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• W2005524812 abstract "Ba2+ current through the L-type Ca2+ channel inactivates essentially by voltage-dependent mechanisms with fast and slow kinetics. Here we found that slow inactivation is mediated by an annular determinant composed of hydrophobic amino acids located near the cytoplasmic ends of transmembrane segments S6 of each repeat of the α1C subunit. We have determined the molecular requirements that completely obstruct slow inactivation. Critical interventions include simultaneous substitution of A752T in IIS6, V1165T in IIIS6, and I1475T in IVS6, each preventing in additive manner a considerable fraction of Ba2+ current from inactivation. In addition, it requires the S405I mutation in segment IS6. The fractional inhibition of slow inactivation in tested mutants caused an acceleration of fast inactivation, suggesting that fast and slow inactivation mechanisms are linked. The channel lacking slow inactivation showed ∼45% of the sustained Ba2+ or Ca2+ current with no indication of decay. The remaining fraction of the current was inactivated with a single-exponential decay (τf ∼ 10 ms), completely recovered from inactivation within 100 ms and did not exhibit Ca2+-dependent inactivation properties. No voltage-dependent characteristics were significantly changed, consistent with the C-type inactivation model suggesting constriction of the pore as the main mechanism possibly targeted by Ca2+ sensors of inactivation. Ba2+ current through the L-type Ca2+ channel inactivates essentially by voltage-dependent mechanisms with fast and slow kinetics. Here we found that slow inactivation is mediated by an annular determinant composed of hydrophobic amino acids located near the cytoplasmic ends of transmembrane segments S6 of each repeat of the α1C subunit. We have determined the molecular requirements that completely obstruct slow inactivation. Critical interventions include simultaneous substitution of A752T in IIS6, V1165T in IIIS6, and I1475T in IVS6, each preventing in additive manner a considerable fraction of Ba2+ current from inactivation. In addition, it requires the S405I mutation in segment IS6. The fractional inhibition of slow inactivation in tested mutants caused an acceleration of fast inactivation, suggesting that fast and slow inactivation mechanisms are linked. The channel lacking slow inactivation showed ∼45% of the sustained Ba2+ or Ca2+ current with no indication of decay. The remaining fraction of the current was inactivated with a single-exponential decay (τf ∼ 10 ms), completely recovered from inactivation within 100 ms and did not exhibit Ca2+-dependent inactivation properties. No voltage-dependent characteristics were significantly changed, consistent with the C-type inactivation model suggesting constriction of the pore as the main mechanism possibly targeted by Ca2+ sensors of inactivation. The voltage-gated inward current of Ca2+ ions is a common mechanism of transient increase in the cytoplasmic free Ca2+ concentration that stimulates a great variety of cellular responses. The rapid and complete inactivation of Ca2+ current is the critical step terminating Ca2+ influx and preventing Ca2+ overloading of the cell. In the case of L-type (α1C) Ca2+channels, two different mechanisms are in control of the Ca2+ current inactivation (1Hadley R.W. Lederer W.J. J. Physiol. (Lond.). 1991; 444: 257-268Crossref Scopus (123) Google Scholar). One mechanism is driven by Ca2+ ions on the cytoplasmic side of the membrane (2Eckert R. Chad J.E. Prog. Biophys. Mol. Biol. 1984; 44: 215-267Crossref PubMed Scopus (410) Google Scholar), whereas the other depends on transmembrane voltage. Replacement of Ca2+ ions by Ba2+ ions in the extracellular medium eliminates Ca2+-dependent inactivation so that Ba2+-conducting Ca2+ channels are inactivated in a voltage-dependent manner. Two major mechanisms have been previously implicated in voltage-dependent inactivation (3Yellen G. Quart. Rev. Biophys. 1998; 31: 239-295Crossref PubMed Scopus (401) Google Scholar). The ball and chain mechanism of an ion pore occlusion by a positively charged segment of the N-terminal tails was first described in the tetramericShaker K+ channel where it supports fastN-type inactivation (4Hoshi T. Zagotta W.N. Aldrich R.W. Science. 1990; 250: 533-538Crossref PubMed Scopus (1277) Google Scholar). Somewhat similarly, thehinged-lid mechanism in the Na+ channel is mediated by the IFM motif of the cytoplasmic linker between repeats III and IV (5West J.W. Patton D.E. Scheuer T. Wang Y. Goldin A.L. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10910-10914Crossref PubMed Scopus (662) Google Scholar). In both Na+ and K+channels, receptor sites for the different inactivation gates are located in S4–S5 intracellular loops (6Isacoff E.Y. Jan Y.N. Jan L.Y. Nature. 1991; 353: 86-90Crossref PubMed Scopus (276) Google Scholar, 7McPhee J.C. Ragsdale D.S. Scheuer T. Catterall W.A. J. Biol. Chem. 1998; 273: 1121-1129Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The second,C-type mechanism of slower K+ channel inactivation (8Choi K. Aldrich R. Yellen G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5092-5095Crossref PubMed Scopus (394) Google Scholar) was found to involve a constriction of the pore by the S6 segments lining the intracellular part of the pore and arranged as an inverted teepee structure (9Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5745) Google Scholar, 10Lopez G.A. Jan Y.N. Jan L.Y. Nature. 1994; 367: 179-182Crossref PubMed Scopus (148) Google Scholar). The voltage-dependent inactivation of α1CCa2+ channels appears to be more complex (for review, see Refs. 11Hering S. Berjukow S. Aczél S. Timin E.N. Trends Pharmacol. Sci. 1998; 19: 439-443Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar,12Hering S. Berjukow S. Sokolov S. Marksteiner R. Weiβ R.G. Kraus R. Timin E.N. J. Physiol. (Lond.). 2000; 528: 237-249Crossref Scopus (117) Google Scholar). Experimental trials of chimeras between α1Cand the faster inactivating Ca2+ channels showed that multiple regions are involved in inactivation, including transmembrane segments IS6 (13Zhang J.F. Ellinor P.T. Aldrich R.W. Tsien R.W. Nature. 1994; 372: 97-100Crossref PubMed Scopus (177) Google Scholar), IIIS6 (14Tang S. Yatani A. Bahinski A. Mori Y. Schwartz A. Neuron. 1993; 11: 1013-1021Abstract Full Text PDF PubMed Scopus (93) Google Scholar, 15Hering S. Aczel S. Kraus R.L. Berjukow S. Striessnig J. Timin E.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13323-13328Crossref PubMed Scopus (62) Google Scholar), IVS5 (16Motoike H.K. Bodi I. Nakayama H. Schwartz A. Varadi G. J. Biol. Chem. 1999; 274: 9409-9420Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), IVS6 (17Döring F. Degtiar V.E. Grabner M. Striessnig J. Hering S. Glossmann H. J. Biol. Chem. 1996; 271: 11745-11749Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 18Berjukow S. Gapp F. Aczel S. Sinnegger M.J. Mitterdorfer J. Glossmann H. Hering S. J. Biol. Chem. 1999; 274: 6154-6160Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 19Berjukow S. Marksteiner R. Gapp F. Sinnegger M.J. Hering S. J. Biol. Chem. 2000; 275: 22114-22120Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), repeats I–II linker (20Spaetgens R.L. Zamponi G.W. J. Biol. Chem. 1999; 274: 22428-22436Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 21Stotz S.C. Hamid J. Spaetgens R.L. Jarvis S.E. Zamponi G.W. J. Biol. Chem. 2000; 275: 24575-24582Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 22Restituito S. Cens T. Barrere C. Geib S. Galas S., De Waard M. Charnet P. J. Neurosci. 2000; 20: 9046-9052Crossref PubMed Google Scholar), as well as the C-terminal determinants, E1537 of EF-hand motif (23Bernatchez G. Talwar D. Parent L. Biophys. J. 1998; 75: 1727-1739Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) and the Ca2+-sensing 80-amino acid domain 1572–1651 (24Soldatov N.M. Zühlke R.D. Bouron A. Reuter H. J. Biol. Chem. 1997; 272: 3560-3566Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In these regions, multiple amino acids were shown to be critical for the rate of inactivation of the Ba2+ current (12Hering S. Berjukow S. Sokolov S. Marksteiner R. Weiβ R.G. Kraus R. Timin E.N. J. Physiol. (Lond.). 2000; 528: 237-249Crossref Scopus (117) Google Scholar). A systematic analysis of these multiple determinants has not been performed. In this work we focused on determinants situated in all four transmembrane segments S6. The approach was based on our earlier observation (25Soldatov N.M. Zhenochin S. AlBanna B. Abernethy D.R. Morad M. J. Membr. Biol. 2000; 177: 129-135Crossref PubMed Scopus (14) Google Scholar) that Ca2+ channel inactivation was impaired by the A752T mutation at a position −2 from the cytoplasmic end of IIS6 identified in the human fibroblast α1CCa2+ channel transcript (26Soldatov N.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4628-4632Crossref PubMed Scopus (97) Google Scholar). Similarly, Val-1504 in IVS6 of the rabbit cardiac α1C was shown to be critical for the channel inactivation (18Berjukow S. Gapp F. Aczel S. Sinnegger M.J. Mitterdorfer J. Glossmann H. Hering S. J. Biol. Chem. 1999; 274: 6154-6160Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The goal of our work was to determine, by single and combined amino acid substitutions, the role and molecular requirements for the involvement of S6 segments in voltage-dependent inactivation. The results are consistent with the C-type inactivation model (4Hoshi T. Zagotta W.N. Aldrich R.W. Science. 1990; 250: 533-538Crossref PubMed Scopus (1277) Google Scholar, 8Choi K. Aldrich R. Yellen G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5092-5095Crossref PubMed Scopus (394) Google Scholar, 12Hering S. Berjukow S. Sokolov S. Marksteiner R. Weiβ R.G. Kraus R. Timin E.N. J. Physiol. (Lond.). 2000; 528: 237-249Crossref Scopus (117) Google Scholar, 13Zhang J.F. Ellinor P.T. Aldrich R.W. Tsien R.W. Nature. 1994; 372: 97-100Crossref PubMed Scopus (177) Google Scholar) and suggest that the slow inactivation of Ca2+ channels is mediated by an annular determinant composed of amino acid residues situated in the cytoplasmic ends of transmembrane segments S6 in repeats I–IV. Complete removal of slow inactivation by a specific set of mutations in all four repeats gave us a unique opportunity to investigate the properties of Ca2+ channels that retain only the fast component of inactivation. pHLCC94 coding for α1C,II was constructed as described previously (25Soldatov N.M. Zhenochin S. AlBanna B. Abernethy D.R. Morad M. J. Membr. Biol. 2000; 177: 129-135Crossref PubMed Scopus (14) Google Scholar). α1C,IL, α1C,IS, α1C,ILS as well as α1C,III and its derivatives (α1C,IIIF, α1C,IIID, α1C,IIIK, α1C,IIIS1, α1C,IIIS2, and α1C,IIIT) were generated by the “megaprimer” method (27Sarkar G. BioTechniques. 1990; 8: 404-407PubMed Google Scholar). To prepare pHLCC250 coding for α1C,III, sense 5′-gctgtacacctgttca-3′ (3162–3177) and mutated antisense primer 5′-gaaaggtgacgatgGTgaagcccacgaagatg-3′ (3477–3508) were amplified in 25 cycles of PCR (40 s at 94 °C, 1 min at 42 °C, 40 s at 72 °C) with 400 ng ofBamHI-linearized pHLCC77 (GenBankTMz34815) using the Ampli-Taq polymerase kit (PerkinElmer Life Sciences). The purified 347-bp megaprimer was subjected to 5 “conditioning” cycles (28Datta A.K. Nucleic Acids Res. 1995; 23: 4530-4531Crossref PubMed Scopus (100) Google Scholar), each composed of 1 min at 94 °C and 3 min at 68 °C. Then antisense primer 5′-tacctcggtgattgctatatcaacaatgctacccacaaca-3′ (3870–3909) was added for 25 cycles of PCR (40 s at 94 °C, 1 min at 65 °C, 2 min at 73 °C). The SfuI/NsiI fragment (3342–3846) of the amplified DNA containing the mutation was incorporated at the respective restriction sites into pHLCC77. Other IIIS6 mutants were prepared in a similar way using the following antisense primers: 5′-gaaaggtgacgatgTcgaagcccacgaag-3′ (3508–3480; V1165D), 5′-cctgaaaggtgacgatTTTgaagcccacgaagatg-3′ (3511–3477; V1165K), 5′-cctgaaaggtgacgatgGGgaagcccacgaagatg-3′ (3511–3477; V1165F), 5′-ctcctgaaaggtgacgGAgacgaagcccacgaag-3′ (3511–3474; I1166S), 5′-ggtgacgatgacgGagcccacgaagatg-3′ (3504–3477; F1164S), 5′-ggtgacgatgacgaagGAcacgaagatgttcatc-3′ (3504–3471; G1163S), and 5′-gtgacgatgacgaagccGGTgaagatgttcatc-3′ (3503–3468; V1162T). To prepare α1C,IL, α1C,IS, and α1C,ILS, megaprimers were amplified using the mutated sense 5′-ctcggtgtgACtagcggagag-3′ (1201–1221), 5′-ggtgtgcttaTcggagagttttcc-3′ (1204–1227), or 5′-ggttctcggtgtgACtaTcggag-3′ (1197–1219) primers, respectively, with the antisense primer 5′-gttgacggactcggtc-3′ (1410–1425). Each of the megaprimers was used with the sense 5′-gcattgtcgaatggaaaccat-3′ (356–376) primer for PCR amplification of the pHLCC77 template. The resulting mutated DNAs were digested by MunI andSphI, and 1-kb fragments (409–1398) were ligated into the respective restriction sites of pHLCC77. α1C,IV-coding pHLCC249 was prepared using the Altered Sites II in vitromutagenesis system (Promega) and antisense mutation primer 5′-gttgtccatgGtgacagctac-3′ (4414–4434) essentially as described earlier (29Soldatov N.M., Oz, M. O'Brien K.A. Abernethy D.R. Morad M. J. Biol. Chem. 1998; 273: 957-963Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Combined II+III (α1C,II,III) and II+IV (α1C,II,IV) mutations were introduced by ligatingPpuMI/NotI fragments of pHLCC250 and pHLCC249, respectively, into pHLCC94. Combined III + IV (α1C,III,IV) and II + III + IV (α1C,II,III,IV) mutations were introduced via ligation of the NsiI/AatII fragment of pHLCC249 into pHLCC250 and the pHLCC94/250 chimera, respectively. All three quadruple mutants (α1C,I–IV) have been generated by a ligation ofMunI/SphI fragment (409–1398) of pHLCC251 or pHLCC252 with the SphI/NsiI fragment (1398–3846) of the pHLCC94/250 chimera and theMunI/NsiI vector part of pHLCC249. All mutations were confirmed by DNA sequencing. Wild-type (α1C,WT) and mutated α1C subunits were co-expressed with β1 (30Ruth P. Rohrkasten A. Biel M. Bosse E. Regulia S. Meyer H.E. Flockerzi V. Hofmann F. Science. 1989; 245: 1115-1118Crossref PubMed Scopus (257) Google Scholar) and α2δ subunits (31Singer D. Biel M. Lotan I. Flockerzi V. Hofmann F. Dascal N. Science. 1991; 253: 1553-1557Crossref PubMed Scopus (441) Google Scholar) as previously described (29Soldatov N.M., Oz, M. O'Brien K.A. Abernethy D.R. Morad M. J. Biol. Chem. 1998; 273: 957-963Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Membrane currents were recorded at 20–22 °C by a two-electrode voltage clamp method using a TEV-200A amplifier (Dagan). The bath solution contained, in millimolar, 40 Ba2+, 50 Na+, 1 K+, 5 HEPES, and 0.3 niflumic acid (pH 7.4 with methanesulfonic acid). Electrodes were filled with 3 m KCl and had resistances between 0.2 and 1 MΩ. One hour before recordings, oocytes were injected with 50 nl of 100 mm BAPTA (1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) (pH 7.4 with CsOH). The duration of test pulses used for the calculation of inactivation kinetics was 1 s to avoid deterioration of the stability of oocyte preparation. Currents were filtered at 1 kHz and sampled at 2.5 kHz. pClamp 8 software (Axon Instruments) was used for data acquisition, and KaleidaGraph software was used for the analysis. The values of the double-exponential fitting are presented only for the purpose of comparison of different mutants; they characterize only the 1-s current kinetics and do not represent the absolute values. Data are given as means ± S.E. Endogenous current, determined in the presence of 5 μm(±)-PN200-110 to block the α1C current, did not exceed 1–2% of the total current. The mutation A752T (26Soldatov N.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4628-4632Crossref PubMed Scopus (97) Google Scholar) that impairs Ca2+ channel inactivation (25Soldatov N.M. Zhenochin S. AlBanna B. Abernethy D.R. Morad M. J. Membr. Biol. 2000; 177: 129-135Crossref PubMed Scopus (14) Google Scholar) is located at the position −2 from the cytoplasmic end of the putative transmembrane segment IIS6 (Table I). The analogous S6 positions in repeats III and IV of α1C are also occupied by hydrophobic amino acids that are essentially conserved among the other Ca2+ channels except in the cases of α1A and α1B, that have an equivalent replacement V1165I (12Hering S. Berjukow S. Sokolov S. Marksteiner R. Weiβ R.G. Kraus R. Timin E.N. J. Physiol. (Lond.). 2000; 528: 237-249Crossref Scopus (117) Google Scholar). To examine the role of these hydrophobic amino acids in Ca2+ channel inactivation, the homologous mutations V1165T in IIIS6 (α1C,III) and I1475T in IVS6 (α1C,IV) have been introduced alone and in combinations with each other or with A752T (α1C,II).Table IMutations for probing the amino acids in transmembrane segments S6 critical for inactivation of the α 1C channelSegmentAmino acid motifMutationα1C constructIS6398NLVLGVLSGE407L404Tα1C,IL NLVLGVLS GES405Iα1C,IS NLVLGVLS GEL404T,S405Iα1C,ILSIIS6746NVFLAIAVDN755A752Tα1C,IIIIIS61159NIFVGFVIVT1168V1165Tα1C,III NIFVGFVIVTV1165Fα1C,IS,II,IIIF,IV NIFVGFVIVTV1165Dα1C,IS,II,IIID,IV NIFVGFVIVTV1165Kα1C,IS,II,IIIK,IV NIFVGFVI VTI1166Sα1C,IS,II,IIIS,IV NIFVGFVIVTF1164Sα1C,IS,II,IIIS1,IV NIFVGFVIVTG1163Sα1C,IS,II,IIIS2,IV NIFVGFVIVTV1162Tα1C,IS,II,IIIT,IVIVS61469NLFVAVIMDN1478I1475Tα1C,IVUnderlined are putative cytoplasmic ends of the predicted transmembrane segments S6 of the four repeats in the L-type α1C Ca2+ channel (26Soldatov N.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4628-4632Crossref PubMed Scopus (97) Google Scholar). Homologous amino acids are delineated by an alignment of the amino acid sequences in the cytoplasmic ends of the S6 regions. In boldfaceare single amino acids of α1C replaced by the indicated mutations. Open table in a new tab Underlined are putative cytoplasmic ends of the predicted transmembrane segments S6 of the four repeats in the L-type α1C Ca2+ channel (26Soldatov N.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4628-4632Crossref PubMed Scopus (97) Google Scholar). Homologous amino acids are delineated by an alignment of the amino acid sequences in the cytoplasmic ends of the S6 regions. In boldfaceare single amino acids of α1C replaced by the indicated mutations. The test pulses to +10 mV eliciting maximum currents were selected to compare the kinetics (Table II and Fig.1 A) and voltagedependence of activation and inactivation of the mutated channels (Fig. 1, B and C). For every trace of Ba2+ current, the steady-state current was determined from the fit (see Table II) and normalized to the peak amplitude. This sustained component of the current (I o) is a measure of the non-inactivating fraction of the Ba2+ current. The inactivating component of the current was further analyzed by double-exponential fitting as a sum of the fast and slow components. Increase of the pulse duration to 2 s did not greatly change the calculated parameters (see TableII, asterisk).Table IIKinetics of Ba 2+ current inactivation in α 1C mutantsMutantFraction ofI oFraction ofI fτfFraction ofI sτs2-bThe slow time constants (τs) are not discussed in this work and in fact require longer pulse intervals for more reliable estimation.n2-cn, number of tested oocytes.%%ms%ms α1C,WT16.9 ± 1.044.4 ± 3.876.0 ± 4.438.7 ± 3.8425 ± 1923Repeats II, III, IV α1C,II25.2 ± 2.047.9 ± 3.071.4 ± 5.526.9 ± 3.0415 ± 2622 α1C,III22.2 ± 1.846.7 ± 3.386.2 ± 9.131.1 ± 3.3404 ± 3214 α1C,IV28.9 ± 2.051.0 ± 1.478.4 ± 2.920.1 ± 1.4395 ± 1619 α1C,II,III52.1 ± 4.331.2 ± 5.369.9 ± 7.816.7 ± 5.3635 ± 839 α1C,II,IV47.0 ± 3.129.4 ± 4.981.0 ± 9.924.0 ± 4.9671 ± 4911 α1C,III,IV50.7 ± 3.132.6 ± 2.863.2 ± 5.416.7 ± 2.8752 ± 7917 α1C,II,III,IV69.7 ± 2.022.9 ± 2.053.8 ± 3.37.4 ± 2.0708 ± 6429Repeat I α1C,IL18.0 ± 2.750.0 ± 4.777.2 ± 7.430.0 ± 4.7507 ± 4917 α1C,IS29.7 ± 3.060.3 ± 1.846.9 ± 3.310.0 ± 1.8337 ± 369 α1C,ILS7.8 ± 2.166.8 ± 3.356.2 ± 2.225.4 ± 3.3354 ± 208All four repeats α1C,IL–IV29.9 ± 2.350.5 ± 4.837.7 ± 3.519.6 ± 4.8641 ± 9112 α1C,ILS–IV43.0 ± 4.956.1 ± 1.014.8 ± 1.80.9 ± 1.0471 ± 2711 α1C,IS-IV45.0 ± 4.855.0 ± 4.810.8 ± 0.4––17Ba2+ currents were elicited by 1-s test pulses2-aIncrease of pulse interval duration to 2 s did not greatly affect the fast time constant (τf) values (88.5 ± 9.0 ms) but somewhat changed the relative distribution of the kinetic components determined from the fit (Io = 13.8 ± 1.0%; If = 56.1 ± 3.9%; Is = 30.1 ± 3.9%) and the τs value (506 ± 30 ms, n = 16. to +10 mV from a holding potential of −90 mV. Inactivation time constants (τ) were determined from the double-exponential fitting of the current decay by equation: I(t) =I ∞ + I f × exp(−t/τf) + I s × exp(−t/(τs), where I ∞ is the steady-state amplitude of the current, I is the amplitude of the initial current, and f and s stand for fast and slow components, respectively. Thus the sum I f +I s represents the apparent inactivating component of the net current. I o is the sustained current component determined as the ratio of steady state to peak current amplitudes.2-a Increase of pulse interval duration to 2 s did not greatly affect the fast time constant (τf) values (88.5 ± 9.0 ms) but somewhat changed the relative distribution of the kinetic components determined from the fit (Io = 13.8 ± 1.0%; If = 56.1 ± 3.9%; Is = 30.1 ± 3.9%) and the τs value (506 ± 30 ms, n = 16.2-b The slow time constants (τs) are not discussed in this work and in fact require longer pulse intervals for more reliable estimation.2-c n, number of tested oocytes. Open table in a new tab Ba2+ currents were elicited by 1-s test pulses2-aIncrease of pulse interval duration to 2 s did not greatly affect the fast time constant (τf) values (88.5 ± 9.0 ms) but somewhat changed the relative distribution of the kinetic components determined from the fit (Io = 13.8 ± 1.0%; If = 56.1 ± 3.9%; Is = 30.1 ± 3.9%) and the τs value (506 ± 30 ms, n = 16. to +10 mV from a holding potential of −90 mV. Inactivation time constants (τ) were determined from the double-exponential fitting of the current decay by equation: I(t) =I ∞ + I f × exp(−t/τf) + I s × exp(−t/(τs), where I ∞ is the steady-state amplitude of the current, I is the amplitude of the initial current, and f and s stand for fast and slow components, respectively. Thus the sum I f +I s represents the apparent inactivating component of the net current. I o is the sustained current component determined as the ratio of steady state to peak current amplitudes. The results, summarized in Table II, show that the mutations to threonine introduced at the −2 positions in any of the S6 segments in repeats II, III, or IV increased the size of the sustained Ba2+ current at the end of a 1-s depolarization pulse ∼1.4- to 1.7-fold compared with the wild-type channel. None of the mutations have markedly altered the kinetics of inactivation. Indeed, the faster inactivating component representing 60–70% of Ba2+ current decay has the time constant τfranging from 71 to 86 ms. The remaining slower component of the Ba2+ current inactivated with a τs from 395 to 415 ms, i.e. in the range characteristic for the α1C, WT channel (Table II). When single mutations were introduced, a 3- to 4-mV shift in both directions in steady-state inactivation curves was observed (Fig.1 C). At the end of the 2-s conditioning pulse, 6.1 ± 1.4% of the maximum Ba2+ current through the wild-type channel remained noninactivated. This fraction increased to 11.5–21.2% by the threonine substitution in repeats II–IV, suggesting that inactivation of the mutated channels was obstructed. The maximum effect of a single mutation was seen with α1C, II, thus confirming the results of our earlier study of this mutant expressed in HEK293 cells (25Soldatov N.M. Zhenochin S. AlBanna B. Abernethy D.R. Morad M. J. Membr. Biol. 2000; 177: 129-135Crossref PubMed Scopus (14) Google Scholar). Thus, the hydrophobic residues of Ala-752 in IIS6, Val-1165 in IIIS6, and Ile-1475 in IVS6 each contribute to the voltage-dependent inactivation of the α1C channel. Mutation of these amino acids to hydrophilic Thr appears to impair the transition from the open to inactivated state and/or destabilizes the inactivated state of the channel. Given the similarity in the effects of the tested single mutations to Thr, our data may suggest that the hydrophobic amino acids in the −2 positions of segments IIS6, IIIS6, and IVS6 are concurrently involved in voltage-dependent inactivation. To test this hypothesis, the S6 mutations in repeats II–IV were combined in the double and triple mutants listed in Table II. We observed that each additional homologous mutation inhibited a considerable fraction of the Ba2+ current inactivation (Fig. 1 A and Table II) without significantly altering current-voltage relationships (Fig.1 B). The voltage dependences of the time constants of inactivation were not strongly affected (Fig. 1 D). Indeed, the ratio τf(−10)/τf(40) of the time constants measured at −10 mV and +40 mV for Ba2+ currents through the α1C,II,IV, α1C,III,IV, and α1C,II,III,IV channels decreased less than 2-fold compared with α1C,WT and other mutants. Steady-state inactivation curves showed an increase of the noninactivating component of Ba2+ current from 6.1 ± 1.4% in α1C,WT to 48.5 ± 3.0% in α1C,II,III,IV (Fig. 1 C). For some of the mutants, these curves were shifted by 3–4 mV toward positive potentials, whereas their slopes in α1C,II,IV and α1C,II,III,IV were less steep than in α1C,WT and other mutants. These data suggest that the combined double and triple mutations to threonine may slightly change the voltage dependence and cooperativity of the voltage sensors for inactivation in these channels. In the three double mutants tested (α1C,II,III, α1C,II,IV, and α1C,III,IV), the sustained component of Ba2+ current increased 2-fold compared with the single mutants, and accounted for 47–52% of the total current (Table II). An augmentation of the sustained current was due to both the fast and slow components of the current, which decreased about proportionally in the analyzed single and double mutants (I f/I s ∼ 2). In the case of α1C,II,III,IV, the fraction of the sustained current increased to ∼70% of the total current (Fig. 1 Aand Table II). It is important to note that the ratio ofI f/I s ∼ 3 determined for the α1C,II,III,IV channel suggests a substantial additional decrease of the slower inactivating component. The triple mutation did not appear to greatly affect the property of Ca2+-dependent inactivation (Fig.2). First, the dominating (87.8 ± 0.2%) fast component of the decay of the Ca2+ current atV t = +10 mV (τf = 16.0 ± 1.2 ms, n = 7; Fig. 2 A) exhibited a 4.6-fold acceleration as compared with the Ba2+ current (Table II) but had kinetics essentially similar to those through the wild-type channel (τf = 15.3 ± 1.5 ms;I f = 95.1 ± 0.5%; n = 16). Second, we observed a U-shape voltage dependence of τf (Fig. 2 B) reflecting the relationship between the inactivation rate and the size of Ca2+ current expected for Ca2+-induced inactivation (32Neely A. Olcese R. Wei X.Y. Birnbaumer L. Stefani E. Biophys. J. 1994; 66: 1895-1903Abstract Full Text PDF PubMed Scopus (85) Google Scholar). However, with Ca2+ as a charge carrier, a sustained component of the current was also observed, but its amplitude (11.6 ± 1.8%,n = 5) was smaller than those measured with Ba2+ current (Table II) and appreciably larger than that in the case of I Ca through α1C,WT(2.8 ± 1.0%; n = 16). One of the possible explanations for the significant residual sustained component of the Ca2+ current in the triple mutant may be partial compensation of the impaired voltage-dependent inactivation in α1C,II,III,IV by the Ca2+-induced inactivation. Although the hydrophobic amino acid mutations were introduced in the presumed pore region of the channel (9Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5745) Google Scholar, 10Lopez G.A. Jan Y.N. Jan L.Y. Nature. 1994; 367: 179-182Crossref PubMed Scopus (148) Google Scholar), the ratio of maximum Ba2+ to Ca2+ currents (∼2.7; n = 3) through the α1C,II,III,IVchannel did not change substantially compared with the wild-type channel, indicating that the ion selectivity was not appreciably affected. Segment IS6 appears to contribute to the voltage-dependent inactivation differently as compared with other S6 segments. The inactivation properties of the L404T mutant α1C,IL are very similar to those of the wild-type channel (Table II). However, incorporation of the L404T mutation into the α1C,II,III,IV channel significantly reversed the effect caused by the combined mutations to threonines in repeats II–IV leading to α1C,II,III,IV (cf. decays of Ba2+ current through the α1C,II,III,IVchannel in Fig. 1 A and those through the α1C,IL–IV channel in Fig.3 A). The sustained component of the Ba2+ current through the α1C,IL–IVchannel was reduced 2.3-fold. Both fast and slow components of the decay increased in size, and the faster inactivating component of the Ba2+ current was accelerated (Table II). Steady-state inactivation curves for α1C,IL and α1C,IL–IV were shifted by 7 and 13 mV toward negative voltages, and their slopes were steeper compared with α1C,WT and α1C,II,III,IV, respectively (Fig. 3 B). In the case of the α1C,IL–IVchannel, the half-maximal activation was also shifted by 12 mV toward negative potentials (Fig. 3 C), but the current-voltage relation for α1C,IL was not appreciably different from those for the other isoforms (Fig. 1 B). Unlike transmembrane segments IIS6–IVS6, segment IS6 contains a hydroxyl amino acid" @default.
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• W2005524812 title "Molecular Determinants of Voltage-dependent Slow Inactivation of the Ca2+ Channel" @default.
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