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Modulation of L-type Ca2+ Channels by Gβγ and Calmodulin via Interactions with N and C Termini of α1C

2000; Elsevier BV; Volume: 275; Issue: 51 Linguagem: Inglês

10.1074/jbc.m005881200

ISSN

1083-351X

Autores

Tatiana Ivanina, Yakov Blumenstein, Elena Shistik, Rachel Barzilai, Nathan Dascal,

Tópico(s)

Cardiac electrophysiology and arrhythmias

Resumo

Neuronal voltage-dependent Ca2+channels of the N (α1B) and P/Q (α1A) type are inhibited by neurotransmitters that activate Gi/o G proteins; a major part of the inhibition is voltage-dependent, relieved by depolarization, and results from a direct binding of Gβγ subunit of G proteins to the channel. Since cardiac and neuronal L-type (α1C) voltage-dependent Ca2+ channels are not modulated in this way, they are presumed to lack interaction with Gβγ. However, here we demonstrate that both Gβγ and calmodulin directly bind to cytosolic N and C termini of the α1C subunit. Coexpression of Gβγ reduces the current via the L-type channels. The inhibition depends on the presence of calmodulin, occurs at basal cellular levels of Ca2+, and is eliminated by EGTA. The N and C termini of α1C appear to serve as partially independent but interacting inhibitory gates. Deletion of the N terminus or of the distal half of the C terminus eliminates the inhibitory effect of Gβγ. Deletion of the N terminus profoundly impairs the Ca2+/calmodulin-dependent inactivation. We propose that Gβγ and calmodulin regulate the L-type Ca2+ channel in a concerted manner via a molecular inhibitory scaffold formed by N and C termini of α1C. Neuronal voltage-dependent Ca2+channels of the N (α1B) and P/Q (α1A) type are inhibited by neurotransmitters that activate Gi/o G proteins; a major part of the inhibition is voltage-dependent, relieved by depolarization, and results from a direct binding of Gβγ subunit of G proteins to the channel. Since cardiac and neuronal L-type (α1C) voltage-dependent Ca2+ channels are not modulated in this way, they are presumed to lack interaction with Gβγ. However, here we demonstrate that both Gβγ and calmodulin directly bind to cytosolic N and C termini of the α1C subunit. Coexpression of Gβγ reduces the current via the L-type channels. The inhibition depends on the presence of calmodulin, occurs at basal cellular levels of Ca2+, and is eliminated by EGTA. The N and C termini of α1C appear to serve as partially independent but interacting inhibitory gates. Deletion of the N terminus or of the distal half of the C terminus eliminates the inhibitory effect of Gβγ. Deletion of the N terminus profoundly impairs the Ca2+/calmodulin-dependent inactivation. We propose that Gβγ and calmodulin regulate the L-type Ca2+ channel in a concerted manner via a molecular inhibitory scaffold formed by N and C termini of α1C. voltage-dependent calcium channel calmodulin C terminus N terminus protein kinase A protein kinase C glutathione S-transferase amino acid(s) N-[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]glycine 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid Voltage-dependent Ca2+ channels (VDCCs)1 are crucial for neuronal and muscular excitability (1Hille B. Ionic Channels of Excitable Membranes. Sinauer, Sunderland1992Google Scholar). Mammalian VDCCs fall into several families distinguished by pharmacological and biophysical properties (L, N, P/Q, T, and R type) and the molecular identity of the main, pore−forming subunit, α1 (2Snutch T.P. Reiner P.B. Curr. Opin. Neurobiol. 1992; 2: 247-253Crossref PubMed Scopus (251) Google Scholar, 3Catterall W.A. Cell Calcium. 1998; 24: 307-323Crossref PubMed Scopus (322) Google Scholar, 4Perez-Reyes E. J. Bioenerg. Biomembr. 1998; 30: 313-318Crossref PubMed Scopus (84) Google Scholar). The neuronal N- and P/Q-type channels, based on α1B and α1A, respectively, are crucial for neurotransmitter release (3Catterall W.A. Cell Calcium. 1998; 24: 307-323Crossref PubMed Scopus (322) Google Scholar). L-type Ca2+ channels containing the "cardiac-type" α1C subunit regulate contraction of cardiac and smooth muscle, and excitability and gene expression in the brain (2Snutch T.P. Reiner P.B. Curr. Opin. 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Chem. 1999; 274: 31145-31149Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 18Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 19Bunemann M. Gerhardstein B.L. Gao T. Hosey M.M. J. Biol. Chem. 1999; 274: 33851-33854Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Neuronal VDCCs are usually inhibited by G protein-coupled receptors byvoltage-independent (mediated by several second messenger pathways (20Bernheim L. Beech D.J. Hille B. Neuron. 1991; 6: 859-867Abstract Full Text PDF PubMed Scopus (186) Google Scholar, 21Diverse-Pierluissi M. Dunlap K. Neuron. 1993; 10: 753-760Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 22Diverse-Pierluissi M. Goldsmith P.K. Dunlap K. Neuron. 1995; 14: 191-200Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 23Shapiro M.S. Wollmuth L.P. Hille B. J. Neurosci. 1994; 14: 7109-7116Crossref PubMed Google Scholar)) and voltage-dependentmechanisms. The latter is fast, membrane−delimited, mediated by Gβγ, and occurs in α1A (P/Q), α1B (N), and α1E (11Zamponi G.W. Snutch T.P. Curr. Opin. Neurobiol. 1998; 8: 351-356Crossref PubMed Scopus (166) Google Scholar, 12Dolphin A.C. J. Physiol. (Lond .). 1998; 506: 3-11Crossref Scopus (233) Google Scholar, 13Ikeda S.R. Dunlap K. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 131-151Crossref PubMed Google Scholar). A hallmark of this modulation is relief of inhibition and acceleration of current activation by depolarization (voltage-dependent facilitation), which reflects a decrease in the affinity of the channel to Gβγ (24Patil P.G. de Leon M. Reed R.R. Dubel S. Snutch T.P. Yue D.T. Biophys. J. 1996; 71: 2509-2521Abstract Full Text PDF PubMed Scopus (107) Google Scholar, 25Zamponi G.W. Snutch T.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4035-4039Crossref PubMed Scopus (110) Google Scholar). In α1A, α1B, and α1E Gβγ binds to L1 loop (15Zamponi G.W. Bourinet E. Nelson D. Nargeot J. Snutch T.P. Nature. 1997; 385: 442-446Crossref PubMed Scopus (406) Google Scholar, 26De Waard M. Liu H. Walker D. Scott V.E. Gurnett C.A. Campbell K.P. Nature. 1997; 385: 446-450Crossref PubMed Scopus (374) Google Scholar, 27Qin N. Platano D. Olcese R. Stefani E. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8866-8871Crossref PubMed Scopus (207) Google Scholar) and CT (27Qin N. Platano D. Olcese R. Stefani E. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8866-8871Crossref PubMed Scopus (207) Google Scholar); NT is also important for the effect of Gβγ, although a direct interaction has not yet been established (28Page K.M. Canti C. Stephens G.J. Berrow N.S. Dolphin A.C. J. Neurosci. 1998; 18: 4815-4824Crossref PubMed Google Scholar, 29Canti C. Page K.M. Stephens G.J. Dolphin A.C. J. Neurosci. 1999; 19: 6855-6864Crossref PubMed Google Scholar, 30Simen A.A. Miller R.J. J. Neurosci. 1998; 18: 3689-3698Crossref PubMed Google Scholar). The relative functional roles of the L1, CT-, and NT-binding sites are still unclear (27Qin N. Platano D. Olcese R. Stefani E. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8866-8871Crossref PubMed Scopus (207) Google Scholar, 31Furukawa T. Nukada T. Mori Y. Wakamori M. Fujita Y. Ishida H. Fukuda K. Kato S. Yoshii M. J. Biol. Chem. 1998; 273: 17585-17594Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 32Herlitze S. Hockerman G.H. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1512-1516Crossref PubMed Scopus (169) Google Scholar). Cytosolic parts of α1 subunits are also involved in inactivation gating. A major part of inactivation of L-type VDCCs is triggered by the entry of Ca2+ (33Eckert R. Chad J.E. Prog. Biophys. Mol. Biol. 1984; 44: 215-267Crossref PubMed Scopus (412) Google Scholar). Calmodulin (CaM) has been recently identified as the Ca2+ sensor indispensable for the Ca2+-dependent inactivation in L (α1C)- and P/Q (α1A)-type channels (34Qin N. Olcese R. Bransby M. Lin T. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2435-2438Crossref PubMed Scopus (251) Google Scholar, 35Lee A. Wong S.T. Gallagher D. Li B. Storm D.R. Scheuer T. Catterall W.A. Nature. 1999; 399: 155-159Crossref PubMed Scopus (1004) Google Scholar, 36Zuhlke R.D. Pitt G.S. Deisseroth K. Tsien R.W. Reuter H. Nature. 1999; 399: 159-162Crossref PubMed Scopus (744) Google Scholar, 37Peterson B.Z. DeMaria C.D. Adelman J.P. Yue D.T. Neuron. 1999; 22: 549-558Abstract Full Text Full Text PDF PubMed Scopus (722) Google Scholar). A CaM-binding site has been identified in the CT of α1C and α1A. Ca2+-dependent CaM interaction with this domain has been found crucial not only for the Ca2+-dependent inactivation, but also for an opposite effect of Ca2+, calledCa 2+ -dependent facilitation (34Qin N. Olcese R. Bransby M. Lin T. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2435-2438Crossref PubMed Scopus (251) Google Scholar, 35Lee A. Wong S.T. Gallagher D. Li B. Storm D.R. Scheuer T. Catterall W.A. Nature. 1999; 399: 155-159Crossref PubMed Scopus (1004) Google Scholar, 36Zuhlke R.D. Pitt G.S. Deisseroth K. Tsien R.W. Reuter H. Nature. 1999; 399: 159-162Crossref PubMed Scopus (744) Google Scholar, 37Peterson B.Z. DeMaria C.D. Adelman J.P. Yue D.T. Neuron. 1999; 22: 549-558Abstract Full Text Full Text PDF PubMed Scopus (722) Google Scholar). Despite the outstanding role of cytosolic segments in channel modulation, our ideas of how these parts affect the gating of the VDCCs are vague. The L-type (α1C) channel is the best studied in this respect; but even here, only the roles of N and C termini have been examined. Removal of the distal half of the CT increases L-type channel currents and open probability by improving the coupling between gating charge (voltage sensor) movement and pore opening (38Wei X. Neely A. Lacerda A.E. Olcese R. Stefani E. Perez-Reyes E. Birnbaumer L. J. Biol. Chem. 1994; 269: 1635-1640Abstract Full Text PDF PubMed Google Scholar). It has been proposed that the CT acts as an inhibitory gate that conformationally restrains the opening of the channel (38Wei X. Neely A. Lacerda A.E. Olcese R. Stefani E. Perez-Reyes E. Birnbaumer L. J. Biol. Chem. 1994; 269: 1635-1640Abstract Full Text PDF PubMed Google Scholar, 39Klockner U. Mikala G. Varadi M. Varadi G. Schwartz A. J. Biol. Chem. 1995; 270: 17306-17310Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Similarly to the CT, removal of the NT also enhances the open probability and the macroscopic currents in L-type channels, and a similar role (of an inhibitory gate) for the NT has been proposed (16Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 17Shistik E. Keren-Raifman T. Idelson G.H. Dascal N. Ivanina T. J. Biol. Chem. 1999; 274: 31145-31149Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The L-type channel is not inhibited by neurotransmitters in a voltage-dependent manner (32Herlitze S. Hockerman G.H. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1512-1516Crossref PubMed Scopus (169) Google Scholar, 40Bourinet E. Charnet P. Tomlinson W.J. Stea A. Snutch T.P. Nargeot J. EMBO J. 1994; 13: 5032-5039Crossref PubMed Scopus (92) Google Scholar, 41Bourinet E. Soong T.W. Stea A. Snutch T.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1486-1491Crossref PubMed Scopus (219) Google Scholar, 42Zhang J.F. Ellinor P.T. Aldrich R.W. Tsien R.W. Neuron. 1996; 17: 991-1003Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), and the L1 loop of α1C does not bind Gβγ (15Zamponi G.W. Bourinet E. Nelson D. Nargeot J. Snutch T.P. Nature. 1997; 385: 442-446Crossref PubMed Scopus (406) Google Scholar, 26De Waard M. Liu H. Walker D. Scott V.E. Gurnett C.A. Campbell K.P. Nature. 1997; 385: 446-450Crossref PubMed Scopus (374) Google Scholar, 27Qin N. Platano D. Olcese R. Stefani E. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8866-8871Crossref PubMed Scopus (207) Google Scholar). Voltage-dependent facilitation has been demonstrated in L-type VDCC, but it was PKA- rather than G protein-dependent (Refs. 40Bourinet E. Charnet P. Tomlinson W.J. Stea A. Snutch T.P. Nargeot J. EMBO J. 1994; 13: 5032-5039Crossref PubMed Scopus (92) Google Scholar and 43Sculptoreanu A. Rotman E. Takahashi M. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10135-10139Crossref PubMed Scopus (164) Google Scholar; however, see Ref. 44Dai S. Klugbauer N. Zong X. Seisenberger C. Hofmann F. FEBS Lett. 1999; 442: 70-74Crossref PubMed Scopus (33) Google Scholar). Therefore, α1C has been assumed to lack any interaction with Gβγ. α1C even served in several studies as a donor of presumably Gβγ-indifferent parts, to create chimeras with other α1 types, in the search for parts of α1 that determine the sensitivity to Gβγ. However, here we demonstrate that Gβγ binds to NT and CT of α1C and inhibits the L-type VDCC in a voltage-independent but calmodulin-dependent manner. We identify a novel CaM-binding site in the NT, which, like the previously identified CT-binding site, is an important determinant of the Ca2+/CaM-dependent inactivation. We propose a model in which NT and CT of the L-type channel form a scaffold that plays a role of an inhibitory gate which integrates the regulatory effects of Gβγ and CaM. The cDNAs of the G protein subunits (bovine Gβ1, bovine Gβ2,human Gβ3, mouse Gβ4 and bovine Gγ2; provided by M. Simon, Caltech) were either amplified by polymerase chain reaction to create EcoRI sites at the 5′ and 3′ ends (Gβ1 and Gγ2) or excised with EcoRI from the original vectors (all the others) and then subcloned into the EcoRI site of the pGEMHE (Gβ1, Gβ2, and Gγ2) and pGEMHJ (Gβ3 and Gβ4) vectors (45Liman E.R. Tytgat J. Hess P. Neuron. 1992; 9: 861-871Abstract Full Text PDF PubMed Scopus (983) Google Scholar, 46Jing J. Chikvashvili D. Singer-Lahat D. Thornhill W.B. Reuveny E. Lotan I. EMBO J. 1999; 18: 1245-1256Crossref PubMed Google Scholar). The cDNAs of CaM and CaM1234 (47Xia X.M. Fakler B. Rivard A. Wayman G. Johnson-Pais T. Keen J.E. Ishii T. Hirschberg B. Bond C.T. Lutsenko S. Maylie J. Adelman J.P. Nature. 1998; 395: 503-507Crossref PubMed Scopus (740) Google Scholar) were provided by J. P. Adelman. cDNAs and RNAs of rabbit Ca2+ channels subunits β2A and α2/δ were as described (48Singer D. Biel M. Lotan I. Flockerzi V. Hofmann F. Dascal N. Science. 1991; 253: 1553-1557Crossref PubMed Scopus (441) Google Scholar). The rabbit heart α1C cDNA (49Mikami A. Imoto K. Tanabe T. Niidome T. Mori Y. Takeshima H. Narumiya S. Numa S. Nature. 1989; 340: 230-233Crossref PubMed Scopus (770) Google Scholar) and all its mutants used here were subcloned intoSalI/HindIII sites of the pGEM-SB vector (16Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The N-terminal deletion mutant of α1C, ΔN2–139, was prepared as described (16Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). To create the α1C C-terminal truncation mutant ΔC1700-end, polymerase chain reaction amplification of a C-terminal part of α1C was performed to create a stop codon after nucleotide 5274 (numbering by Ref. 49Mikami A. Imoto K. Tanabe T. Niidome T. Mori Y. Takeshima H. Narumiya S. Numa S. Nature. 1989; 340: 230-233Crossref PubMed Scopus (770) Google Scholar) followed by aHindIII site. The truncated cDNA was subcloned back into pGEM-SB. The ΔNΔC mutant was constructed by cutting and ligating the appropriate parts of ΔN2–139 and ΔC1700-end mutants. The RNAs were prepared using a standard procedure (50Dascal N. Lotan I. Longstaff A. Revest P. Protocols in Molecular Neurobiology. Humana Press, Totowa, NJ1992: 205-225Google Scholar). cDNAs designed to create glutathione S-transferase (GST) fusion proteins were constructed using polymerase chain reaction strategy, with primers containing the desired restriction sites and linked in-frame to GST, as described (16Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The cDNA constructs encoded the GST fusion proteins of the following segments of α1C: whole N terminus (N1–154); CT and three C-terminal cDNA fragments (C, aa 1505–2171; C0, aa 1505–1846; C1, aa 1664–1845; and C2, aa 1841–2171); L1, aa 438–550; L2, aa 783–930; and L3, aa 1196–1249. Xenopus laevisfrogs were maintained and dissected as described (50Dascal N. Lotan I. Longstaff A. Revest P. Protocols in Molecular Neurobiology. Humana Press, Totowa, NJ1992: 205-225Google Scholar). Oocytes were injected with equal amounts (by weight) of the mRNAs of α1C or its mutants with of α2/δ, with or without β2A, and incubated for 3–5 days at 20–22 °C in NDE96 solution (96 mm NaCl, 2 mm KCl, 1 mm MgCl2, 1 mm CaCl2, 2.5 mm Na pyruvate, 50 μg/ml gentamycine, 5 mm HEPES, pH 7.5). Whole cell currents were recorded using the Gene Clamp 500 amplifier (Axon Instruments, Foster City, CA) using the two-electrode voltage clamp technique in a solution containing 40 mm Ba(OH)2 or 40 mmCa(OH)2, 50 mm NaOH, 2 mm KOH, and 5 mm HEPES, titrated to pH 7.5 with methanesulfonic acid. In some cases, a solution with 2 mm Ba2+ was used (2 mm Ba(OH)2, 96 mm NaOH, 2 mm KOH, 5 mm Hepes, pH titrated to 7.5 with methanesulfonic acid). Stimulation, data acquisition, and analysis were performed using pCLAMP software (Axon Instruments). Ba2+currents were measured by a 200 or 400 ms step to 20 mV from a holding potential of −80 mV. The procedures were essentially as described (16Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In brief, [35S]Met/Cys-labeled Gβ1, Gγ2, CaM, and CaM1234 were translated on the template of in vitro synthesized RNAs using a rabbit reticulocyte translation kit (Promega). The fusion proteins were synthesized and extracted from Escherichia coli using the Amersham Pharmacia Biotech kit. Purified GST fusion proteins (5–10 μg) or purified GST (10 μg) were incubated with 5 μl of the lysate, containing the 35S-labeled proteins in 500 μl of phosphate-buffered saline with 0.05% Tween 20, for 2 h at room temperature, with gentle rocking. In some experiments the incubation was done in the presence of 1 mm CaCl2 or 5 mm EGTA. In the experiments shown in Fig. 5 C, the incubation was done in the same buffer but with varying concentrations of free Ca2+, in the presence of 2 mm EGTA; free Ca2+ concentration was calculated using the MAXC program. Then 30 μl of glutathione-Sepharose beads (Amersham Pharmacia Biotech) were added, and the mixture was incubated for 30 min at 4 °C and washed four times in 1 ml of the same buffer. Following washing, GST fusion proteins were eluted with 30 μl of 20 mm reduced glutathione in elution buffer (120 mm NaCl, 100 mm Tris-HCl, pH 8). CaM was analyzed on 15%, Gβ, on 12% SDS-polyacrylamide gels. Gγ was analyzed on Tricine ready-made gels (Bio-Rad). The labeled products were identified and quantified by autoradiography using a PhosphorImager (Molecular Dynamics) as described (51Ivanina T. Perets T. Thornhill W.B. Levin G. Dascal N. Lotan I. Biochemistry. 1994; 33: 8786-8792Crossref PubMed Scopus (60) Google Scholar). Phosphorylation of the N-terminal GST fusion protein was performed as described (17Shistik E. Keren-Raifman T. Idelson G.H. Dascal N. Ivanina T. J. Biol. Chem. 1999; 274: 31145-31149Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). This was performed essentially as described (51Ivanina T. Perets T. Thornhill W.B. Levin G. Dascal N. Lotan I. Biochemistry. 1994; 33: 8786-8792Crossref PubMed Scopus (60) Google Scholar, 52Shistik E. Ivanina T. Puri T. Hosey M. Dascal N. J. Physiol. (Lond .). 1995; 489: 55-62Crossref Scopus (128) Google Scholar). Oocytes were injected with mRNAs and incubated in NDE solution containing 0.5 mCi/ml [35S]methionine/cysteine (Amersham Pharmacia Biotech) for 4 days at 22 °C. Plasma membranes were separated manually (51Ivanina T. Perets T. Thornhill W.B. Levin G. Dascal N. Lotan I. Biochemistry. 1994; 33: 8786-8792Crossref PubMed Scopus (60) Google Scholar) from the rest of the oocyte (designated as internal fraction). 10–20 membranes and 3–5 internal fractions, or 5 whole oocytes, were homogenized, proteins were solubilized, immunoprecipitated as described (16Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 52Shistik E. Ivanina T. Puri T. Hosey M. Dascal N. J. Physiol. (Lond .). 1995; 489: 55-62Crossref Scopus (128) Google Scholar), and electrophoresed on 6 or 3–8% polyacrylamide-SDS gels. Card-I antibody was kindly provided by M. M. Hosey (Northwestern University, Chicago) (53Chien A.J. Zhao X. Shirokov R.E. Puri T.S. Chang C.F. Sun D. Rios E. Hosey M.M. J. Biol. Chem. 1995; 270: 30036-30044Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). The data are presented as mean ± S.E. To overcome the problem of batch-to-batch variability in current amplitudes, the results were normalized as follows (54Sharon D. Vorobiov D. Dascal N. J. Gen. Physiol. 1997; 109: 477-490Crossref PubMed Scopus (137) Google Scholar): in each oocyte, IBawas normalized to the mean amplitude of IBa in the control group of oocytes of the same donor. These normalized values were averaged across all oocyte batches tested. Comparisons between two groups (e.g. control and Gβγ-expressing groups) were tested for statistically significant differences (p < 0.05 or better) using two-tailed unpaired t tests. Comparison between several groups was done using one-way analysis of variance (ANOVA) followed by Dunnet's or Tukey's tests, using the SigmaStat software (SPSS Corp.). Direct interaction between intracellular segments of α1C with Gβγ was studied in vitro using fragments of α1C fused to GST, covering all the large intracellular segments of α1C (Fig. 1 A). They included the whole NT (N1–154), the full-length CT (C), three subdivisions of the C terminus (C0, C1, and C2, as shown in Fig. 1 A, inset), and three interdomain linker loops (L1, L2, and L3). The binding of Gβγ to NT, CT, or its parts, and L3 has not been examined in the past. The GST fusion proteins were immobilized on glutathione-agarose beads and assayed for interaction with in vitro translated, 35S-labeled Gβ1γ2 subunits. Unexpectedly, NT and CT bound Gβ1γ2, whereas GST alone and loops L1, L2, and L3 did not bind Gβγ (Fig. 1 B, upper panel). The results of all experiments were quantitated by normalizing the 35S-Gβγ signal obtained from each GST fusion protein to that of the NT obtained in the same experiment (Fig. 1 B, lower panel). The CT exhibited the strongest interaction with Gβγ, mainly localized to its proximal half (C0; aa residues 1505–1846). The distal half of the CT, C2, showed a weak but reproducible Gβγ binding. Since there is no binding in the middle of CT (C1), it is plausible that Gβγ binds to two separate sites in the CT, roughly in its first quarter (from the beginning of C0 to the beginning of C1, between aa 1505 and 1664) and the last half. NT showed intermediate Gβγ binding. Fig. 1 C shows that the binding of Gβγ to NT and CT was Ca2+-independent: it was identical in the presence of 1 mm Ca2+ or 5 mm EGTA, or with no additions (control). The binding of Gβγ to NT (which is a PKC substrate: see Ref. 17Shistik E. Keren-Raifman T. Idelson G.H. Dascal N. Ivanina T. J. Biol. Chem. 1999; 274: 31145-31149Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) was not affected by phosphorylation by PKC or by the presence of the Ca2+ channel β2A subunit (data not shown). Of the 5 known Gβ proteins (55Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (704) Google Scholar), the highly homologous Gβ1 through Gβ4 often show considerable selectivity in modulating the voltage-dependent Ca2+ channels (Refs. 56Kleuss C. Scherubl H. Hescheler J. Schultz G. Wittig B. Nature. 1992; 358: 424-426Crossref PubMed Scopus (333) Google Scholar, 57Hescheler J. Schultz G. Ann. N. Y. Acad. 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Nukada T. Mori Y. Wakamori M. Fujita Y. Ishida H. Fukuda K. Kato S. Yoshii M. J. Biol. Chem. 1998; 273: 17585-17594Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 32Herlitze S. Hockerman G.H. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1512-1516Crossref PubMed Scopus (169) Google Scholar, 40Bourinet E. Charnet P. Tomlinson W.J. Stea A. Snutch T.P. Nargeot J. EMBO J. 1994; 13: 5032-5039Crossref PubMed Scopus (92) Google Scholar, 41Bourinet E. Soong T.W. Stea A. Snutch T.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1486-1491Crossref PubMed Scopus (219) Google Scholar, 42Zhang J.F. Ellinor P.T. Aldrich R.W. Tsien R.W. Neuron. 1996; 17: 991-1003Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). It seemed the Gβγ, when released from G proteins after activation of the relevant neurotransmitters by agonists, did not directly regulate the L-type channel. This leaves open the question of the functional consequences of the interaction between α1C and Gβγ revealed by experiments of Fig. 1. To address this problem, we used coexpression methodology. L-type channels were expressed in Xenopus oocytes in full subunit composition (α1C, α2δ and β2A), or without the β subunit (α1α2δ combination). Currents were measured using the two-electrode voltage clamp technique. At standard levels of expression of the channel used here (1 ng of RNA/oocyte for α1α2δβ, 2.5 ng/oocyte for α1α2δ), the averageIBa was 1064 ± 47 nA (n = 58 oocytes, N = 8 batches) and 324 ± 15 nA (n = 149, N = 19), respectively. In accord with previous reports, activation by acetylcholine of a coexpressed muscarinic m2 receptor (which couples to Gi/oproteins) did not cause any consistent modulation ofIBa (data not shown). However, upon additional coexpression of Gαo, which is indispensable for muscarinic inhibition of L-type Ca2+ channels

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