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• W2055544513 abstract "The hydrophobic locus VAVIM is conserved in the S6 transmembrane segment of domain IV (IVS6) in CaV1 and CaV2 families. Herein we show that glycine substitution of the VAVIM motif in CaV2.3 produced whole cell currents with inactivation kinetics that were either slower (A1719G ≈ V1720G), similar (V1718G), or faster (I1721G ≈ M1722G) than the wild-type channel. The fast kinetics of I1721G were observed with a ≈+10 mV shift in its voltage dependence of activation (E0.5,act). In contrast, the slow kinetics of A1719G and V1720G were accompanied by a significant shift of ≈-20 mV in their E0.5,act indicating that the relative stability of the channel closed state was decreased in these mutants. Glycine scan performed with Val 349 in IS6, Ile701 in IIS6, and Leu1420 in IIIS6 at positions predicted to face Val1720 in IVS6 also produced slow inactivating currents with hyperpolarizing shifts in the activation and inactivation potentials, again pointing out a decrease in the stability of the channel closed state. Mutations to other hydrophobic residues at these positions nearly restored the channel gating. Altogether these data indicate that residues at positions equivalent to 1720 exert a critical control upon the relative stability of the channel closed and open states and more specifically, that hydrophobic residues at these positions promote the channel closed state. We discuss a three-dimensional homology model of CaV2.3 based upon Kv1.2 where hydrophobic residues at positions facing Val1720 in IS6, IIS6, and IIIS6 play a critical role in stabilizing the closed state in CaV2.3. The hydrophobic locus VAVIM is conserved in the S6 transmembrane segment of domain IV (IVS6) in CaV1 and CaV2 families. Herein we show that glycine substitution of the VAVIM motif in CaV2.3 produced whole cell currents with inactivation kinetics that were either slower (A1719G ≈ V1720G), similar (V1718G), or faster (I1721G ≈ M1722G) than the wild-type channel. The fast kinetics of I1721G were observed with a ≈+10 mV shift in its voltage dependence of activation (E0.5,act). In contrast, the slow kinetics of A1719G and V1720G were accompanied by a significant shift of ≈-20 mV in their E0.5,act indicating that the relative stability of the channel closed state was decreased in these mutants. Glycine scan performed with Val 349 in IS6, Ile701 in IIS6, and Leu1420 in IIIS6 at positions predicted to face Val1720 in IVS6 also produced slow inactivating currents with hyperpolarizing shifts in the activation and inactivation potentials, again pointing out a decrease in the stability of the channel closed state. Mutations to other hydrophobic residues at these positions nearly restored the channel gating. Altogether these data indicate that residues at positions equivalent to 1720 exert a critical control upon the relative stability of the channel closed and open states and more specifically, that hydrophobic residues at these positions promote the channel closed state. We discuss a three-dimensional homology model of CaV2.3 based upon Kv1.2 where hydrophobic residues at positions facing Val1720 in IS6, IIS6, and IIIS6 play a critical role in stabilizing the closed state in CaV2.3. Voltage-dependent Ca2+ channels (VDCC) 3The abbreviation used is: VDCC, voltage-dependent Ca2+ channel. 3The abbreviation used is: VDCC, voltage-dependent Ca2+ channel. are membrane proteins that play a key role in promoting Ca2+ influx in response to membrane depolarization in excitable cells. VDCCs arise from the multimerization of distinct subunits: CaVα1, CaVβ, and CaVα2δ, and sometimes CaVγ (1Arikkath J. Campbell K.P. Curr. Opin. Neurobiol. 2003; 13: 298-307Crossref PubMed Scopus (421) Google Scholar). To this date, molecular cloning has identified the primary structures for 10 distinct calcium channel CaVα1 subunits (2Snutch T.P. Reiner P.B. Curr. Opin. Neurobiol. 1992; 2: 247-253Crossref PubMed Scopus (251) Google Scholar, 3Zhang J.F. Randall A.D. Ellinor P.T. Horne W.A. Sather W.A. Tanabe T. Schwarz T.L. Tsien R.W. Neuropharmacology. 1993; 32: 1075-1088Crossref PubMed Scopus (570) Google Scholar, 4Birnbaumer L. Campbell K.P. Catterall W.A. Harpold M.M. Hofmann F. Horne W.A. Mori Y. Schwartz A. Snutch T.P. Tanabe T. Neuron. 1994; 13: 505-506Abstract Full Text PDF PubMed Scopus (316) Google Scholar, 5Perez-Reyes E. Cribbs L.L. Daud A. Lacerda A.E. Barclay J. Williamson M.P. Fox M. Rees M. Lee J.H. Nature. 1998; 391: 896-900Crossref PubMed Scopus (638) Google Scholar, 6Cribbs L.L. Lee J.H. Yang J. Satin J. Zhang Y. Daud A. Barclay J. Williamson M.P. Fox M. Rees M. Perez-Reyes E. Circ. Res. 1998; 83: 103-109Crossref PubMed Scopus (515) Google Scholar, 7Randall A. Benham C.D. Mol. Cell. Neurosci. 1999; 14: 255-272Crossref PubMed Scopus (73) Google Scholar, 8Catterall W.A. Annu. Rev. Cell Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1935) Google Scholar) that are classified into three main subfamilies according to their gating properties (CaV1, CaV2, and CaV3). The CaVα1 subunit is the main pore-forming subunit that carries the channel activation gating among other functions. The CaVα1 subunits of VDCCs are evolutionarily related to the α subunit of Kv channels with a single polypeptidic chain carrying four domains of six transmembrane segments (S1–S6) (9Hille B. Ion Channels of Excitable Membranes, Third Ed. 2001; Google Scholar). Although the overall identity at the primary sequence level is very low between CaV and Kv channels, it goes up to 10–25% when comparing the S6 transmembrane segments. As in Kv channels, the S6 transmembrane segments of CaVα1 are believed to line the channel pore and form the channel inner vestibule. It was inferred from the three-dimensional structures of KcsA, MthK, KvAP, KirBac, and Kv1.2 channels that the M2/S6 transmembrane segments include the activation gate that controls channel opening (10Doyle D.A. Cabral J.M. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5702) Google Scholar, 11Jiang Y. Lee A. Chen J. Cadene M. Chait B.T. MacKinnon R. Nature. 2002; 417: 515-522Crossref PubMed Scopus (1203) Google Scholar, 12Jiang Y. Lee A. Chen J. Ruta V. Cadene M. Chait B.T. MacKinnon R. Nature. 2003; 423: 33-41Crossref PubMed Scopus (1633) Google Scholar, 13Kuo A. Gulbis J.M. Antcliff J.F. Rahman T. Lowe E.D. Zimmer J. Cuthbertson J. Ashcroft F.M. Ezaki T. Doyle D.A. Science. 2003; 300: 1922-1926Crossref PubMed Scopus (732) Google Scholar, 14Long S.B. Campbell E.B. MacKinnon R. Science. 2005; 309: 897-903Crossref PubMed Scopus (1835) Google Scholar). In the Shaker K+ channels, the residue hydrophobicity in this region could alter the channel closed-open equilibrium (15Hackos D.H. Chang T.H. Swartz K.J. J. Gen. Physiol. 2002; 119: 521-532Crossref PubMed Scopus (150) Google Scholar, 16Yifrach O. MacKinnon R. Cell. 2002; 111: 231-239Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). To study the functional importance of S6 residues in the gating of CaV2.3, we searched for conserved motifs of hydrophobic residues. The VAVIM motif is conserved in the S6 transmembrane segment of domain IV (IVS6) of high voltage-activated CaV1 and CaV2 families (Fig. 1A). Numerous algorithms align the PVPVIV activation locus in Shaker Kv channels with the FVAVIM (50% identity) (Fig. 1B) suggesting that this locus could play a role in the activation gating of HVA VDCCs. Precious clues regarding the role of the VAVIM motif in channel function came from genetic diseases. Mutation of the conserved Ile to Leu in CaV2.1 was identified in patients suffering from familial hemiplegic migraine (17Kraus R.L. Sinnegger M.J. Glossmann H. Hering S. Striessnig J. J. Biol. Chem. 1998; 273: 5586-5590Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 18Terwindt G.M. Ophoff R.A. Haan J. Sandkuijl L.A. Frants R.R. Ferrari M.D. Eur. J. Hum. Genet. 1998; 6: 297-307Crossref PubMed Scopus (86) Google Scholar). I1811L altered the channel gating properties by shifting the voltage dependence of activation by -5 to -12 mV, depending upon the expression system (17Kraus R.L. Sinnegger M.J. Glossmann H. Hering S. Striessnig J. J. Biol. Chem. 1998; 273: 5586-5590Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 19Hans M. Luvisetto S. Williams M.E. Spagnolo M. Urrutia A. Tottene A. Brust P.F. Johnson E.C. Harpold M.M. Stauderman K.A. Pietrobon D. J. Neurosci. 1999; 19: 1610-1619Crossref PubMed Google Scholar, 20Cao Y.Q. Tsien R.W. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2590-2595Crossref PubMed Scopus (68) Google Scholar, 21Tottene A. Fellin T. Pagnutti S. Luvisetto S. Striessnig J. Fletcher C. Pietrobon D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13284-13289Crossref PubMed Scopus (219) Google Scholar). Altogether, these experiments suggested that Ile could be strictly required for normal gating of HVA VDCC. Herein we show that substitutions of the Ile1721 residue in the human CaV2.3, corresponding to Ile1811 in the human CaV2.1, by residues of different structural properties (hydrophobicity, charge, polarity, and size) yielded whole cell currents with altered gating properties. Mutations of Ile1721 (Ala, Glu, Gly, His, Leu, Pro, Thr, and Val) including the conservative I1721L mutation, shifted the voltage dependence of inactivation to more negative potentials (up to -30 mV shifts) compared with the wild-type CaV2.3 channel. Glycine mutants A1719G, V1720G, I1721G, and M1722G within the VAVIM motif significantly altered the channel whole cell kinetics as well as the voltage dependence of channel activation and inactivation. The equilibrium between the channel closed and open states was modified in favor of the closed state in I1721G and M1722G. In contrast, A1719G and V1720G shifted the open/closed equilibrium toward the channel open state. Glycine mutations introduced at positions equivalent to Val1720 in S6 of Domains I (Val349), II (Ile701), and III (Leu1420) yielded similar results prompting the suggestion that the open state was stabilized in these mutants. Altogether our findings identify S6 hydrophobic residues in Domains I to IV that play a unique role in controlling the relative stability of the open and closes states in CaV2.3. Recombinant DNA Techniques-The human CaV2.3 (GenBank™ L27745) (22Schneider T. Wei X. Olcese R. Costantin J.L. Neely A. Palade P. Perez-Reyes E. Qin N. Zhou J. Crawford G.D. Smith R.G. Appel S.H. Stefani E. Birnbaumer L. Receptors & Channels. 1994; 2: 255-270PubMed Google Scholar), the rat CaVβ3 (GenBank M88751) (23Castellano A. Wei X. Birnbaumer L. Perez-Reyes E. J. Biol. Chem. 1993; 268: 3450-3455Abstract Full Text PDF PubMed Google Scholar), and the rat brain CaVα2bδ (GenBank NM_000722) (24Williams M.E. Feldman D.H. McCue A.F. Brenner R. Velicelebi G. Ellis S.B. Harpold M.M. Neuron. 1992; 8: 71-84Abstract Full Text PDF PubMed Scopus (438) Google Scholar) were used. Point mutations were produced with the QuikChange XL-mutagenesis kit (Stratagene) using 39-mer primers on the full-length CaV2.3 clone as described elsewhere (25Raybaud A. Dodier Y. Bissonnette P. Simoes M. Bichet D.G. Sauve R. Parent L. J. Biol. Chem. 2006; 281: 39424-39436Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Constructs were screened by automated double-stranded sequence analysis (BioST, Lachine, Quebec, Canada). Run-off transcripts were prepared using the T7 RNA polymerase mMessage mMachine® transcription kit (Ambion, Austin, TX) and stored at -20 °C before use. Functional Expression of CaV2.3-Oocytes were obtained from female Xenopus laevis clawed frogs as described previously (25Raybaud A. Dodier Y. Bissonnette P. Simoes M. Bichet D.G. Sauve R. Parent L. J. Biol. Chem. 2006; 281: 39424-39436Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 26Berrou L. Dodier Y. Raybaud A. Tousignant A. Dafi O. Pelletier J.N. Parent L. J. Biol. Chem. 2005; 280: 494-505Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 27Dafi O. Dodier Y. Berrou L. Raybaud A. Sauve R. Parent L. Biophys. J. 2004; 87: 3181-3192Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Oocytes were injected with 46 nl of a solution containing cRNA coding for the CaVα1, CaVα2bδ, and CaVβ3 subunits in a 3:1:2 weight ratio. Co-expression with the ancillary CaVβ3, which is predominant in brain tissues like Cav2.3 (28Birnbaumer L. Qin N. Olcese R. Tareilus E. Platano D. Costantin J. Stefani E. J. Bioenerg. Biomembr. 1998; 30: 357-375Crossref PubMed Scopus (200) Google Scholar, 29Serikov V. Bodi I. Koch S.E. Muth J.N. Mikala G. Martinov S.G. Haase H. Schwartz A. Biochem. Biophys. Res. Commun. 2002; 293: 1405-1411Crossref PubMed Scopus (13) Google Scholar), promotes strong functional expression (30Parent L. Schneider T. Moore C.P. Talwar D. J. Membr. Biol. 1997; 160: 127-140Crossref PubMed Scopus (51) Google Scholar, 31Meza U. Thapliyal A. Bannister R.A. Adams B.A. Mol. Pharmacol. 2007; 71: 284-293Crossref PubMed Scopus (18) Google Scholar), and emphasizes closed-state inactivation (32Patil P.G. Brody D.L. Yue D.T. Neuron. 1998; 20: 1027-1038Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) of CaV2.3. Functional expression of mutants was deemed significant with whole cell Ba2+ currents >300 nA. Electrophysiological Recordings in Oocytes-Wild-type and mutant channels were screened at room temperature for macroscopic Ba2+ currents, 2–4 days after RNA injection, using a two-electrode voltage-clamp amplifier (OC-725C, Warner Instruments) as described earlier (25Raybaud A. Dodier Y. Bissonnette P. Simoes M. Bichet D.G. Sauve R. Parent L. J. Biol. Chem. 2006; 281: 39424-39436Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 26Berrou L. Dodier Y. Raybaud A. Tousignant A. Dafi O. Pelletier J.N. Parent L. J. Biol. Chem. 2005; 280: 494-505Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 27Dafi O. Dodier Y. Berrou L. Raybaud A. Sauve R. Parent L. Biophys. J. 2004; 87: 3181-3192Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 33Berrou L. Bernatchez G. Parent L. Biophys. J. 2001; 80: 215-228Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Oocytes were routinely injected with 23 nl of a 50 mm EGTA (Sigma) 0.5–2 h before the experiments. Data Acquisition and Analysis-PClamp software 8.2 (Molecular Devices, Sunnyvale, CA) was used for on-line data acquisition and analysis. A series of 450-ms voltage pulses (5 mV steps) were applied at a frequency of 0.2 Hz from a holding potential of -120 mV to take into account the negative shift of the Ile1721 mutants in the inactivation curve. Activation parameters were estimated from the peak I-V curves obtained for each channel combination and are reported as the mean of individual measurements ± S.E. Briefly, the I-V relationships were normalized to the maximum amplitude and were fitted to a Boltzmann equation with E0.5,act being the mid-potential of activation as described elsewhere (25Raybaud A. Dodier Y. Bissonnette P. Simoes M. Bichet D.G. Sauve R. Parent L. J. Biol. Chem. 2006; 281: 39424-39436Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 26Berrou L. Dodier Y. Raybaud A. Tousignant A. Dafi O. Pelletier J.N. Parent L. J. Biol. Chem. 2005; 280: 494-505Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 27Dafi O. Dodier Y. Berrou L. Raybaud A. Sauve R. Parent L. Biophys. J. 2004; 87: 3181-3192Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The estimation of E0.5,act using non-stationary measurements rests upon the assumption that the transition to the open state is much faster than the transition to the inactivated state. The measure of E0.5,act yields an estimation of the free energy differences between closed (C) and open (O) states as explained previously (16Yifrach O. MacKinnon R. Cell. 2002; 111: 231-239Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Changes in E0.5,act can thus be interpreted as a modification of the ratio between the open and closed states. Time constants of whole cell current traces were estimated with the predefined Equation 1 in Clampfit 8.2 that uses the Chebyshev routine and a 4-point smoothing filter with n = 1 for deactivation time constants and n = 3 for inactivation time constants. Under the latter, the inactivation time course requires two time constants τinact,fast and τinact,slow. f(t)=nΣi=1Aie−t/τi+C (Eq. 1) As the number of exponential functions needed to account for the inactivation process varied between mutants, inactivation kinetics were quantified using r50 values. The r50 ratio is defined as the ratio of peak whole cell currents remaining 50 ms later (I50 ms/IPeak). The voltage dependence of inactivation was obtained after a series of 5-s prepulses (h5000 for h measured at 5000 ms) that varied from -120 to +30 mV at a frequency of 0.02 Hz with E0.5,inact being the mid-potential of inactivation (25Raybaud A. Dodier Y. Bissonnette P. Simoes M. Bichet D.G. Sauve R. Parent L. J. Biol. Chem. 2006; 281: 39424-39436Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 26Berrou L. Dodier Y. Raybaud A. Tousignant A. Dafi O. Pelletier J.N. Parent L. J. Biol. Chem. 2005; 280: 494-505Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 27Dafi O. Dodier Y. Berrou L. Raybaud A. Sauve R. Parent L. Biophys. J. 2004; 87: 3181-3192Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Most mutations inactivated completely within the 5-s pulse with less than 2% of the peak currents remaining at the end of the inactivating pulse. However, the phrase “isochronal inactivation” is more accurate when estimating the voltage dependence of inactivation of the I701G mutant that displayed 15% residual currents. Statistical analyses were performed using the one-way analysis of variance fitting routine for two independent populations included in Origin 7.0. Data were considered statistically significant at p < 0.01. Homology Modeling of S6 Regions in CaV2.3-An analysis based on the SAMT02 algorithm confirmed Kv1.2 as a suitable template to model the S5 and S6 segments in each of the 4 domains of CaV2.3. The identity of Kv1.2 with CaV2.3 at the primary sequence varies from 11% for IIIS6 to up to 25% for IVS6 with values of 17% for IS6 and 22% for IIS6. The primary sequence of each of the four S6 segments of CaV2.3 was aligned with the S6 segment of Kv1.2 by using LALIGN without any gap in the structure. Introducing gaps in the alignment could in certain cases (such as in IIS6) improve local alignments while decreasing the overall homology. For each S6, the score for the alignment of the distal S6 was systematically higher than for the beginning of the S6 segment. A Kv1.2-based model of the selectivity filter and pore helix could not, however, be generated as the linkers were generally longer in CaV than in Kv1.2. Hence, the computer-based molecular model of these regions without the selectivity filter and the pore helix was achieved with Modeler 9v1 (salilab.org/modeler) using the molecular coordinates of Kv1.2 (Protein Data Bank 2A79). 100 models were built and the models with the lowest objective function were further checked for internal consistency using Procheck (biotech.ebi.ac.uk/cgi-bin/sendquery). The precision of the models decreased significantly for the residues located at the C-terminal end in the absence of structural constraints. The global score of the final model was 0.10, whereas values higher than -0.50 are generally considered to be acceptable. The model was minimized with the DISCOVER module of INSIGHTII (Accelrys, San Diego, CA) with a dielectric constant of 80 to simulate the presence of solvent. The three-dimensional representations were generated with the INSIGHTII software as described elsewhere (34Simoes M. Garneau L. Klein H. Banderali U. Hobeila F. Roux B. Parent L. Sauve R. J. Gen. Physiol. 2002; 120: 99-116Crossref PubMed Scopus (18) Google Scholar, 35Dodier Y. Banderali U. Klein H. Topalak O. Dafi O. Simoes M. Bernatchez G. Sauve R. Parent L. J. Biol. Chem. 2004; 279: 6853-6862Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The Role of IVS6 in the Gating of CaV2.3-The I1811L mutation is one of the four original mutations of the CaVα1 subunit of the human CaV2.1 that is associated with familial hemiplegic migraine (17Kraus R.L. Sinnegger M.J. Glossmann H. Hering S. Striessnig J. J. Biol. Chem. 1998; 273: 5586-5590Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 36Ophoff R.A. Terwindt G.M. Vergouwe M.N. van E.R. Oefner P.J. Hoffman S.M. Lamerdin J.E. Mohrenweiser H.W. Bulman D.E. Ferrari M. Haan J. Lindhout D. van Ommen G.J. Hofker M.H. Ferrari M.D. Frants R.R. Cell. 1996; 87: 543-552Abstract Full Text Full Text PDF PubMed Scopus (2103) Google Scholar). As the Ile residue is conserved in HVA VDCCs, we hypothesized that it might be important for the normal gating properties of HVA channels such as CaV2.3. Most Ile1721 mutants yielded high voltage-activated inward Ba2+ currents (Fig. 2, supplemental Fig. S1 and Table S1) with the exception of I1721D, I1721R, and I1721K that failed to express functional channels. The observation that Ile1721 tolerated a wide range of point mutations suggests a minimum of structural constraints in its local environment. Other than I1721L and I1721A, functional Ile1721 mutants produced currents with faster inactivation kinetics than the wild-type channel (supplemental Fig. S1). At +10 mV, r50 decreased from wild-type ≅I1721L ≅ I1721A > I1721H > I1721E > I1721V ≅ I1721T ≅ I1721P > I1721G. This is the first report of mutations speeding up the inactivation kinetics of CaV2.3 because mutations in the I-II linker decreased the inactivation kinetics (33Berrou L. Bernatchez G. Parent L. Biophys. J. 2001; 80: 215-228Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Our results with I1721T in CaV2.3 contrast with reports that the equivalent mutation I1475T in IVS6 of the L-type CaV1.2 did not markedly alter the kinetics of whole cell current inactivation (37Shi C. Soldatov N.M. J. Biol. Chem. 2002; 277: 6813-6821Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Charged residues at position 1721 produced non-functional mutants save for I1721E that showed little difference in activation and inactivation potentials despite faster inactivation kinetics. I1721T displayed faster inactivation kinetics without any significant changes in E0.5,act or E0.5,inact. Both the activation and inactivation gating of I1721L and I1721A were significantly shifted by ≅-5 to -10 mV as compared from the wild-type channel (supplemental Table S1) but inactivated with kinetics similar to the wild-type channel. In the case of I1721V, I1721P, and I1721G, the activation potentials were significantly shifted toward positive voltages, whereas inactivation potentials were shifted in the negative direction. These observations coupled with the faster inactivation kinetics of these mutants suggest that the fraction of channels in the open state at any given voltage was decreased in I1721V, I1721P, and I1721G. This conclusion holds true even when taking into account the changes in the slope factor Z. As estimated from the ΔGappaa scale calculated by the group of Stephen White (38Hessa T. Kim H. Bihlmaier K. Lundin C. Boekel J. Andersson H. Nilsson I. White S.H. Nature. 2005; 433: 377-381Crossref PubMed Scopus (783) Google Scholar), hydrophobicity decreases in the following order: Ile ≅ Leu > Val > Ala > Thr > Gly > His > Pro > Glu, whereas volume decreases from Ile ≅ Leu > His > Val ≅ Glu > Thr > Pro > Ala > Gly. As a result, no single parameter such as hydrophobicity, charge, or volume can account for the altered channel gating of Ile1721 mutants. V1720G and I1721G Yield Clues Regarding the Position of the Channel Activation Gate in IVS6-With I1721V and I1721P, I1721G produced the strongest changes in channel gating. To minimize steric and structural constraints, we chose to identify residues that are critical for channel activation gating by performing a glycine scan of the conserved VAVIM locus in the distal portion of IVS6 (Fig. 3A). Glycine mutants decreased the inactivation kinetics of neighboring A1719G and V1720G residues but sped up the kinetics of I1721G and M1722G as compared with the wild-type and V1718G channels. The r50 values thus decreased from A1719G ≅ V1720G > wild-type ≅ V1718G > I1721G ≅ M1722G (Fig. 3B). The fast inactivation kinetics of the latter were accompanied by a faster recovery from inactivation, suggesting that the inactivated state was not more stable in I1721G and M1722G (supplemental Fig. S2A). The activation potentials of the latter were either not significantly different (M1722G) or more positive (I1721G, p < 0.001) than the wild-type channel. Coupled with the fact that these 2 mutants inactivated at potentials ≈-30 mV more negative than the wild-type channel (Fig. 3C), these observations altogether indicate that glycine mutations introduced at positions 1721 and 1722 result in a decreased channel open state. In all these mutants, activation and inactivation potentials were typically shifted toward negative voltages by CaVβ3 (supplemental Fig. S2B). For I1721G, this means that E0.5,act = 6.9 ± 0.4 mV (n = 6) and E0.5,inact = -64 ± 3 mV (n = 3) in the absence of CaVβ3, values that remain significantly different from E0.5,act = -1.4 ± 0.4 mV (n = 29) and E0.5,inact = -33.7 ± 0.8 mV (n = 36) for the wild-type channel under the same conditions. The faster kinetics of I1721G and M1722G contrast with the slower kinetics produced by G352A in IS6 and R378E mutations in the I-II linker (33Berrou L. Bernatchez G. Parent L. Biophys. J. 2001; 80: 215-228Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 39Bernatchez G. Berrou L. Benakezouh Z. Ducay J. Parent L. Biochim. Biophys. Acta. 2001; 1514: 217-229Crossref PubMed Scopus (14) Google Scholar). It was then proposed that the slow kinetics of these mutants resulted from either a disruption of the fast inactivated state or from a stabilization of the channel open state. To investigate whether the increased inactivation gating of I1721G and M1722G could antagonize the effects of the slow mutants, the double mutants G352A/M1722G and R378E/I1721G were produced. Unfortunately, neither double mutant expressed functional whole cell currents, leaving this question unanswered. In contrast to I1721G and M1722G, A1719G and V1720G displayed slow inactivating kinetics accompanied with negative shifts of ≈-20 mV in their activation potentials. This change in gating departs from our observations with G352A in IS6 (25Raybaud A. Dodier Y. Bissonnette P. Simoes M. Bichet D.G. Sauve R. Parent L. J. Biol. Chem. 2006; 281: 39424-39436Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and the charged mutations of Arg378 in the I-II linker (33Berrou L. Bernatchez G. Parent L. Biophys. J. 2001; 80: 215-228Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 39Bernatchez G. Berrou L. Benakezouh Z. Ducay J. Parent L. Biochim. Biophys. Acta. 2001; 1514: 217-229Crossref PubMed Scopus (14) Google Scholar) where the decreased inactivation kinetics occurred without any change in the channel activation potential of CaV2.3. The significant shifts in the E0.5,act toward negative voltages were accompanied by similar increases in the slope factor Z. Hence, introducing glycine residues at positions 1719 and 1720 appears to shift the open/closed equilibrium toward the open state. Mutations of Val1720 to Ala and Ile nearly restored the normal channel gating with smaller shifts in the mid-potentials of activation and inactivation than measured with V1720G (Fig. 4; supplemental Table S1) suggesting that hydrophobic residues at this position could shift the closed/open state equilibrium toward the channel closed state. Glycine Mutations in Positions Equivalent to Val1720 Promote the Channel Open State-As the primary structure of VDCCs identifies 4 distinct S6 domains, domain-specific effects were investigated at positions equivalent to Val1720 (Fig. 5) and Ile1721 (L350 in IS6, Ala702 in IIS6, and Ile1421 in IIIS6) (supplemental Fig. S3). As seen for I1721G (IVS6), I1421G (IIIS6) displayed inactivation kinetics significantly faster than the wild-type channel at all voltages (supplemental Fig. S3). Unlike I1721G, however, the activation potential of I1421G was not significantly altered, which suggests that the mutation did not affect the relative stability of the open and closed states. L350G (IS6) behaves mostly like the wild-type channel, whereas the slower inactivation kinetics of A702G (IIS6) were observed in the absence of significant shifts in its activation and inactivation potentials (supplemental Fig. S3). In contrast, glycine mutations at positions Val349 (IS6), Ile701 (IIS6), and Leu1420 (IIIS6) equivalent to Val1720 (IVS6) significantly altered the biophysical properties of the channel. In particular, all mutations slowed down the channel inactivation kinetics (Fig. 5B). At 0 mV, the r50 values decreased from wild-type ≅ V349G (IS6) < L1420G (IIIS6) ≅ V1720G (IVS6) < I701G (IIS6). The decrease in channel kinetics in these mutants was achieved through the progressive elimination of the fast inactivation time constant up to the point where the contribution of the fast time constant becomes negligible in I701G. The mutations also significantly hyperpolarized the activation potentials by -5 to -35 mV and the inactivation potentials by -18 to -24 mV (supplemental Tables S1 and S2 and Fig. S4). The most spectacular alterations in channel properties were, however, observed for I701G in IIS6. Indeed, the hyperpolarization of its activation curve was stronger than the negative shift in its mid-potential of inactivation" @default.
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• W2055544513 title "The Role of Distal S6 Hydrophobic Residues in the Voltage-dependent Gating of CaV2.3 Channels" @default.
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