Differential Role of the α1C Subunit Tails in Regulation of the Cav1.2 Channel by Membrane Potential, β Subunits, and Ca2+ Ions
2005; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês
10.1074/jbc.m412140200
ISSN1083-351X
AutoresEvgeny Kobrinsky, Swasti Tiwari, Victor A. Maltsev, Jo Beth Harry, Edward G. Lakatta, Darrell R. Abernethy, Nikolai M. Soldatov,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoVoltage-gated Cav1.2 channels are composed of the pore-forming α1C and auxiliary β and α2δ subunits. Voltage-dependent conformational rearrangements of the α1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the α1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the α1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both α2δ and β subunits remain essential for the functional channel. In contrast, if α1C subunits with are expressed α2δ but in the absence of a β subunit, plasma membrane anchoring of the α1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the α1C N-tail/β interaction: (i) co-expression of β restores inactivation properties, (ii) release of the α1C N terminus inhibits the β-deficient channel, and (iii) voltage-gated mobility of the α1C N-tail vis à vis the plasma membrane is increased in the β-deficient (silent) channel. Together, these data argue that both the α1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as β subunit modulation of the channel. The α1C N-tail may have a role in the channel trafficking and is a target of the β subunit modulation. The β subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the α1C C and N termini, β subunit, and the cytoplasmic pore region confers the multifactorial regulation of Cav1.2 channels. Voltage-gated Cav1.2 channels are composed of the pore-forming α1C and auxiliary β and α2δ subunits. Voltage-dependent conformational rearrangements of the α1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the α1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the α1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both α2δ and β subunits remain essential for the functional channel. In contrast, if α1C subunits with are expressed α2δ but in the absence of a β subunit, plasma membrane anchoring of the α1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the α1C N-tail/β interaction: (i) co-expression of β restores inactivation properties, (ii) release of the α1C N terminus inhibits the β-deficient channel, and (iii) voltage-gated mobility of the α1C N-tail vis à vis the plasma membrane is increased in the β-deficient (silent) channel. Together, these data argue that both the α1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as β subunit modulation of the channel. The α1C N-tail may have a role in the channel trafficking and is a target of the β subunit modulation. The β subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the α1C C and N termini, β subunit, and the cytoplasmic pore region confers the multifactorial regulation of Cav1.2 channels. Cav1.2 channels are known for their key role in triggering Ca2+ signaling in a wide variety of cells. Calmodulin (CaM) 1The abbreviations used are: CaM, calmodulin; ADSI, the annular determinant of slow inactivation; CDI, Ca2+-dependent inactivation; I–V, current-voltage; PH, pleckstrin homology; PIP2, phosphatidylinositol bisphosphate; ECFP, enhanced cyan fluorescent protein; EYFP, enhanced yellow fluorescent protein; FRET, fluorescence resonance energy transfer. regulates Cav1.2 conductance by responding to Ca2+ binding that shuttles it between two CaM-binding sites in the proximal half of the α1C subunit C-terminal tail (1.Zühlke R.D. Pitt G.S. Deisseroth K. Tsien R.W. Reuter H. Nature. 1999; 399: 159-162Crossref PubMed Scopus (740) Google Scholar, 2.Pate P. Mochca-Morales J. Wu Y. Zhang J.-Z. Rodney G.G. Serysheva I.I. Williams B.Y. Anderson M.E. Hamilton S.L. J. Biol. Chem. 2000; 275: 39786-39792Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 3.Romanin C. Gamsjaeger R. Kahr H. Schaufler D. Carlson O. Abernethy D.R. Soldatov N.M. FEBS Lett. 2000; 487: 301-306Crossref PubMed Scopus (66) Google Scholar, 4.Dzhura I. Wu Y. Zhang R. Colbran R.J. Hamilton S.L. Anderson M.E. J. Physiol. (Lond.). 2003; 550: 731-738Crossref Scopus (17) Google Scholar). CaM signals Ca2+ for transcription activation (5.Kobrinsky E. Schwartz E. Abernethy D.R. Soldatov N.M. J. Biol. Chem. 2003; 278: 5021-5028Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) or Ca2+-induced intracellular Ca2+ release (6.Woo S.-H. Soldatov N.M. Morad M. J. Physiol. (Lond.). 2003; 552: 437-447Crossref Scopus (28) Google Scholar) by the voltage-gated rearrangement of the α1C subunit C terminus, thus linking Ca2+-dependent inactivation (CDI) and Ca2+ signal transduction (7.Soldatov N.M. Trends Pharmacol. Sci. 2003; 24: 167-171Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). With these voltage- and Ca2+-gated rearrangements, the role of the Cav1.2 cytoplasmic termini may be further defined when the α1C subunit tails are uncoupled from the channel regulation by transient immobilization in the plasma membrane. The association of α1C with the auxiliary α2δ and β subunits is important for the functional expression of Cav1.2 channels. The cytoplasmic β subunit binds to a conserved "α-interaction domain" in the α1C subunit cytoplasmic linker between transmembrane repeats I and II (8.Pragnell M. De Waard M. Mori Y. Tanabe T. Snutch T.P. Campbell K.P. Nature. 1994; 368: 67-70Crossref PubMed Scopus (552) Google Scholar, 9.De Waard M. Pragnell M. Campbell K.P. Neuron. 1994; 13: 495-503Abstract Full Text PDF PubMed Scopus (225) Google Scholar). The extracellular α2 subunit is bound via an SS bridge to its post-translationally cleaved transmembrane δ peptide (10.De Jongh K.S. Warner C. Catterall W.A. J. Biol. Chem. 1990; 265: 14738-14741Abstract Full Text PDF PubMed Google Scholar, 11.Jay S.D. Sharp A.H. Kahl S.D. Vedvick T.S. Harpold M.M. Campbell K.P. J. Biol. Chem. 1991; 266: 3287-3293Abstract Full Text PDF PubMed Google Scholar) that renders association with α1C. Both α2δ (12.Gurnett C.A. Dewaard M. Campbell K.P. Neuron. 1996; 16: 431-440Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 13.Felix R. Gurnett C.A. De Waard M. Campbell K.P. J. Neurosci. 1997; 17: 6884-6891Crossref PubMed Google Scholar, 14.Shirokov R. Ferreira G. Yi J. Ríos E. J. Gen. Physiol. 1998; 111: 807-823Crossref PubMed Scopus (49) Google Scholar) and β subunits (15.Birnbaumer L. Qin N. Olcese R. Tareilus E. Platano D. Costantin J. Stefani E. J. Bioenerg. Biomembr. 1998; 30: 357-375Crossref PubMed Scopus (201) Google Scholar, 16.Yamaguchi H. Hara M. Strobeck M. Fukasawa K. Schwartz A. Varadi G. J. Biol. Chem. 1998; 273: 19348-19356Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 17.Colecraft H.M. Alseikhan B. Takahashi S.X. Chaudhuri D. Mittman S. Yegnasubramanian V. Alvania R.S. Johns D.C. Marban E. Yue D.T. J. Physiol. (Lond.). 2002; 541: 435-452Crossref Scopus (187) Google Scholar, 18.Hullin R. Khan I.F.Y. Wirtz S. Mohacsi P. Varadi G. Schwartz A. Herzig S. J. Biol. Chem. 2003; 278: 21623-21630Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 19.Takahashi S.X. Mittman S. Colecraft H.M. Biophys. J. 2003; 84: 3007-3021Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) modulate the channel. In particular, β subunits affect the time course of the Ba2+ current decay up to 3-fold depending on the type of the β subunit. To study conformational rearrangements in the channel in response to depolarization, measurements of differential changes in fluorescence resonance energy transfer (FRET) between the cyan (ECFP) and yellow (EYFP) fluorescent proteins fused to the α1C and β subunit termini have been an effective approach. The current findings begin to specify the central features of conformational rearrangements associated with the transition of the channel from the resting (–90 mV) to the inactivated state of Cav1.2. With the (EYFP)N-α1C,77-(ECFP)C/β1a/α2δ channel as a model, FRET microscopy showed reversible voltage-gated rearrangements between the α1C,77 tails and pointed to a role for the C-terminal mobile tail in intracellular Ca2+ signal transduction (5.Kobrinsky E. Schwartz E. Abernethy D.R. Soldatov N.M. J. Biol. Chem. 2003; 278: 5021-5028Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Another study (20.Kobrinsky E. Kepplinger K.J.F. Yu A. Harry J.B. Kahr H. Romanin C. Abernethy D.R. Soldatov N.M. Biophys. J. 2004; 87: 844-857Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) characterized the voltage-gated rearrangements between the N-terminal tails of the α1C and β subunits associated with differential β subunit modulation of inactivation and demonstrated limited rearrangements of both N-tails with regard to the plasma membrane. To investigate further the role of voltage-gated mobility of the α1C,77 N-terminal tail for function of the Cav1.2 channel, here we have investigated the effects of N-terminal deletion or plasma membrane immobilization on Ca2+- and voltage-dependent inactivation, as well as β subunit regulation of the channel. Most interestingly, if the α1C/α2δ channel is assembled without a β subunit, either deletion or anchoring of the α1C,77 N-terminal tail results in high amplitude non-inactivating Ba2+ or Ca2+ currents. This provides a mechanism to explain how limited mobility of the α1C subunit N-terminal tail integrates the β subunit and the α1C subunit C terminus in inactivation of the channel. Molecular Biology—Reverse transcription-PCR cloning of human hippocampal α1C subunit (see Supplemental Material) showed substantial diversity of the transcripts because of alternative splicing. The exon-22 isoform (GenBank™ accession number Z34815) of the human hippocampus α1C subunit, known as α1C,77 (21.Soldatov N.M. Bouron A. Reuter H. J. Biol. Chem. 1995; 270: 10540-10543Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), was selected for this study because it was identified in other human tissues and cells. (EYFP)N-α1C,77, (PH-EYFP)N-α1C,77, α1C,77-(PH-ECFP)C, and (ECFP)N-PH and (EYFP)N-PH (22.van der Wal J. Habets R. Varnai P. Balla T. Jalink K. J. Biol. Chem. 2001; 276: 15337-15344Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) expression plasmids (N and C indicate the N and C terminus of a subunit, respectively) were prepared in the pcDNA3 vector for eukaryotic expression as described earlier (5.Kobrinsky E. Schwartz E. Abernethy D.R. Soldatov N.M. J. Biol. Chem. 2003; 278: 5021-5028Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) by using pEYFP and pECFP vectors (Clontech). To delete the N-terminal amino acids 2–120 of the α1C,77 subunit, the PCR product obtained by amplification of pHLCC77 (21.Soldatov N.M. Bouron A. Reuter H. J. Biol. Chem. 1995; 270: 10540-10543Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) with the sense 5′-tggatccgccaccATGGTCGAATGGAAACCATTTG-3′ and antisense 5′-AGCCATGATCCCATCATACATCAC-3′ primers (noncoding nucleotides are shown in lowercase letters) was digested by BamHI and SgrAI and ligated at the respective sites into 77-pcDNA3 (23.Soldatov 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). Nucleotide sequences of all PCR and ligation products were confirmed at the DNA sequencing facility of the University of Maryland. The β1a and α2δ subunits in pcDNA3 vector were prepared as described by Soldatov et al. (24.Soldatov N.M. Zhenochin S. AlBanna B. Abernethy D.R. Morad M. J. Membr. Biol. 2000; 177: 129-135Crossref PubMed Scopus (14) Google Scholar). Transient Expression in COS1 Cells—COS1 cells were maintained at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum (Invitrogen). For transient Ca2+ channel expression, cells were plated on poly-d-lysine-coated coverslips (MatTek) 18 h before transfection with cDNAs coding for the indicated α1C, β1a, and α2δ subunits of the Ca2+ channel (1:1:1, w/w) using an Effectene kit (Qiagen) according to the protocol described previously (20.Kobrinsky E. Kepplinger K.J.F. Yu A. Harry J.B. Kahr H. Romanin C. Abernethy D.R. Soldatov N.M. Biophys. J. 2004; 87: 844-857Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Transfected cells were visualized by ECFP or EYFP tags genetically fused to the α1C subunits. In some experiments, the Gαq Q209L mutant (The Guthrie Research Institute, Sayre, PA) was co-expressed with Ca2+ channel subunits. Electrophysiological Experiments—Whole-cell patch clamp recordings were performed at room temperature (20–22 °C) using the Axopatch 200B amplifier (Axon Instruments) 48–72 h after transfection. The extracellular bath solution contained the following (in mm): 100 NaCl, 20 BaCl2, 1 MgCl2, 10 glucose, 10 HEPES, adjusted to pH 7.4, with NaOH. Borosilicate glass pipettes were fire-polished and showed a typical resistance of 3–6 megohms when filled with pipette solution containing the following (in mm): 110 CsCl, 5 MgATP, 10 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, 20 tetraethylammonium, 0.2 cAMP, and 20 HEPES, adjusted to pH 7.4 with CsOH (24.Soldatov N.M. Zhenochin S. AlBanna B. Abernethy D.R. Morad M. J. Membr. Biol. 2000; 177: 129-135Crossref PubMed Scopus (14) Google Scholar). Capacitance compensation and series resistance were set at 60%. Currents were sampled at 2.5–5 kHz and filtered at 1 kHz. Voltage protocols were generated, and data were digitized, recorded, and analyzed using pClamp 8.1 software (Axon Instruments). The holding potential (Vh) was –90 mV, and test pulses were applied at 30-s intervals. Current-voltage (I-V) curves were obtained by step depolarizations to test potentials in the range of –60 to +50 mV (with 10-mV increments) applied from the holding potential. Statistical values are given as means ± S.E. Single channel recordings were carried out in cell-attached configurations at 24 °C in the bath solution containing the following (in mm): 110 l-aspartic acid, 20 KCl, 2 MgCl2, 2 EGTA, and 20 HEPES, adjusted to pH 7.4 with KOH. The pipette solution contained the following (in mm): 110 BaCl2, 5 HEPES, adjusted to pH 7.4 with NaOH. The pipettes were heat-polished and showed resistances of 3–4.5 megohms. Step depolarizations to the indicated potentials were applied from Vh = –90 mV for 1 s followed by inter-pulse intervals of 5 s. Currents were sampled at 40 kHz and low pass-filtered with a gauss filter at 500 Hz. Histograms with a bin size of 0.03 pA were fit with a sum of two gauss functions. Single channel data were analyzed using Clampfit 9.2 software (Axon Instruments) FRET measurements were combined with whole-cell patch clamp as described in detail by Kobrinsky et al. (5.Kobrinsky E. Schwartz E. Abernethy D.R. Soldatov N.M. J. Biol. Chem. 2003; 278: 5021-5028Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 20.Kobrinsky E. Kepplinger K.J.F. Yu A. Harry J.B. Kahr H. Romanin C. Abernethy D.R. Soldatov N.M. Biophys. J. 2004; 87: 844-857Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Images were recorded with a Hamamatsu digital camera C4742-95 mounted on the Nikon epifluorescent microscope TE200 (60 × 1.2 N.A. objective) equipped with an excitation 75-watt xenon lamp and multiple filter sets (Chroma Technology). Acquisition and analysis of FRET images were carried out with C-Imaging (Compix) and MetaMorph (Universal Imaging) software packages. Conditions for the measurements of corrected images of FRET between (ECFP)N-PH and (EYFP)N-α1C,77 in the absence or on the presence of the β1a subunit closely matched those described recently (20.Kobrinsky E. Kepplinger K.J.F. Yu A. Harry J.B. Kahr H. Romanin C. Abernethy D.R. Soldatov N.M. Biophys. J. 2004; 87: 844-857Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Our goal in this study was to reveal the role of the α1C,77 subunit C- and N-terminal tails in inactivation of the Cav1.2 channel and its regulation by β subunits. To simplify the interpretation of the results, in most of this series of experiments we used the protein kinase C-insensitive Cav1.2 assembled with the α1C,77 subunit identified in human hippocampus (see Supplemental Material). Our previous experiments (5.Kobrinsky E. Schwartz E. Abernethy D.R. Soldatov N.M. J. Biol. Chem. 2003; 278: 5021-5028Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 20.Kobrinsky E. Kepplinger K.J.F. Yu A. Harry J.B. Kahr H. Romanin C. Abernethy D.R. Soldatov N.M. Biophys. J. 2004; 87: 844-857Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 25.Harms G.S. Cognet L. Lommerse P.H.M. Blab G.A. Kahr H. Gamsjager R. Spaink H.P. Soldatov N.M. Romanin C. Schmidt T. Biophys. J. 2001; 81: 2639-2646Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) provided evidence that electrophysiological properties of the channel remained essentially unaltered by the N- and/or C-terminal fusion of the green fluorescent protein analogs to the α1C,77 subunit. Measurements of state-dependent FRET, as an indicator of the voltage-gated rearrangements of the ECFP/EYFP-labeled channel, have demonstrated significant mobility of the α1C subunit C-terminal tail and its crucial role for Ca2+ signal transduction, contrasting with a relatively weak β subunit dependent mobility of the α1C N terminus. Effect of Plasma Membrane Immobilization of the α1C C Terminus on CDI, Voltage-dependent Inactivation, and Ion Selectivity of the Channel—In the first set of experiments, the C terminus of the α1C,77 subunit was immobilized to the plasma membrane via the pleckstrin homology (PH) domain of phospholipase Cδ1. To prepare the plasmid encoding α1C,77-(PH-ECFP)C, the PH domain was genetically fused to the last C-terminal Leu-2138 residue of the α1C,77 subunit, and the reading frame was completed with the ECFP-coding sequence. The β subunit-deficient α1C,77-(ECFP)C/α2δ channel was predominantly localized in the cytoplasm and did not show substantial membrane targeting (for details, see Harry et al. (26.Harry J.B. Kobrinsky E. Abernethy D.R. Soldatov N.M. J. Biol. Chem. 2004; 279: 46367-46372Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar)). The C-terminal fusion of the PH domain was sufficient to direct the plasma membrane insertion of the channel, as one can see from the distribution of the ECFP fluorescence across the cell (Fig. 1A, inset). When co-expressed in COS1 cells with the α2δ subunit but in the absence of β subunits (Fig. 1A), the α1C,77-(PH-ECFP)C/α2δ channel generated only a minute Ba2+ current in response to a +10-mV depolarization applied from Vh = –90 mV. However, when α2δ and α1C,77-(PH-ECFP)C were co-expressed with the β1a subunit (Fig. 1B), the amplitude of the Ba2+ current increased severalfold, and the current decay exhibited a distinctly prolonged plateau at approximately half-maximum of the current. Fig. 1C shows a set of the representative traces of the Ba2+ current evoked by 600-ms test pulses in the range of 0 to +50 mV (10-mV increments) applied from Vh = –90 mV. The corresponding averaged I-V relation is presented in Fig. 1D. The most prominent feature of the Ba2+ current through the channel with the plasma membrane-anchored α1C subunit C terminus was the sustained component of the current (Fig. 1D, open circles in the I-V curve) that, in a wide range of the current-evoking potentials, composed 35.5 ± 3.5% (n = 7) of the total Ba2+ current (from 31.4% at –10 mV to 56.1% at +50 mV). The sustained current was preceded by a rapidly inactivating component with the fast time constant τf of 20.0 ± 5.0 ms (at +20 mV; n = 9). Overall, the anchoring of the α1C subunit C terminus in the plasma membrane accelerated fast inactivation and inhibited slow inactivation of the Ba2+ current. Release of the α1C,77 subunit C-tail could be stimulated by the hydrolysis of the PH domain PIP2 upon activation of phospholipase C. In previous studies, we demonstrated that activation of PIP2 hydrolysis by epidermal growth factor-mediated stimulation of the co-expressed epidermal growth factor receptors helped to fully restore the CDI and voltage-dependent slow inactivation of the channel (5.Kobrinsky E. Schwartz E. Abernethy D.R. Soldatov N.M. J. Biol. Chem. 2003; 278: 5021-5028Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Because the epidermal growth factor-mediated recovery was transient and additionally complicated by inhibition of the channel activity (27.Wu L. Buer C.S. Zhen X.-G. Xie C. Yang J. Nature. 2002; 419: 947-952Crossref PubMed Scopus (189) Google Scholar), we analyzed the effects of the co-expression of the constitutively active mutant (Q209L) of the Gαq protein that depletes the plasma membrane PIP2 (e.g. see Howes et al. (28.Howes A.L. Arthur J.F. Zhang T. Miyamoto S. Adams J.W. Dorn II, G.W. Woodcock E.A. Brown J.H. J. Biol. Chem. 2003; 278: 40343-40351Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar)). Fig. 1E (inset) shows the fluorescence image of a COS1 cell co-expressing the α1C,77-(PH-ECFP)C, α2δ, and β1a subunits, the Gαq Q209L mutant, and representative traces of the Ba2+ current generated in response to step depolarizations in the range of +10 to +40 mV applied from Vh = –90 mV. Co-expression of the Gαq Q209L mutant shifted inactivation of the channels to the normal phenotype with a prominent slow component. Single exponential fitting shows the time constant of the Ba2+ current decay of 200 ± 38 ms (at +10 mV; n = 9) and the lack of sustained component. The inability of the channel with the immobilized C-terminal tail to complete inactivation was corroborated by the analysis of steady-state inactivation properties (Fig. 1F). Approximately 70% of the Ba2+ current through the α1C,77-(PH-ECFP)C/α2δ/β1a channel evoked by a +10-mV depolarization remained non-inactivated after a depolarizing prepulse in a range of –10 to +50 mV was applied from Vh = –90 mV prior to the +10-mV test pulse (Fig. 1F, closed circles). The release of the α1C,77 C-tail restored inactivation of the channel, as can be seen from a comparison of the Ba2+ current decay (Fig. 1E) and steady-state inactivation curves recorded in the absence and in the presence of the constitutively active Gαq Q209L mutant (Fig. 1F). Cav1.2 channels classically inactivate by a combination of the voltage- and Ca2+-dependent mechanisms. One of the main consequences of the replacement of Ba2+ for Ca2+ as the charge carrier is an acceleration of the macroscopic current decay or CDI (29.Zhou J. Olcese R. Qin N. Noceti F. Birnbaumer L. Stefani E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2301-2305Crossref PubMed Scopus (85) Google Scholar). However, this was not found to hold true for the C-terminal tail-anchored Ca2+ channel. Indeed, the representative traces of the Ca2+ current through the α1C,77-(PH-ECFP)C/α2δ/β1a channel (Fig. 1G), recorded from the same cell as Ba2+ currents in Fig. 1C, show both inactivating and sustained components of the decay. Similar to the Ba2+ current, the large sustained Ca2+ current components lasted for the duration of depolarization at all indicated test pulses (+20 to +50 mV). Although the fast component of inactivation was not as prominent as in the case of the Ba2+ current, the average fraction of the sustained currents (44.9 ± 2.3%, from 33.3% at –10 mV to 48.6% at +50 mV, Fig. 1H) was essentially the same as with Ba2+ as the charge carrier. All these features of the C-terminal tail-anchored α1C,77-(PH-ECFP)C channel closely matched the phenotype of the α1C,IS-IV channel, which has the slow inactivation mechanism inhibited by the specific mutation in the cytoplasmic pore region (30.Shi C. Soldatov N.M. J. Biol. Chem. 2002; 277: 6813-6821Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The I-V relations for the Ba2+- and Ca2+-conducting α1C,77-(PH-ECFP)C channel show a number of common patterns (Fig. 1, D and H). Immobilization of the α1C,77 subunit C-terminal tail caused a shift in activation of ion conductance by 10–15 mV to more positive potentials. Currents reached the peak of the I-V relationship at +20 (Ca2+) or +30 mV (Ba2+), exhibiting a 10–20-mV shift of the maximum toward more positive voltages as compared with the α1C,77-(ECFP)C/α2δ/β1a channel. The apparent reversal potential was also notably changed. Both the Ba2+ and Ca2+ currents reversed direction at much higher voltages than in the wild-type channel. Although this effect was not investigated in detail, it may be due to altered ion selectivity and/or decreased permeability to Cs+ ions (introduced in electrodes) in the outward direction that contributed significantly to the apparent reversal potential of the wild-type channel (≈ +65 mV) (31.Zhou Z. Bers D.M. J. Physiol. (Lond.). 2000; 523: 57-66Crossref Scopus (17) Google Scholar). Release of the α1C,77-(PH-ECFP)C subunit C-terminal tail by co-expression of the Gαq Q209L mutant reversed these changes in the I-V relationship to parameters that are more characteristic for the α1C,77-(ECFP)C/α2δ/β1a channel, including ion selectivity and the peak current voltage (Fig. 1D, filled squares). The permeability of the wild type α1C,77 channel to Ba2+ is on average 2.8 times greater than that of the Ca2+ ions (24.Soldatov N.M. Zhenochin S. AlBanna B. Abernethy D.R. Morad M. J. Membr. Biol. 2000; 177: 129-135Crossref PubMed Scopus (14) Google Scholar, 30.Shi C. Soldatov N.M. J. Biol. Chem. 2002; 277: 6813-6821Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Immobilization of the α1C subunit C-tail reduced the difference between the maximum amplitudes of the Ba2+ and Ca2+ currents (compare traces in Fig. 1, C and G), again indicating that the anchoring of the α1C subunit C-terminal tail may affect the ion conductance of the channel. Taken together, these data support the model (7.Soldatov N.M. Trends Pharmacol. Sci. 2003; 24: 167-171Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) that links CDI and slow inactivation of the Cav1.2 channel to specific folding of the α1C subunit C terminus vis à vis the cytoplasmic pore region. β Subunit Facilitation of the Cav1.2 Channel Gating Is Revealed by Immobilization of the α1C Subunit N-terminal Tail—In the second set of experiments, the N-terminal tail of the (EYFP)N-α1C,77 subunit was anchored to the plasma membrane via the PH domain. The (PH-EYFP)N-α1C,77 channel was expressed in COS1 cells in different combinations with auxiliary β1a and α2δ subunits. Fig. 2 shows a collection of representative traces of the Ba2+ current elicited by depolarization to +10 mV from Vh = –90 mV in a set of COS1 cells of approximately similar size. The insets in Fig. 2 are fluorescent images of the expressing cells showing subcellular localization of the EYFP-tagged α1C subunits. Confirming earlier data (32.Meir A. Bell D.C. Stephens G.J. Page K.M. Dolphin A.C. Biophys. J. 2000; 79: 731-746Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 33.Cornet V. Bichet D. Sandoz G. Marty I. Brocard J. Bourinet E. Mori Y. Villaz M. De Waard M. Eur. J. Neurosci. 2002; 16: 883-895Crossref PubMed Scopus (45) Google Scholar), COS1 cells did not show appreciable endogenous expression of Cav1.2 (Fig. 2A). Transfection of COS1 cells with a mixture of cDNAs coding for the (EYFP)N-α1C,77, β1a and α2δ subunits (Fig. 2B) renders the current with characteristics (Fig. 3, curves 3) closely resembling those of the wild-type channel (24.Soldatov N.M. Zhenochin S. AlBanna B. Abernethy D.R. Morad M. J. Membr. Biol. 2000; 177: 129-135Crossref PubMed Scopus (14) Google Scholar). Anchoring of the α1C subunit N terminus by co-expression of (PH-EYFP)N-α1C,77 with the β1a and α2δ subunits (Fig. 2C) accelerated the kinetics of inactivation of the Ba2+ current by ∼15% (5.Kobrinsky E. Schwartz E. Abernethy D.R. Soldatov N.M. J. Biol. Chem. 2003; 278: 5021-5028Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). An acceleration of inactivation was also seen when the PH domain was separately co-expressed with the (EYFP)N-α1C,77, β1a, and α2δ subunits (Fig. 2D). Making no assumptions about the nature of these variations, we investigated the effect of the deletion of the β1a subunit from the expressed constituents of the channel.Fig. 3Effect of the β subunit on inactivation properties of the Ca2+ channel with the N terminus of the α1C subunit immobilized in the plasma membrane. The (PH-EYFP)N-α1C,77 and α2δ subunits were co-expressed in COS1 cells with (curve 1) or without β1a (curve 2). The channel assembled of the (EYFP)N-α1C,77, α2δ and β1a subunits was used as control (curve 3). A, superimposed curves of the Ba2+ current evoked by stepwise depolarization to +20 mV applied from Vh = –90 mV. Traces were normalized to the same current amplitude. B, the I-V relationships for the channels composed of (curve 1) (PH-EYFP)N-α1C,77/α2δ/β1a (n = 6), (curve 2 (PH-EYFP)N-α1C,77/α2δ (n = 13), and (curve 3) (EYFP)N-α1C,77/α2δ/β1a (n = 6). Ba2+ currents were measured with 15-s intervals between 600-ms test pulses in the range of –60 to +50 mV applied from Vh = –90 mV. C, an averaged steady-state curves recorded with the channels composed of (curve 1) (PH-EYFP)N-α1C,77/α2δ/β
Referência(s)