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J. Biol. Chem., Vol. 280, Issue 13, 12474-12485, April 1, 2005

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Differential Role of the _1C Subunit Tails in Regulation of the Ca_v1.2 Channel by Membrane Potential, Subunits, and Ca²+ Ions^*

Evgeny Kobrinsky, Swasti Tiwari, Victor A. Maltsev, Jo Beth Harry, Edward Lakatta, Darrell R. Abernethy, and Nikolai M. Soldatov¶

From the Laboratory of Clinical Investigation and Cardiovascular Science, NIA, National Institutes of Health, Baltimore, Maryland 21224

Received for publication, October 26, 2004 , and in revised form, January 18, 2005.

	ABSTRACT

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Voltage-gated Ca_v1.2 channels are composed of the pore-forming _1C and auxiliary and ₂ subunits. Voltage-dependent conformational rearrangements of the _1C subunit C-tail have been implicated in Ca²⁺ signal transduction. In contrast, the _1C 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 _1C subunit C-tail in the plasma membrane inhibits Ca²⁺-dependent and slow voltage-dependent inactivation. Both ₂ and subunits remain essential for the functional channel. In contrast, if _1C subunits with are expressed ₂ but in the absence of a subunit, plasma membrane anchoring of the _1C N terminus or its deletion inhibit both voltage- and Ca²⁺-dependent inactivation of the current. The following findings all corroborate the importance of the _1C N-tail/ interaction: (i) co-expression of restores inactivation properties, (ii) release of the _1C N terminus inhibits the -deficient channel, and (iii) voltage-gated mobility of the _1C N-tail vis à vis the plasma membrane is increased in the -deficient (silent) channel. Together, these data argue that both the _1C N- and C-tails have important but different roles in the voltage- and Ca²⁺-dependent inactivation, as well as subunit modulation of the channel. The _1C N-tail may have a role in the channel trafficking and is a target of the subunit modulation. The subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the _1C C and N termini, subunit, and the cytoplasmic pore region confers the multifactorial regulation of Ca_v1.2 channels.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Ca_v1.2 channels are known for their key role in triggering Ca²⁺ signaling in a wide variety of cells. Calmodulin (CaM)¹ regulates Ca_v1.2 conductance by responding to Ca²⁺ binding that shuttles it between two CaM-binding sites in the proximal half of the _1C subunit C-terminal tail (1–4). CaM signals Ca²⁺ for transcription activation (5) or Ca²⁺-induced intracellular Ca²⁺ release (6) by the voltage-gated rearrangement of the _1C subunit C terminus, thus linking Ca²⁺-dependent inactivation (CDI) and Ca²⁺ signal transduction (7). With these voltage- and Ca²⁺-gated rearrangements, the role of the Ca_v1.2 cytoplasmic termini may be further defined when the _1C subunit tails are uncoupled from the channel regulation by transient immobilization in the plasma membrane.

The association of _1C with the auxiliary ₂ and subunits is important for the functional expression of Ca_v1.2 channels. The cytoplasmic subunit binds to a conserved "-interaction domain" in the _1C subunit cytoplasmic linker between transmembrane repeats I and II (8, 9). The extracellular ₂ subunit is bound via an SS bridge to its post-translationally cleaved transmembrane peptide (10, 11) that renders association with _1C. Both ₂ (12–14) and subunits (15–19) modulate the channel. In particular, subunits affect the time course of the Ba²⁺ current decay up to 3-fold depending on the type of the 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 _1C and 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 Ca_v1.2. With the (EYFP)_N-_1C,77-(ECFP)_C/_1a/₂ channel as a model, FRET microscopy showed reversible voltage-gated rearrangements between the _1C,77 tails and pointed to a role for the C-terminal mobile tail in intracellular Ca²⁺ signal transduction (5). Another study (20) characterized the voltage-gated rearrangements between the N-terminal tails of the _1C and subunits associated with differential 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 _1C,77 N-terminal tail for function of the Ca_v1.2 channel, here we have investigated the effects of N-terminal deletion or plasma membrane immobilization on Ca²⁺- and voltage-dependent inactivation, as well as subunit regulation of the channel. Most interestingly, if the _1C/₂ channel is assembled without a subunit, either deletion or anchoring of the _1C,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 _1C subunit N-terminal tail integrates the subunit and the _1C subunit C terminus in inactivation of the channel.

	MATERIALS AND METHODS

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Molecular Biology—Reverse transcription-PCR cloning of human hippocampal _1C subunit (see Supplemental Material) showed substantial diversity of the transcripts because of alternative splicing. The exon-22 isoform (GenBank^TM accession number Z34815 [GenBank] ) of the human hippocampus _1C subunit, known as _1C,77 (21), was selected for this study because it was identified in other human tissues and cells. (EYFP)_N-_1C,77, (PH-EYFP)_N-_1C,77, _1C,77-(PH-ECFP)_C, and (ECFP)_N-PH and (EYFP)_N-PH (22) 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) by using pEYFP and pECFP vectors (Clontech). To delete the N-terminal amino acids 2–120 of the _1C,77 subunit, the PCR product obtained by amplification of pHLCC77 (21) 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). Nucleotide sequences of all PCR and ligation products were confirmed at the DNA sequencing facility of the University of Maryland. The _1a and ₂ subunits in pcDNA3 vector were prepared as described by Soldatov et al. (24).

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 _1C, _1a, and ₂ subunits of the Ca²⁺ channel (1:1:1, w/w) using an Effectene kit (Qiagen) according to the protocol described previously (20). Transfected cells were visualized by ECFP or EYFP tags genetically fused to the _1C subunits. In some experiments, the G_q 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). 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 (V_h) 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 V_h = –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, 20). Images were recorded with a Hamamatsu digital camera C4742-95 mounted on the Nikon epifluorescent microscope TE200 (60 x 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-_1C,77 in the absence or on the presence of the _1a subunit closely matched those described recently (20).

	RESULTS

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Our goal in this study was to reveal the role of the _1C,77 subunit C- and N-terminal tails in inactivation of the Ca_v1.2 channel and its regulation by subunits. To simplify the interpretation of the results, in most of this series of experiments we used the protein kinase C-insensitive Ca_v1.2 assembled with the _1C,77 subunit identified in human hippocampus (see Supplemental Material). Our previous experiments (5, 20, 25) 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 _1C,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 _1C subunit C-terminal tail and its crucial role for Ca²⁺ signal transduction, contrasting with a relatively weak subunit dependent mobility of the _1C N terminus.

Effect of Plasma Membrane Immobilization of the _1C C Terminus on CDI, Voltage-dependent Inactivation, and Ion Selectivity of the Channel—In the first set of experiments, the C terminus of the _1C,77 subunit was immobilized to the plasma membrane via the pleckstrin homology (PH) domain of phospholipase C1. To prepare the plasmid encoding _1C,77-(PH-ECFP)_C, the PH domain was genetically fused to the last C-terminal Leu-2138 residue of the _1C,77 subunit, and the reading frame was completed with the ECFP-coding sequence. The subunit-deficient _1C,77-(ECFP)_C/₂ channel was predominantly localized in the cytoplasm and did not show substantial membrane targeting (for details, see Harry et al. (26)). 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 ₂ subunit but in the absence of subunits (Fig. 1A), the _1C,77-(PH-ECFP)_C/₂ channel generated only a minute Ba²⁺ current in response to a +10-mV depolarization applied from V_h = –90 mV. However, when ₂ and _1C,77-(PH-ECFP)_C were co-expressed with the _1a 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 V_h = –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 _1C 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 _f of 20.0 ± 5.0 ms (at +20 mV; n = 9). Overall, the anchoring of the _1C subunit C terminus in the plasma membrane accelerated fast inactivation and inhibited slow inactivation of the Ba²⁺ current.

View larger version (31K): [in this window] [in a new window]   FIG. 1. Inhibition of CDI and slow voltage-dependent inactivation by immobilization of the 1C subunit C-terminal tail in the plasma membrane. The 1C,77-(PH-ECFP)C and 2 subunits were co-expressed in COS1 cells in the absence (A) or in the presence (B) of the 1a subunit. Epifluorescent images of the expressing cells (insets) showing distribution of 1C,77-(PH-ECFP)C were obtained with the cyan fluorescent protein filter. Representative Ba2+ currents were recorded in cells of approximately the same diameter in response to a +10-mV test pulse applied for 600 ms from Vh = –90 mV. Dotted lines indicate zero current. C, typical traces of the Ba2+ current through the 1C,77-(PH-ECFP)C/2/1a channel evoked by indicated test pulses applied from Vh = –90 mV. D, I-V relations (n = 13) measured for the peak Ba2+ currents (closed circles) and sustained currents (mean values determined in the range of 400–500 ms, open circles). The averaged curves were normalized to the maximum current. Control (filled squares) for the channel co-expressed with the Gq Q209L mutant was fitted (smooth line) by equation IBa = Gmax (V – Erev)/{1 + exp((V – V0.5)/kI-V)}, where Gmax is maximum conductance; Erev = 68.9 ± 4.6 mV is the reversal potential; V0.5 = –9.5 ± 2.4 mV is voltage at 50% of IBa activation; and kI-V =–10.2 ± 1.2 is slope factor. E, epifluorescent image of the expressing cell and representative traces of the Ba2+ current evoked by indicated test pulses applied from Vh = –90 mV. F, steady-state inactivation curves for Ba2+ currents through the 1C,77-(PH-ECFP)C/2/1a channel assembled in the absence (filled circles; n = 3) and presence (filled squares; n = 5) of the constitutively active Gq Q209L mutant. A 1-s conditioning prepulse was applied from Vh =–90 mV (10-mV increments up to +60 mV) followed by a 100-ms test pulse to +10 mV. The intervals between each cycle were 15 s. The peak current amplitudes in each curve were normalized to the maximum value determined in the range of –60 to +50 mV. The Gq Q209L mutant control (smooth line) was fitted by Boltzmann function: IBa = A + B/{1 + exp((V – V0.5)/k)}, were A and B are fractions of non-inactivating (15.1 ± 2.4%) and inactivating channels, respectively; V is the conditioning prepulse voltage; V0.5 = –8.6 ± 1.7 mV is the voltage at half-maximum of inactivation, and k = 15.7 ± 1.9 is a slope factor. G, typical traces of Ca2+ currents through the 1C,77-(PH-ECFP)C/2/1a channel activated by indicated test pulses applied from Vh =–90 mV. H, averaged I-V relationships (normalized to the maximum current) for the peak (filled circles) and sustained (determined as in D, open circles) Ca2+ currents (n = 10). Scaling bars, 3 µm.

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 _1C,77-(PH-ECFP)_C/₂/_1a 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 V_h = –90 mV prior to the +10-mV test pulse (Fig. 1F, closed circles). The release of the _1C,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 G_q Q209L mutant (Fig. 1F).

Ca_v1.2 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). 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 _1C,77-(PH-ECFP)_C/₂/_1a 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 _1C,77-(PH-ECFP)_C channel closely matched the phenotype of the _1C,IS-IV channel, which has the slow inactivation mechanism inhibited by the specific mutation in the cytoplasmic pore region (30).

The I-V relations for the Ba²⁺- and Ca²⁺-conducting _1C,77-(PH-ECFP)_C channel show a number of common patterns (Fig. 1, D and H). Immobilization of the _1C,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 _1C,77-(ECFP)_C/₂/_1a 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). Release of the _1C,77-(PH-ECFP)_C subunit C-terminal tail by co-expression of the G_q Q209L mutant reversed these changes in the I-V relationship to parameters that are more characteristic for the _1C,77-(ECFP)_C/₂/_1a channel, including ion selectivity and the peak current voltage (Fig. 1D, filled squares). The permeability of the wild type _1C,77 channel to Ba²⁺ is on average 2.8 times greater than that of the Ca²⁺ ions (24, 30). Immobilization of the _1C 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 _1C subunit C-terminal tail may affect the ion conductance of the channel. Taken together, these data support the model (7) that links CDI and slow inactivation of the Ca_v1.2 channel to specific folding of the _1C subunit C terminus vis à vis the cytoplasmic pore region.

Subunit Facilitation of the Ca_v1.2 Channel Gating Is Revealed by Immobilization of the _1C Subunit N-terminal Tail—In the second set of experiments, the N-terminal tail of the (EYFP)_N-_1C,77 subunit was anchored to the plasma membrane via the PH domain. The (PH-EYFP)_N-_1C,77 channel was expressed in COS1 cells in different combinations with auxiliary _1a and ₂ subunits. Fig. 2 shows a collection of representative traces of the Ba²⁺ current elicited by depolarization to +10 mV from V_h = –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 _1C subunits. Confirming earlier data (32, 33), COS1 cells did not show appreciable endogenous expression of Ca_v1.2 (Fig. 2A). Transfection of COS1 cells with a mixture of cDNAs coding for the (EYFP)_N-_1C,77, _1a and ₂ subunits (Fig. 2B) renders the current with characteristics (Fig. 3, curves 3) closely resembling those of the wild-type channel (24). Anchoring of the _1C subunit N terminus by co-expression of (PH-EYFP)_N-_1C,77 with the _1a and ₂ subunits (Fig. 2C) accelerated the kinetics of inactivation of the Ba²⁺ current by 15% (5). An acceleration of inactivation was also seen when the PH domain was separately co-expressed with the (EYFP)_N-_1C,77, _1a, and ₂ subunits (Fig. 2D). Making no assumptions about the nature of these variations, we investigated the effect of the deletion of the _1a subunit from the expressed constituents of the channel.

View larger version (32K): [in this window] [in a new window]   FIG. 2. Effect of immobilization of the 1C N terminus on Ba2+ currents in Cav1.2 of different subunit composition. Shown are representative traces of the Ba2+ current activated by +10-mV test pulses applied from Vh = –90 mV. On the right from each trace are epifluorescent images of the expressing cells showing distribution of (EYFP)N-1C,77 and obtained with the yellow fluorescent protein filter. A, nontransfected COS1 cell. B, EYFP-tagged prototype of the wild type conventional channel composed of the (EYFP)N-1C,77, 1a, and 2 subunits. C, effect of the 1C N-terminal tail anchoring to the plasma membrane via fused PH domain on the (PH-EYFP)N-1C,77/1a/2 channel activity. D, effect of co-expression of (EYFP)N-PH on Cav1.2 composed of the 1C,77, 1a, and 2 subunits. E–G, lack of the Ca2+ channel activity in COS1 cells expressing the (EYFP)N-1C,77 (E), (PH-EYFP)N-1C,77 (F), or (EYFP)N-1C,77 + 2 subunits (G) in the absence of the subunit. H, non-inactivating Ba2+ current rendered in the presence of 2 by a deletion of the subunit and immobilization of the 1C,77 N terminus. I, inhibition of the (PH-EYFP)N-1C,77/2 channel activity by constitutive expression of the Gq Q209L mutant precluding immobilization of the 1C,77 N terminus. Scaling bars, 3 µm.

View larger version (14K): [in this window] [in a new window]   FIG. 3. Effect of the subunit on inactivation properties of the Ca2+ channel with the N terminus of the 1C subunit immobilized in the plasma membrane. The (PH-EYFP)N-1C,77 and 2 subunits were co-expressed in COS1 cells with (curve 1) or without 1a (curve 2). The channel assembled of the (EYFP)N-1C,77, 2 and 1a 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-1C,77/2/1a (n = 6), (curve 2 (PH-EYFP)N-1C,77/2 (n = 13), and (curve 3) (EYFP)N-1C,77/2/1a (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-1C,77/2/1a (n = 3), (curve 2 (PH-EYFP)N-1C,77/2 (n = 6), and (curve 3) (EYFP)N-1C,77/2/1a (n = 6). The indicated 1-s conditioning pulses were applied from Vh = –90 mV and followed by the 100-ms test pulse to +10 mV. Interval between pulses was 15 s. Recorded peak currents were normalized to the maximum value determined in the range between –60 and +40 mV. Curves 1 and 3 were fitted by Boltzmann function.

Activation and Inactivation Properties of the (PH-EYFP)_N-_1C,77/₂ Channel—The effects of the _1C subunit N-tail immobilization in the plasma membrane on electrophysiological properties of the channel are presented in Fig. 3. For comparison with the channel that has the _1C subunit N-terminal tail EYFP-tagged but not anchored, Fig. 3, A–C, includes data for Ba²⁺ currents through the (EYFP)_N-_1C,77/_1a/₂ channel (curves 3). Fig. 3A shows superimposed traces of Ba²⁺ currents (normalized to the same amplitude) through the (PH-EYFP)_N-_1C,77/_1a/₂ channel (trace 1), (PH-EYFP)_N-_1C,77/₂ channel (trace 2), and the control channel (trace 3). When co-expressed with the _1a and ₂ subunits, the (PH-EYFP)_N-_1C,77 channel showed notable acceleration of inactivation (Fig. 3A, trace 1) compared with the control. However, expression of the same channel in the absence of the subunit (Fig. 3A, trace 2) significantly delayed activation and inhibited inactivation of the current. A single-exponential fit of the activation time course (+20 mV) showed a substantial increase in the time constant of activation (_a) of the Ba²⁺ current through the subunit-deficient (PH-EYFP)_N-_1C,77/₂ channel (34.0 ± 10.5 ms, n = 13) as compared with the (PH-EYFP)_N-_1C,77/_1a/₂ channel (_a = 2.4 ± 0.3 ms; n = 6; p < 0.005). The respective I-V relationships are shown in Fig. 3B. The voltage-dependent characteristics, obtained from the fit of the I-V curve for the control (EYFP)_N-_1C,77/_1a/₂ channel, were typical for the L-type Ca²⁺ channel current, including E_rev = 60 ± 2 mV, V_0.5 = 0.02 ± 0.95 mV, and k_I-V = –7.8 ± 0.5 (n = 6). The plasma membrane immobilization of the _1C,77 subunit N-terminal tail shifted the voltage dependence of activation (V_0.5) to more negative potentials by –15.5 ± 2.4 (+_1a; n = 6) and –23 ± 4.5 mV (–_1a; n = 13). The threshold of activation of the Ba²⁺ current was shifted by 10 mV toward more negative voltages in the -deficient channel (Fig. 3B, filled circles) as compared with the (PH-EYFP)_N-_1C,77/_1a/₂ channel (open circles). In the -deficient channel, the voltage that elicited the maximum Ba²⁺ current (+10 mV) was shifted by 10 mV to more positive potentials. However, the most notable change was the increase of the apparent reversal potential in both (PH-EYFP)_N-_1C,77/₂± _1a channels. A similar effect was also induced by immobilization of the _1C subunit C-terminal tail (Fig. 1) and may be a result of altered ion selectivity.

To characterize further the inactivation characteristics of the Ba²⁺ current through the (PH-EYFP)_N-_1C,77/₂± _1a channels, we measured steady-state inactivation curves by using a two-step voltage clamp protocol (Fig. 3C). Steady-state inactivation properties point to differences in voltage dependence of inactivation of the Ba²⁺ current as compared with the control (EYFP)_N-_1C,77/_1a/₂ channel (Fig. 3C, curve 3). Voltage at the half-maximum of inactivation was shifted from –7.9 ± 0.7 mV (n = 6) in control to –15.9 ± 3.4 (n = 3) in the (PH-EYFP)_N-_1C,77/_1a/₂ channel (Fig. 3C, curve 1). Although the voltage dependence of availability (20.1 ± 1.5%) was not significantly changed by the plasma membrane immobilization of the _1C,77 N-terminal tail (21.0 ± 5.9%), the slope k of the steady-state inactivation curve (fitted by the Boltzmann equation) decreased from 6.3 ± 0.6 to 13.5 ± 3.8 (p < 0.05). Thus, cooperativity in voltage gating leading to inactivation is significantly affected by the plasma membrane immobilization of the _1C subunit N-terminal tail. Steady-state inactivation analysis also confirmed that the voltage dependence of inactivation of the subunit-deficient channel is inhibited in the range of conditioning pulses of up to +60 mV (Fig. 3C, curve 2).

The single channel analysis corroborated the macroscopic data (Fig. 4, A and B). Recordings of the Ba²⁺ current in the cell-attached configuration (110 mM Ba²⁺) revealed rarely occurring activations of the (PH-EYFP)_N-_1C,77/₂ channel that were characterized by low probabilities of both opening and closing (Fig. 4B). Openings of the channel were preceded by a large number of blank traces. The loss of inactivation of this channel was evidenced by continuous bursting of a single Ca²⁺ channel activity during long lasting depolarizations (mean burst length was 154 ± 18 ms, n = 39). In the presence of the subunit (Fig. 4A), the appearance of bursts was as low as 1 burst for 100 traces, whereas in the absence of the subunit, we observed bursts almost in every trace with the channel activity (Fig. 4B). The first latency was significantly prolonged ( = 55.4 ms) compared with the control (EYFP)_N-_1C,77/_1a/₂ channel ( = 28.4 ms). This explains the delay in activation of the whole-cell current in the anchored (PH-EYFP)_N-_1C,77/₂ channel.

View larger version (55K): [in this window] [in a new window]   FIG. 4. Effects of the 1C,77 subunit N terminus uncoupling by anchoring or deletion on single channel Ba2+ currents through the subunit-deficient Ca2+ channels expressed in COS1 cells. Ba2+ currents were measured in 110 mM Ba2+ and high K+ solution at a stimulation rate of 0.1 Hz. A and B, effect of anchoring of the 1C,77 subunit N terminus in the plasma membrane with (A) and without 1a (B) subunit expressed. (PH-EYFP)N-1C,77/2 channel current was activated by a test pulse to 0 mV. Insets show detailed single channel activity. Single channel averages are depicted in gray under the original traces in A and B with exponential fit and mean level, respectively, (n = 74 and n = 34). Respective first latency histograms are also shown with their exponential fits. C and D, effect of the 1C,77 subunit N terminus deletion with (C) and without 1a (D) subunit expressed. The N-1C,77/2 channel current was activated by a test pulse to –10 mV. Length of bursts was 234.7 ± 31.6 ms, (n = 40) (C); and 361.9 ± 70.3 ms, (n = 23, p < 0.05) (D).

View larger version (21K): [in this window] [in a new window]   FIG. 5. Lack of inactivation of the Ca2+ current through the subunit-deficient Cav1.2 channel with the N-terminal tail of the 1C subunit immobilized in the plasma membrane. The (PH-EYFP)N-1C,77 and 2 subunits were co-expressed in COS1 cells in the absence (A–C) or in the presence (D) of the constitutively active Gq Q209L mutant to enforce (A and B) or to prevent (D) the PH domain-mediated plasma membrane-anchoring of the 1C,77 subunit N-tail. In control (C), co-expression of the (PH-EYFP)N-1C,77 and 2 subunits with 1a restored inactivation. Ca2+ currents were elicited by a series of 600-ms test pulses to the indicated voltages in the range of –20 to +50 mV (A) or by a +20-mV depolarization for 30 s (B) or 600 ms (C and D) applied from Vh = –90 mV.

View larger version (21K): [in this window] [in a new window]   FIG. 6. Effect of the subunit on Ca2+ currents through the Ca2+ channel with a genetically deleted N terminus of the 1C subunit. The N-N 1C,77 and 2 subunits were co-expressed in COS1 in cells the presence (A) or in the absence (B) of the 1a subunit. Ca2+ currents were evoked by a +10-mV test pulse applied from Vh = –90 mV. C, the averaged normalized I-V relationships of the Ca2 h+current through the -1C,77/2 channel in the presence (filled n, = 11) or absence (open circles circles, n = 7) of the 1a subunit. Currents were measured with 30-s intervals between 0.6-s test pulses in the range of –60 to +50 mV applied with 10-mV increments from Vh = –90 mV. Fluorescent images were recorded with (ECFP)N-1a (A, panel a), N-1C,77-(ECFP)C (A and B, panels b), and (ECFP)N-2 (B, panel a) that retain full functional activity (for details see Ref. 20). Scale bars, 3 µm.

Effect of the Subunit on the Voltage-gated Mobility of the _1C Subunit N-terminal Tail—Finally, we assessed whether the subunit affects the voltage-gated mobility of the _1C subunit N-terminal tail. The (EYFP)_N-_1C,77/₂ channel was co-expressed with the (ECFP)_N-PH in the absence (Fig. 7A) or in the presence (Fig. 7B) of the _1a subunit (for arrangement of fluorophores and subunits, see panels b). In this experiment, the EYFP-labeled N-terminal tail of the _1C,77 subunit reports its state-dependent position to the FRET partner, ECFP, fused to the plasma membrane-trapped PH domain. FRET was confined to the plasma membrane region (Fig. 7, yellow boxes in panels a) and was set to be recorded within the time windows, marked by black or red bars under the actual Ba²⁺ current traces on panels c, simultaneously with acquisition of the currents. An image recorded at the resting state (–90 mV, Fig. 7, black bars corresponding to panels d) was followed by an image recorded at the end of a +20 mV depolarization test pulse (red bars corresponding to panels e). One can see that independent of the presence of the _1a subunit, a corrected FRET signal of different intensity was observed in Fig. 7, panels d and e. Ratios (Fig. 7, panels f) between the two consecutive images recorded at –90 and +20 mV reflect voltage-dependent conformational rearrangements of the N-tail fluorophore fused to the _1C,77 subunit with regard to the relatively small membrane-trapped PH domain probe (5). In the presence of the _1a subunit (Fig. 7B), the channel generated a Ba²⁺ current in response to a +20-mV depolarization (V_h =–90 mV) that showed complete inactivation within 500 ms (Fig. 7B, panel c). Thus, the +20-mV corrected FRET image recorded at the end of the 600-ms depolarizing pulse (Fig. 7B, panel e) corresponds to a predominantly inactivated state of the channel. However, the ratio (Fig. 7B, panel f) shows very little if any change in corrected FRET between –90 and +20 mV (0.90 ± 0.06; n = 6). A similar result was observed with another type of subunit, the rabbit ₂ (20).

View larger version (30K): [in this window] [in a new window]   FIG. 7. Voltage-gated rearrangements between the fluorophores of (ECFP)N-PH and (EYFP)N-1C,77 revealed in the absence of a subunit. The (EYFP)N-1C,77 and 2 subunits of the Ca2+ channel were co-expressed in COS1 cells with (ECFP)N-PH in the absence (A) or presence (B) of the 1a subunit. Panels a show phase-contrast cell images with a shadow of patch pipette. Schematic diagram (panel b) depicts the arrangement of fluorophores. Panels c show Ba2+ currents evoked by 600-ms (A) or 1-s (B) depolarizations to +20 mV from Vh = –90 mV. Applied voltage protocols are presented above the current traces. Panels d and e show corrected images of FRET between (ECFP)N-PH and (EYFP)N-1C,77 in the plasma membrane regions (marked by yellow boxes in panels a) recorded, respectively, at the resting (–90 mV) and inactivated states of the channel (+20 mV). The time windows for the –90 and +20-mV acquisitions are marked by black and red horizontal bars below Ba2+ current traces, respectively. Left scaling bars apply to both images. Panels f show ratios of images d/e corresponding to the voltage-dependent changes of corrected FRET associated with a reversible voltage-gated rearrangement between the fluorophores (A) or lack of it (B). Scale bars, 3 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Ca_v1.2 channels play an important role in initiation of Ca²⁺ signaling in many cells, including neurons (39). Here we have investigated differential roles of the C- and N-terminal tails of one of the neuronal "short" (exon 1) _1C isoforms in voltage-, Ca²⁺-, and subunit-dependent regulation of the Ca_v1.2 channel.

Our study is an important step forward in understanding Ca_v1.2 channel regulation as a multifactorial process. Here for the first time we have analyzed differential regulation of the Ca²⁺ channel by the C- and N-terminal tails of the _1C subunit with simultaneous assessment of the role of subunits. To deconvolute the contribution of these parts, transient anchoring to the plasma membrane via the PH domain fused to the _1C subunit tails has been used to uncouple the _1C tails from the channel regulation. To assess the potential interplay between the tails and a subunit, we compared properties of the channel expressed in the absence and in the presence of . The accumulated body of data shows that inactivation of the Ca_v1.2 channel is mediated by a combinatorial input of these distinct parts, in which CDI is essentially additive to voltage-dependent inactivation.

Voltage-dependent slow inactivation and CDI were previously co-identified with the calmodulin-binding regions in the middle part of the _1C subunit C-tail (for review see Ref. 40). A recent picture (41) still includes an EF hand-like motif of the C-tail proximal region, although involvement of this motif in CDI has been contested by experimental evidence (29). Our findings show that CDI is not mediated solely by determinants of the _1C C-terminal tail. Investigation of other cytoplasmic constituents of the channel complex have been crucial in identifying the roles of the N-tail, the cytoplasmic pore region of _1C, and subunits for inactivation and in understanding how slow and fast voltage-dependent inactivation and CDI evolve from their interplay. A distinct advantage of COS1 cells lacking endogenous Ca²⁺ channel subunits (32) over other expression systems, such as HEK293 cells or Xenopus oocytes, permits an unambiguous interpretation of our data on subunit modulation of the channel. Because no appreciable Ca²⁺ channel activity was exhibited by the _1C,77/₂ channel (Fig. 2G), unless a subunit was co-expressed, we define the subunit modulation as a facilitation of voltage gating of the channel. Recognizing that the kinetics of inactivation of the Ba²⁺ current through the "wild-type" _1C,77/_1a/₂ channel is best fit by a sum of two (fast and slow) exponentials, the respective fractional components of the Ba²⁺ current measured at the peak of I-V curves were analyzed to assess the voltage-dependent fast and slow inactivation mechanisms.

We have shown previously that four highly conserved amino acids of the transmembrane segment S6, constituting the cytoplasmic end of the pore, jointly form the ADSI. Their simultaneous mutation (S405I in IS6, A752T in IIS6, V1165T in IIIS6, and I1475T in IVS6) generates the _1C,IS-IV channel, which does not show the slow inactivation, CDI, or differential subunit modulation (30). Our findings have shown that the _1C subunit C-terminal tail is subject to voltage-gated conformational rearrangements that specifically deliver Ca²⁺ signaling to downstream targets involved in cAMP-response element-binding protein-dependent transcription activation (5). The plasma membrane anchoring of the C-tail interrupts Ca²⁺ signal transduction despite robust Ca²⁺ current through the channel. Thus, a specific position of the C-terminal tail vis à vis the polypeptide packing of the cytoplasmic channel constituents (including the _1C subunit tails and a subunit) is crucial for CDI and slow voltage-dependent inactivation.

We now find that plasma membrane immobilization of the _1C,77 C-terminal tail alters inactivation of the channel similar to the mutation of the ADSI (Fig. 1). An interesting distinction from the electrophysiological phenotype of the _1C,IS-IV channel, however, is a shift of the I-V relationship to more positive potentials. Because these dramatic changes do not involve structural alterations of the ADSI or the calmodulin-binding regions, and are completely reversible upon the release of the _1C,77-(PH-ECFP)_C subunit C-tail, stimulated by PIP₂ hydrolysis, it is reasonable to assume that the plasma membrane anchoring of the _1C C-terminal tail interferes with the functional folding of the cytoplasmic polypeptide bundle in the pore region, including ADSI. This may directly affect the pore-forming transmembrane segment IVS6, connected to the _1C C-tail, and, indirectly, the ensemble of the pore conformations defining the ion selectivity of the channel.

A very different role was found for the _1C subunit N-terminal tail. FRET microscopy combined with the patch clamp (20) revealed limited voltage-dependent mobility of the _1C N terminus in relationship with the plasma membrane (Fig. 7B). This may correspond to the restricted local dynamics of the adjacent S1 segment observed in other channels (42). Here we find that in the subunit-deficient (silent) channel, voltage-gated conformational rearrangements of the _1C subunit N terminus vis à vis the plasma membrane are significantly increased (Fig. 7B). The functional importance of the conformational "rigidity" to voltage gating conferred by a subunit to the _1C subunit N terminus was confirmed here by anchoring of the N-tail to the plasma membrane, stabilized by the fused PH domain.

The subunit confers conformational rigidity to the _1C subunit N-tail in a manner that facilitates the channel response to voltage gating. We have found that the plasma membrane-anchoring of the _1C subunit N terminus, in the absence of , completely inhibited inactivation of the Ba²⁺ (or Ca²⁺) current. At the same time, activation of the channel current in response to depolarization becomes slow independently of the charge carrier (Fig. 2H). The single channel study pointed to low probability of both opening and closing of the channel (Fig. 4B). Uncoupling of the N-terminal tail by its deletion from the _1C subunit also inhibited inactivation of the channel (Fig. 7). However, in this case retardation of the channel activation was enhanced to a greater extent, probably because of long lasting episodes of rare openings (Fig. 4D). Release of the plasma membrane-anchored _1C N terminus essentially blocked the channel current (Fig. 2I), leaving only some residual Ca²⁺ channel activity that may be generated by the remaining small fraction of channels with immobilized _1C N-tails. Overall, these data correspond to the result obtained with the -deficient Ca_v1.2 channel (Fig. 2G). Conversely, co-expression of with ₂ subunits re-established both the voltage- and Ca²⁺-dependent inactivation of the channels despite uncoupling of the _1C subunit N-terminal tail from the regulation by its plasma membrane anchoring. These observations shed new light on the role of the subunit. It appears that there is a subunit-dependent desensitization of the _1C subunit N-tail to voltage, which is essential for the fast activation, CDI, and voltage-dependent inactivation of the channel.

Our data support the conclusion of Dascal and co-workers (34) that subunits prevent inhibition of the channel by the N-tail. The results of our work demonstrate that the N-terminal tail of the _1C subunit is a channel silencer, competing with the subunit facilitation of the channel gating. This may account for CDI and voltage-dependent inactivation and activation of the current.

Our findings add to the understanding of the subunit regulation of the Ca_v1.2 channel in a number of ways. The "chaperon" hypothesis implies that subunits ease functional expression of the Ca_v1.2 channel by binding to the -interaction domain resulting in inhibition of an endoplasmic reticulum retention signal of the _1C subunit (43). The present study does not support this view. In COS1 cells, free of endogenous Ca²⁺ channel subunits, an uncoupling of the _1C subunit N-terminal tail (by membrane anchoring or deletion) completely eliminates the requirement of subunits for the robust expression of functional Ca_v1.2 channels, but co-expression of ₂ is critical.

Recent investigation of the human cardiac short _2f and _2g subunits (26) identified the C-terminal 153-amino acid sequence as essential for the functional modulation of the channel and interaction with the _1C subunit. This region includes Ser-574 that is subject to phosphorylation by phosphatidylinositol 3-kinase as necessary and sufficient to promote Ca²⁺ channel trafficking to the plasma membrane (44). Although the mechanism of this effect is unknown, our data put the _1C subunit N-terminal tail in play as a possible determinant of the channel trafficking and a target of the subunit modulation.

Thus a new picture of the subunit modulation of the Ca_v1.2 channel has emerged (Fig. 8), showing how acts as a "molecular wedge" that prevents the N terminus of the _1C subunit from blocking the pore. Dependence of CDI on interaction between the _1C subunit N-tail and the subunit led us to a hypothesis that the subunit and the _1C C-tail may have an integrating role for the cytoplasmic polypeptide packing underlying the pore.

View larger version (37K): [in this window] [in a new window]   FIG. 8. Hypothetical molecular arrangement of the identified regulatory determinants of the Cav1.2 channel in the resting (–90 mV) and inactivated (+20 mV) states. Shown are multifactorial molecular correlates of the Cav1.2 channel regulation assembled in a cytoplasmic polypeptide packing (gray area) underlying the pore. The correlates include the following: (i) the ADSI as a combination of four amino acids (boldface letters) at the cytoplasmic end of transmembrane segments S6; (ii) a subunit, associated, via its C-terminal (dark) region (26), with the linker between the repeats I and II of 1C. Variable proximal region of the 2 subunit (striped) is not essential for the 1C- interaction (26) but may contribute to differential modulation of Cav1.2 inactivation (20). The N termini of the 1C and subunits are subject to voltage-gated rearrangements (double arrows) associated with differential subunits modulation of slow inactivation; (iii) the 1C subunit N-terminal that is blocked by from inhibiting the channel. The N-tails of both the 1C and subunits do not show voltage-dependent conformational rearrangements vis à vis the plasma membrane unless the slow inactivation is inhibited by ADSI mutation (20); (iv) the 1C subunit C-terminal tail with the locus LA (amino acids 1571–1599) of apo-CaM binding (2, 3), and a post-LA (IQ) site of Ca2+-CaM binding (1). CaM is involved in CDI as an important stabilizing component of this packing, because the Ca2+-free CaM may cross-link the LA site to the interior of the packing, where it is hidden from the cytosolic Ca2+ and available only to the permeating Ca2+ (for details see Ref. 7). In the resting state (–90 mV), the subunit may stabilize an intercalation of the apo-CaM·LA complex in close vicinity of the ADSI, thus exposing CaM to the Ca2+ influx at the intracellular opening of the pore. Mutation of ADSI, plasma-membrane anchoring of the 1C subunit C terminus, as well as uncoupling (by deletion or anchoring) of the 1C subunit N-tail in a subunit-deficient channel, all deprive the channel of CDI, despite the presence of the C-terminal tail CaM-binding determinants LA and IQ. Inactivation of the channel (+20 mV) causes a voltage-gated rearrangement of the 1C C terminus (double arrow) that serves as a mobile carrier of the Ca2+ signal for cAMP-response element-binding protein transcriptional activation (5).

	FOOTNOTES

 
^* This work was supported by the National Institutes of Health, NIA Intramural Research Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Methods, Results and Discussion, Refs. 1–7, Fig. 1, and Table 1.

^¶ To whom correspondence should be addressed: Laboratory of Clinical Investigation, National Institute on Aging, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8343; Fax: 410-558-8318; E-mail: soldatovN{at}grc.nia.nih.gov.

¹ The 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.

	ACKNOWLEDGMENTS

 
We thank Mike Shi and Larissa Maltseva for technical help with electrophysiology and Yasir Kazmi for help with fluorescent images. We also thank Drs. Mark Mattson and Katsutoshi Furukawa for critically reading the manuscript.

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