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), GFP-tagged nonfunctional proline mutants localized predominantly at the plasma membrane (data not shown). These findings demonstrate that the lack of current observed for these constructs is unlikely to be caused by a failure of the α1-subunits to reach the plasma membrane. Threonine Mutations Shift the Voltage Dependence of CaV1.2 Gating—More than 90% of the proline mutants in segment IS6 did not conduct barium currents (Table 1). This distinguishes this segment from IIS6, where 8 of 14 (almost 60%) proline substitutions resulted in functional channels (12Hohaus A. Beyl S. Kudrnac M. Berjukow S. Timin E.N. Marksteiner R. Maw M.A. Hering S. J. Biol. Chem. 2005; 280: 38471-38477Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). We have recently shown that gradual changes in hydrophobicity in selected positions of transmembrane segment IIS6 gradually shift the activation and inactivation curves of CaV1.2 (see Ref. 11Hering S. Beyl S. Stary A. Kudrnac M. Hohaus A. Guy H.R. Timin E. Channels. 2008; 2: 61-69Crossref PubMed Scopus (12) Google Scholar). To elucidate a potential role of hydrophobic interactions in segment IS6, we systematically substituted the putative bundle crossing region (residues 429–434) by the small and polar threonine. Constructs L429T and L434T conducted barium currents, whereas other threonine mutations did not result in functional channels (Fig. 2 and Table 1). L429T and L434T shifted the voltage dependence of channel activation in the hyperpolarizing direction and slightly slowed activation and deactivation kinetics. The largest shift in channel activation and the correspondingly slowest kinetics of current activation and deactivation were observed, however, for L434T suggesting a potentially important role of this residue in CaV1.2 gating. In line with our previous observations in segment IIS6, the changes in the voltage dependences of activation and inactivation occurred in parallel, which can be seen in Fig. 8 where midpoint voltages of activation curves are plotted versus midpoint voltages of inactivation curves. However, such a correlation is not observed for all CaV channel constructs (e.g. Spaetgens and Zamponi (30Spaetgens R.L. Zamponi G.W. J. Biol. Chem. 1999; 274: 22428-22436Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), Stotz and Zamponi (31Stotz S.C. Zamponi G.W. J. Biol. Chem. 2001; 276: 33001-33010Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), see also the IIS6 mutations A780P in Hohaus et al. (12Hohaus A. Beyl S. Kudrnac M. Berjukow S. Timin E.N. Marksteiner R. Maw M.A. Hering S. J. Biol. Chem. 2005; 280: 38471-38477Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar)), and the structural basis is currently unknown. Paired Mutations in Segments IS6 and IIS6 Inducing Additive Shifts of the Activation Curve—After identification of “gating sensitive” residues in segments IS6, we made use of previously characterized mutants in segment IIS6 to study if these structural changes contribute to channel gating independently or in a coupled fashion. First we analyzed the combined impact of the IIS6 mutations L779T (-9-mV shift) and I781T (-28-mV shift, see Ref. 12Hohaus A. Beyl S. Kudrnac M. Berjukow S. Timin E.N. Marksteiner R. Maw M.A. Hering S. J. Biol. Chem. 2005; 280: 38471-38477Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) and the newly identified residues in segment IS6 L429T and L434T (causing shifts of about -7 and -12 mV, see Table 1). According to our homology model of the closed conformation, all four residues are located in close proximity (Fig. 9), where residues Leu-434 (IS6) and residue Leu-779 (IIS6) interact directly with each other and residues Ser-435 and Ile-781 interact with residues from neighboring S6 helices. It was therefore interesting to elucidate if the mutations in these regions of IS6 and IIS6 would have additive (independent) or nonadditive effects on the channel activation and inactivation curves. As shown in Fig. 3, the impact of all four mutations in segments IS6 and IIS6 on the activation curves was additive. The voltage of half-maximal channel activation of construct L434T/I781T was shifted by -40 mV (-12 to 28 mV) and of construct L429T/I781T by -34 mV (-7 to 28 mV). Less prominent shifts were observed for L429T/L779T (≈-12 mV) and L434T/L779T (≈-22 mV) constructs. Double mutants L429T/I781T and L434T/I781T displayed slow activation at hyperpolarized potentials (Fig. 4). The large shift of the activation curve of L434T/I781T was associated with a slower activation (τact" @default.
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• W1991438270 title "Coupled and Independent Contributions of Residues in IS6 and IIS6 to Activation Gating of CaV1.2" @default.
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