Voltage-gated Mobility of the Ca2+ Channel Cytoplasmic Tails and Its Regulatory Role*

Evgeny Kobrinsky, Elena Schwartz, Darrell R. Abernethy, and Nikolai M. SoldatovDagger

From the National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224

Received for publication, November 4, 2002, and in revised form, November 26, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transient increase in intracellular free Ca2+ concentration generated by the voltage-gated Cav1.2 channels acts as an important intracellular signal. By using fluorescence resonance energy transfer combined with patch clamp in living cells, we present evidence for voltage-gated mobility of the cytoplasmic tails of the Cav1.2 channel and for its regulatory role in intracellular signaling. Anchoring of the C-terminal tail to the plasma membrane caused an inhibition of its state-dependent mobility, channel inactivation, and CREB-dependent transcription. Release of the tail restored these functions suggesting a direct role for voltage-gated mobility of the C-terminal tail in Ca2+ signaling.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The ion conductance of voltage-dependent ion channels is tightly regulated by the gating mechanism encoded in membrane voltage sensors and cytoplasmic gates of the pore-forming alpha 1 subunit (1). This voltage gating is due to the displacement of membrane charges (2) that has been documented as gating current (3) and further characterized as conformational rearrangements detected by spectroscopy of the attached fluorescent groups (4-6). However, little is known about a functional role for state-dependent mobility of the channel cytoplasmic tails (7). Such motion of the L-type (Cav1.2) Ca2+ channel C-terminal tail that binds calmodulin (CaM)1 may serve to transfer a regulatory signal (8, 9). Thus, investigation of the mobility of cytoplasmic regions of Ca2+ channels associated with their gating is important for understanding of the molecular correlates of channel regulation and mechanisms of Ca2+ signaling.

Spectral properties of the enhanced cyan (ECFP) and yellow fluorescent proteins (EYFP) (10) are well suited for measurements of molecular rearrangements by FRET (11). Here we genetically fused the cytoplasmic N and C termini of the human Cav1.2 channel alpha 1C pore-forming subunits with EYFP and ECFP, respectively. The labeled channels were then functionally expressed in COS1 cells. The rearrangements of the tails due to transition into the distinct functional states of the channel were monitored using FRET (12) in living cells under voltage clamp conditions. A regulatory role for the voltage-gated mobility of the C-terminal tail was then characterized.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Molecular Biology-- (EYFP)N-alpha 1C,77 and (EYFP)N-alpha 1C,IS-IV expression plasmids were prepared in pcDNA3 vector essentially as described earlier (13, 14) using pEYFP vector (Clontech). To prepare the PH-EYFP and PH-ECFP expression plasmids, the 518-bp EcoRI/BamHI fragment of pPLCPH-enhanced green fluorescent protein (15) was ligated into the pECFP-1 and pEYFP-1 plasmids, respectively, at EcoRI/BamHI sites. To prepare the alpha 1C,77-(ECFP)C expression plasmid, the 1029-bp AatII/NotI fragment of 77pcDNA3 (13) was replaced by the coding sequence of the pECFP vector amplified by PCR using 5'-terminal AatII linker and 3'-terminal NotI linker containing stop codon. (EYFP)N-alpha 1C,77F-(ECFP)C and (EYFP)N-alpha 1C,IS-IV-(ECFP)C expression plasmids were prepared in a similar way using (EYFP)N-alpha 1C,77 and (EYFP)N-alpha 1C,IS-IV expression plasmids. To prepare (PH-EYFP)N-alpha 1C,77 expression plasmid, BsrGI (-13), MfeI (412) PCR segment of pHLCC77 (16) containing 5'-terminal linker (5'-TGTACAAGGCCACC-3') was ligated with the 6115-bp MfeI/NotI fragment of 77pcDNA3 into the NotI and BsrGI-cut PH-EYFP plasmid. To prepare (PH-EYFP)N-alpha 1C,77-(ECFP)C expression plasmid, the 3474-bp NotI/PpuMI fragment of 77CFPpcDNA3 was replaced into the respective sites of (PH-EYFP)N-alpha 1C,77. To prepare the alpha 1C,77-(PH-ECFP)C expression plasmid, a 927-nucleotide 3'-terminal AatII/XhoI PCR fragment with stop codon replaced by the XhoI linker was ligated with the XhoI/NotI fragment of open reading frame of the PH-CFP plasmid into the AatII, NotI-cut pHLCC77 plasmid. Its 5096-nucleotide PpuMI/NotI fragment was then subcloned into 77pcDNA3 or (EYFP)N-alpha 1C,77pcDNA3 to give the alpha 1C,77-(PH-ECFP)C and (EYFP)N-alpha 1C,77-(PH-ECFP)C expression plasmids, respectively.

Electrophysiology-- COS1 cells were grown on poly-D-lysine-coated coverslips (MatTek) in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were transfected with pcDNA3 vectors coding for alpha 1C, beta 1 (17) (or beta 2a (18)), and alpha 2delta (19) subunits using the Effectene kit (Qiagen). Effectene was used for transfection to minimize autofluorescence added by many other transfection reagents. EGF receptor was co-expressed in tail-anchoring FRET experiments. To release pleckstrin homology (PH) domain, serum-deprived (24 h) cells were exposed to EGF (100 ng/ml). Ion currents were recorded using the Axopatch 200B amplifier (Axon Instruments) at 20-22 °C by the whole-cell patch clamp method 48-72 h after transfection of COS1 cells. Voltage protocols were generated, and data were digitized, recorded, and analyzed using pClamp 8.1 software (Axon). The extracellular bath solution contained (in mM) NaCl, 100; BaCl2, 20; MgCl2, 1; glucose, 10, HEPES, 10, pH 7.4. The electrodes had resistance 3-6 megohms and were filled with pipette solution containing (in mM) CsCl, 110; MgATP, 5, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate, 10; tetraethylammonium, 20; cAMP, 0.2; HEPES, 20, pH 7.4. Currents were sampled at 2.5-5 kHz and filtered at 1 kHz.

FRET Imaging-- Images were recorded with the Hamamatsu digital camera C4742-95 mounted on a Nikon epifluorescent microscope TE200 equipped with multiple filter sets (Chroma Technology). C-Imaging (Compix) and MetaMorph (Universal Imaging) software were used to obtain and analyze images. The photobleaching experiments were conducted with the 100-watt mercury lamp. For all other experiments, a 75-watt xenon lamp was used. FRET was quantified with three filter sets as follows: for EYFP cube, excitation filter 500/20 nm, dichroic beam splitter 515 nm, and emission filter 535/30 nm; for FRET (ECFP/EYFP) cube, excitation 436/20 nm, dichroic beam splitter 455 nm, and emission filter 540/30 nm; for ECFP cube, excitation filer 436/20 nm, dichroic beam splitter 455 nm, and emission filter 480/40 nm. Regions of interest were selected using the C-Imaging software where intensity (I) from three filter sets was determined after background subtraction. Corrected intensity of FRET (IFRETc) was calculated (20) as (IFRET - IECFP × 58.5% - IEYFP × 11.5%). In the patch clamp experiments, the acquisitions of fluorescence ranging from 50 to 300 ms were obtained with simultaneous recording of the current at the indicated conditioning voltages under steady state. In some cases, the images were adjusted pixel-by-pixel using the reference channel of regions of interest. With the acceptor photobleaching, we used an excitation filter 436/10 nm and an emission filter 470/30 nm for ECFP and an excitation filter 500/10 nm and emission filter 535/20 nm for EYFP. The apparent efficiency of FRET was calculated as (IECFP* - IECFP)/IECFP*, where IECFP and IECFP* - intensities of ECFP fluorescence before and after acceptor (EYFP) photobleaching, respectively. Normalization of the corrected FRET ratios against EYFP or ECFP fluorescence showed very similar values (Table I). Corrected FRET and bleed-through values were obtained by standard approach (20, 21) according to Equation 1,

                              
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Table I
Comparison of the corrected FRET ratios with and without normalization against EYFP or ECFP fluorescence (24)


<UP>I</UP><SUB><UP>FRET</UP><SUP><UP>c</UP></SUP></SUB>=(<UP>I</UP><SUB><UP>FRET</UP></SUB>−<UP>I</UP><SUB><UP>ECFP</UP></SUB>×a−<UP>I</UP><SUB><UP>EYFP</UP></SUB>×b) (Eq. 1)
where a and b are, respectively, the norm of the percentage of ECFP and EYFP bleed-through under the FRET filter set, and IFRET, IECFP and IEYFP are intensities in each region of interest under FRET, ECFP, and EYFP filter sets, respectively. For the double-labeled channels, we have experimentally determined that a = 58.5% and b = 11.5%. Background subtraction was carried out using the standard approach (e.g. Ref. 22). In each image, we determined the average fluorescence from the cell-free regions. Under our experimental conditions, the average fluorescence measured from these specified regions was not different from the fluorescence measured with unused substrate covered with the bath solution only. Variability of the background fluorescence ranged from 9 to 14, whereas those of the experimental cell fluorescence varied from 50 to 250 units of the same arbitrary scale. To obtain ratio of images, 10 was added to each pixel of the image corrected by the background subtraction to avoid division by zero.

Plotting of corrected FRET images was carried out according to standard and well established procedures (for example, see Refs. 20 and 23). Because our images were obtained from the same cells with the same level of ECFP expression, there was no need to additionally normalize FRET images against ECFP. Under these conditions, we focused only on the relative changes of fluorescence at different holding potentials. Comparison of the ratios of corrected FRET (+40 mV/-90 mV) with those additionally normalized against EYFP, ECFP, and square root of their product according to Xia and Liu (24) (see Table I) clearly demonstrates that additional normalization did not significantly change the results of corrected FRET ratio. This result was obtained probably because the ratio of ECFP to EYFP in experiments with the double-labeled channels was always equal to 1.

CREB-dependent Transcription Activation-- The plasmids YKIDN and KIXCN coding for KID and KIX domains (25), respectively, were co-expressed (1:1) in COS1 cells with other cDNAs at 2.5:1 ratio to cDNA coding for an alpha 1C subunit. To monitor transcriptional activation under voltage clamp conditions, we used the perforated patch clamp technique (26). beta -Escin (20 µM, Sigma) was added to the pipette solution containing potassium gluconate 120, NaCl 10, MgATP 2.5, HEPES 5, and KCl, 20; pH 7.2. External solution contained (in mM) NaCl, 140; KCl, 5.4, MgCl2 1, HEPES 5, CaCl2 2.0, and glucose 5.5, pH 7.4. Depolarization to +20 mV from the holding potential of -90 mV was applied to elicit maximum Ca2+ current. To monitor changes of free Ca2+ concentration, cells were loaded with Fluo-4 by incubation for 20 min at 37 °C in 5 µM Fluo-4 AM (Molecular Probes) added to the external solution. For de-esterification of the probe, cells were incubated for another 20 min before experiment. Ca2+ measurements were performed at 20-22 °C using confocal microscope PCM2000 (Nikon, Inc.) with excitation by an argon laser at 488 nm and recording of the fluorescence emission at >515 nm.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Acceptor Photobleaching Assay-- Confirming earlier findings (13, 27), fusion of the green fluorescent protein variants to the cytoplasmic N and/or C termini did not compromise channel function. The electrophysiological properties of the (EYFP)N-alpha 1C,77-(ECFP)C channel were similar to those of the wild-type channel (13) except of the acceleration by ~15% of the Ba2+ current inactivation (Table II). A direct FRET acceptor photobleaching assay provided strong evidence of FRET in the (EYFP)N-alpha 1C,77-(ECFP)C channel (Fig. 1). Illumination of the transfected COS1 cells for 15 min with a mercury lamp caused on average >90% photobleaching of EYFP. The resulting increase in donor fluorescence (shown as yellow-green and yellow-red signals, Fig. 1A, c and e, respectively) is a direct demonstration of FRET (11). As a positive control, under the same conditions, FRET was observed (Fig. 1A, d and f) with the co-expressed mixture of the EYFP-PH and ECFP-PH domains of phospholipase Cdelta 1 (28). In both cases FRET was confined to the plasma membrane region where the functional channel molecules reside. We found that the mixture of co-expressed (1:1) single N- and C-tail-labeled channel isoforms did not show substantial intermolecular FRET (Fig. 1B) indicating that with the double-labeled alpha 1C channels we recorded predominantly intramolecular FRET.

                              
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Table II
Comparison of electrophysiological properties of the wild-type alpha 1C,77 (A) and fluorescent labeled (EYFP)N-alpha 1C,77-(ECFP)C (B), and (EYFP)N-alpha 1C,IS-IV-(ECFP)C (C) Ca2+ channels
The alpha 1C subunits were co-expressed with beta 1 and alpha 2delta accessory subunits. Ba2+ currents were elicited by 600-ms test pulses to +10 mV from a holding potential of -90 mV. The bath medium contained 20 mM Ba2+. The time constants of the Ba2+ current inactivation tau fast and tau slow were determined by two-exponential fitting. (The approximated tau slow values are presented solely to reflect the fact that slow inactivation is completely inhibited in the labeled alpha 1C,IS-IV channel.) Vmax, voltage for the peak current, and V0.5, voltage at 50% of the Ba2+ current activation were determined from the current-voltage relationship. V0.5in, the voltage at half-maximum of inactivation, and Isust, fraction of non-inactivating current, were determined from the fitting of steady-state inactivation curves by Boltzmann function. Steady-state inactivation curves were measured using a two-step voltage clamp protocol. A 1-s conditioning pulse was applied at 30-s intervals with 10-mV increments from the holding potential Vh = -90 mV followed by a 250-ms test pulse to +10 mV. Peak current amplitudes were normalized to maximum value. All fittings were performed according to Ref. 33. Number of tested cells is shown in parentheses.


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Fig. 1.   Direct FRET measurements in the (EYFP)N-alpha 1C-(ECFP)C channels. A, FRET with acceptor photobleaching in the (EYFP)N-alpha 1C,77-(ECFP)C channel containing the beta 1 accessory subunit (left panels; scale bar, 7 µm) and in the equimolar mixture of EYFP-PH and ECFP-PH domains co-expressed as positive control (right panels; scale bar 14 µm). EYFP was attached to the 124-amino acid N-terminal tail of alpha 1C,77. 306 amino acids of the 662-amino acid C-terminal tail of the alpha 1C subunit were replaced by ECFP so that the overall length of the C-terminal tail of the fusion protein was 652 amino acids. a and b, phase contrast images of the expressing COS1 cells. c and d, ratios of ECFP fluorescence intensity before and after photobleaching. e and f, corresponding three-dimensional images. B, increase of apparent efficiency of FRET due to acceptor photobleaching in cells expressing the indicated double-labeled channels or EYFP-PH/ECFP-PH mixture but not the mixtures of single-labeled channels (n, number of cells; *, p < 0.0001, unpaired t test). In the schematics, Y and C stand for EYFP and ECFP, respectively. Co-expressed beta -subunits are indicated at the lower panel. Error bars, S.E.

Voltage-dependent FRET in the Inactivated State-- FRET is the result of long range dipole-dipole interactions between fluorophores. FRET depends on the distance (r6) between the donor and acceptor fluorophore and, to a lesser extent, the relative orientation (kappa 2) of the dipoles (12). To determine the differences in relative proximity and/or angular orientation of the tagged alpha 1C tails in the functionally distinct resting, conducting, and inactivated states of the channel, we combined FRET imaging with voltage clamp. We used quantitative analysis of reversibly changing FRET in place of acceptor photobleaching as the fluorophores became irreversibly damaged by photobleaching. Simultaneous monitoring of the Ba2+ current provided a direct assessment of the Cav1.2 channel's transition into the resting, conducting, or inactivated state prior to FRET imaging. The images were acquired with a three-filter set system, successfully used elsewhere (20, 29), for sensitized acceptor emission. The recombinant channel, composed of the wild-type alpha 1C,77 and accessory beta 1 and alpha 2delta subunits, was stabilized in either the resting or inactivated state by whole-cell voltage clamp at -90 or +40 mV, respectively. Depolarization to +40 mV was selected to evoke maximum activation of the channels under conditions when the state of inactivation could be monitored. Thus, the amplitudes of the Ba2+ currents (Fig. 2d) were less than 50% of the maximum ones. The analysis of FRET images of a cell expressing the (EYFP)N-alpha 1C,77-(ECFP)C channel (Fig. 2A) showed that the sequential transitions between the resting (-90 mV, Fig. 2A, c) and the inactivated states of the channel (+40 mV, Fig. 2A, b) produced a fully reversible increase in FRET: (IFRETc)+40 mV/(IFRETc)-90 mV = 2.9 ± 0.7 (n = 24). The steady state inactivation analysis indicated that ~7% of the Ba2+-conducting (EYFP)N-alpha 1C,77-(ECFP)C channels remain available (Table II), suggesting that >= 93% of the channels are inactivated and <= 7% remain in the resting closed state at the end of the depolarization pulse shown in Fig. 2A. Since the resting state is characterized by low FRET, this fraction has only slightly contributed to the measurement of FRET. Taken together, these data indicate that the cytoplasmic tails of the channel in the inactivated state are rearranged into a conformation that gives greater FRET as compared with the closed resting state.


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Fig. 2.   Reversible changes of FRET associated with voltage gating of Ca2+ channels. Right panel, phase-contrast images of expressing cells with the shadow of patch pipette (a) and schematic diagrams depicting the arrangement of fluorophores (below). A, shown are the ratios of corrected FRET images (b and c) sequentially recorded at the indicated voltages with FRET cube and their three-dimensional presentations (below) for (EYFP)N-alpha 1C,77-(ECFP)C channel in b inactivated (+40 mV) and c resting (-90 mV) states; B, shown are the ratios when inactivation of the same channel was slowed down by the replacement of beta 1 with beta 2a subunit, in b predominantly conducting (+40 mV) and c resting states; and C, shown are the ratios for the non-inactivating (EYFP)N-alpha 1C,IS-IV-(ECFP)C channel in b conducting (+40 mV) and c resting states. Scale bars, 8 µm. Image areas indicated by arrows were digitally magnified to demonstrate confinement of FRET to the plasma membrane. Note that in every case the Ba2+ current recordings (d) provide evidence of the channel state achieved prior to FRET image acquisitions (marked by red bars). FRET at holding potential Vh = -90 mV was recorded for the same duration of time before and after the depolarization pulse.

The electrophysiological properties of the alpha 1C channel are not significantly changed by the deletion of the N-terminal tail (30). In contrast, the proximal half of the C-terminal tail is essential for channel function (13, 30, 31). A fusion of ECFP to the full size, 662-amino acid C-terminal tail of alpha 1C,77 did not alter the electrophysiological properties of the (EYFP)N-alpha 1C,77F-(ECFP)C channel (data not shown), but reduced efficiency of FRET determined in the acceptor photobleaching assay to 0.19 ± 0.02 (n = 29), thus indicating that apparent separation of the fluorophores has increased. However, the intensity of FRET in the inactivated state (+40 mV) of this full-length (EYFP)N-alpha 1C,77F-(ECFP)C channel was not significantly different as compared with the truncated channel.

Voltage-dependent FRET in the Conducting State of the Cav1.2 Channel-- Rapid spontaneous channel inactivation complicates direct FRET imaging of the channel in the transient conducting state. Replacement of the cytoplasmic accessory beta 1 subunit with beta 2a (32) significantly slowed inactivation (compare Ba2+ current traces on d in Fig. 2, A and B) and thus allowed the channel to be maintained in a predominantly conducting state. Voltage-dependent FRET was measured with the (EYFP)N-alpha 1C,77-(ECFP)C/beta 2a channel at the end of a 3-s depolarization at +40 mV. Under these conditions, the Ba2+ current activation was almost maximal, whereas inactivation was minimal with respect to the time of FRET acquisition (marked by the red bar above the current traces). The voltage-dependent increase of FRET (3.8 ± 0.8, n = 15, see Fig. 2B) resulting from the transition of the channels from the resting (-90 mV) to a predominantly conducting state (+40 mV) was found not to be significantly different from that determined for the inactivated state (2.9 ± 0.7, Fig. 2A). Thus, the folding of the channel tails characterized by FRET in the ensemble of predominantly conducting channels was on average closer to those determined for the inactivated than those determined for the resting state of the same channel.

This finding was independently confirmed by the study of the alpha 1C,IS-IV channel that is deprived of slow inactivation by mutations introduced in the pore region (14). The (EYFP)N-alpha 1C,IS-IV-(ECFP)C channel expressed in COS1 cells showed the characteristic sustained Ba2+ current (Fig. 2C, d). The amplitude of the Ba2+ current through the alpha 1C,IS-IV channel was smaller than those through the alpha 1C,77 channel. Similar changes were observed earlier for other isoforms of the Cav1.2 channel with impaired Ca2+-induced inactivation (33). This was found to be due to a lower open probability and a 10-15% reduction in single channel conductance (13). In the stable conducting (+40 mV) state of the alpha 1C,IS-IV channel (Fig. 2C, b), the corrected FRET increased 7.4 ± 0.9-fold (n = 19; p < 0.0005, compared with Fig. 2A, b) suggesting further reduction in the apparent distance between the labeled tails and/or dipole reorientation of the fluorophores (12) leading to an increase of FRET as compared with the resting state (-90 mV, see Fig. 2C, c). Taken together, these data point to distinct rearrangements of the alpha 1C tails associated with voltage gating.

Role of the Voltage-gated Mobility of the C-terminal Tail in the Cav1.2 Channel Inactivation-- To investigate a regulatory role of the voltage-gated rearrangements of the alpha 1C C-terminal tail, the tail was anchored to the plasma membrane via the PH domain of phospholipase Cdelta 1 (Fig. 3). This domain binds specifically to phosphatidylinositol bisphosphate in the inner leaflet of the plasma membrane. Hydrolysis of phosphatidylinositol bisphosphate by activation of phospholipase C induces redistribution of the PH domain to the cytoplasm (15, 34).


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Fig. 3.   Effect of immobilization and release of the C-terminal tail of Ca2+ channel on state-dependent FRET. A, direct FRET imaging. Phase contrast (a) and FRET images of COS1 cell expressing the (EYFP)N-alpha 1C,77-(PH-ECFP)C channel before (b) and after (c) release of the membrane-anchoring PH domain. Upper panels, digitally magnified image areas pointed by arrows. d, ratio of images (c and b) showing increased FRET in the membrane region in response to the C-tail release. Upper panel, three-dimensional image. B, inhibition of inactivation and state-dependent FRET in cell expressing the (EYFP)N-alpha 1C,77-(PH-ECFP)C channel with the membrane-trapped C-terminal tail. a, phase-contrast image of the expressing cell and schematic diagram of the alpha 1C channel construct (below). b, the Ba2+ current trace elicited by depolarization to +40 mV from Vh = -90 mV. c and d, ratio of corrected FRET images recorded at the indicated membrane potentials. C, restoration of inactivation and state-dependent FRET after release of the C-tail. a, schematic diagram of the alpha 1C channel construct. b, Ba2+ current trace elicited by depolarization to +40 mV from Vh = -90 mV. c and d, ratio of corrected FRET images recorded at the indicated potentials. Left panels, digital magnifications of the membrane region. Low panels, corresponding three-dimensional images. Scale bars, 8 µm. Voltage protocol was similar to Fig. 2.

The (EYFP)N-alpha 1C,77-(PH-ECFP)C channel with anchored C-tail was co-expressed in COS1 cells with epidermal growth factor (EGF) receptor to permit a release of the PH-tagged C-terminal tail by activating phospholipase gamma  and phosphatidylinositol bisphosphate hydrolysis in response to exposure of the cell to EGF (Fig. 3A). The Ba2+ current through the channel with the anchored C-terminal tail was activated in the characteristic range of membrane potentials but exhibited a very slowly inactivating component of the Ba2+ current (Fig. 3B, b). Membrane trapping of the N-terminal tail did not alter the inactivation properties of the channel (data not shown).

Very little, if any, FRET was observed with the anchored C-terminal tail in both the conducting (Fig. 3B, c) and the resting (d) states of the channel. The release of the tail by EGF treatment irreversibly restored the ability of the channel to inactivate fully as can be seen from the complete decay of the current (Fig. 3C, b) and increased FRET in the inactivated state (Fig. 3C, c). The ratio (IFRETc)+40 mV/(IFRETc)-90 mV = 1.9 ± 0.9 (n = 5, p < 0.05) was not different to that observed with the (EYFP)N-alpha 1C,77-(ECFP)C (Fig. 2A) and (EYFP)N-alpha 1C,77F-(ECFP)C (not shown) channels. Thus, limitations imposed on free movement of the C-terminal tail of the alpha 1C channel affect inactivation properties as well as the associated FRET. When released, the C-tail appears to assume a functional conformation as determined by both the return of the normal inactivation properties of the channel and voltage-dependent FRET. This precludes a re-insertion of the PH domain into the membrane. Even a 30-min washout period, the longest that we were able to achieve without losing the voltage clamp, did not restore the membrane association of the PH domain distinguished by the properties of the channel with the trapped tail.

Role of the Voltage-gated Mobility of the C-terminal Tail in Regulation of CREB-dependent Transcription-- The amplitude of the voltage-gated mobility of the Cav1.2 channel C-terminal tail may be sufficient to have a role in Ca2+ signal transduction. It has been shown previously (8) that Cav1.2 channels are important for Ca2+-induced activation of cAMP-responsive element-binding protein (CREB)-dependent transcription. In this work, to study the role of the voltage-gated mobility of the Ca2+ channel C-terminal tail for transcriptional activation, we investigated the interaction between KID and KIX domains of CREB and co-activator CREB-binding protein under voltage clamp conditions by monitoring FRET between (EYFP)-KID and (ECFP)-KIX, both containing nuclear localization sequences (25). Use of perforated patch clamp technique (26) allowed us to preserve the integrity of the cytoplasmic content of the cell, crucial in retaining components of signaling cascade involved in CREB-dependent transcription. We found that when the C-terminal tail of the alpha 1C,77-(PH)C channel was anchored via the PH domain to the plasma membrane (Fig. 4A, a), activation of the channel by depolarization to +20 mV increased intracellular free Ca2+ concentration ([Ca2+]i) detected by the free Ca2+ indicator Fluo-4 (Fig. 4A, a, see image on the lower panel). However, stimulation of the sustained Ca2+ conductance of the anchored channel by depolarization (Fig. 4A, d) did not cause strong activation of CREB-dependent transcription despite the presence of the CaM-binding IQ motif of the C-tail 1624-1635 (35), previously identified as important for CREB activation (8). Release of the C-terminal tail of the channel, induced at -90 mV by ACh stimulation of the co-expressed type 1 muscarinic acetylcholine receptor (M1AChR), was accompanied by activation of the inositol 1,4,5-trisphosphate-dependent Ca2+ release but did not induce substantial activation of CREB-dependent transcription (Fig. 4A, b). It was the release of the C-terminal tail of the channel combined with the activation of Ca2+ conductance by depolarization that was essential to re-establish the voltage-gated signaling sufficient for CREB-dependent transcription activation (Fig. 4A, c). The fact that this response was inhibited by co-expression of CaM1234 (Fig. 4B), a Ca2+-insensitive analogue of CaM retaining the affinity to CaM-binding sites (36), points to a critical role of CaM in signal transduction by the voltage-gated mobile C-terminal tail of the alpha 1C channel. Selective disruption of the apo-CaM-binding site 1572-1598 (37-39) on the C-terminal tail of the alpha 1C channel (33) (Fig. 4C) abolished activation of CREB-dependent transcription despite unrestricted mobility of the C-terminal tail and the presence of CaM and IQ motif (8). These results, for the first time, demonstrated the role of the Ca2+ channel C-tail as a voltage-gated mobile carrier of the signal for CREB-dependent transcriptional activation (Fig. 4D). This signal appears to be associated with apo-CaM binding to the C-tail and may involve its Ca2+ loading (9) and translocation to the IQ motif (8, 36), a Ca2+-filled CaM-binding site on the C-tail.


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Fig. 4.   CREB-dependent activation of transcription. CREB activation was examined under perforated patch conditions by FRET between the EYFP-labeled KID domain of CREB and ECFP-labeled KIX domain of CREB-binding protein as interaction partners. A, CREB activation in COS1 cells expressing M1AChR and the (EYFP)N-alpha 1C,77-(PH-ECFP)C channel with the membrane-trapped C-tail, before (a) and after (b and c) its release by ACh activation of M1AChR. d, representative trace of the Ca2+ current (20 mM Ca2+ in bath medium) showing the sustained component of Ca2+ conductance due to the C-tail anchoring, which was completely eliminated by the C-tail release (not shown). The CREB activation (A, c), shown by intensive FRET signal in the nucleus, was abolished (B) by the co-expression of the Ca2+-insensitive analogue of CaM (36) (CaM1234) or (C) by disruption of the apoCaM-binding site in the free C-tail of the alpha 1C,77L channel (33), which lost Ca2+-dependent inactivation property as the result of this mutation. Upper panels show voltage and ACh application protocol. Twelve steps of depolarization from -90 mV to +20 mV, 10-mV increment for 1-s pulse with 10-s intervals were applied, and a 100-ms FRET images a-c were recorded at the end of the last depolarization before (a) or after (c) 5-min application of 5 µM ACh. b shows effect of 5 µM ACh applied at -90 mV. Left panels, phase-contrast images of expressing cells with the shadow of patch pipette; scale bars, 4 µm. Free Ca2+ measurements with Fluo-4 (lower panels) were carried out under the same conditions and show that the activation of CREB-induced transcription did not depend strongly on free intracellular Ca2+ unless the C-tail was released. Schematic diagrams depict positions of the alpha 1C C-tail vis-à-vis the plasma membrane. D, histogram of FRET intensity ratio of images c and b (n = 3, p < 0.01).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this report, we provide strong evidence that the C-terminal cytoplasmic tail is a functionally important moving part of the voltage-gated Ca2+ channel. Previously implicated as cytoplasmic channel elements that respond to voltage gating were the cytoplasmic domain between repeat II-III of the skeletal muscle alpha 1S subunit (40), inactivation gates of Na+ channel (41), and the ball-and-chain (N-type) inactivation determinant of K+ channel (7, 42). The state dependence of FRET in the alpha 1C channel is associated with conformational refolding of the cytoplasmic parts in the resting, conducting, and inactivated states. FRET increased upon transition from the resting to the inactivated or conducting states of the Ca2+ channel suggesting gating-dependent conformational mobility of the channel tails. The anchoring of the C-terminal tail to the plasma membrane impaired both the channel inactivation and FRET until fully recovered with its release. Thus, the state-dependent mobility of the cytoplasmic C-terminal tail is essential for regulation of the Ca2+ channel.

In addition to relative proximity of the tagged alpha 1C tails, their angular orientation may contribute to FRET because the environment of the fluorophores in the (EYFP)N-alpha 1C-(ECFP)C channels may be structured and their segmental motions may not independently randomize the orientations (kappa 2 <=  2/3) (12). The kappa 2 factor adds to the uncertainties complicating interpretation of the FRET measurements in terms of translational distances and is of particular concern when dipoles become oriented perpendicular to one another (kappa 2 = 0). Because FRET was consistently observed, such perpendicular orientation seems unlikely. In fact the corresponding voltage-dependent movement of the alpha 1C cytoplasmic C-tail is sufficient to reach other targets and be involved in signal transduction. An important regulatory signal associated with the C-terminal tail is CaM, which supports Ca2+-induced inactivation of the Ca2+ channel (35, 37, 38). It is possible that the C-terminal tail mobility may, in a state-dependent manner, provide a coordinated transfer of CaM between the channel pore inner mouth, where it is loaded by Ca2+ when the channel opens, and protein targets like CaM-activated protein kinase (8), where Ca2+/CaM exerts signaling. This hypothesis may be generalized to the role of CaM-carrying mobile tails in other Ca2+ channels (29, 43).

    ACKNOWLEDGEMENTS

The discussions with C. Romanin, M. Morad and H. Reuter motivated much of the early work of this study. The help of A. Blatt and O. Carlson in molecular biology and A. Yu in image analysis and preparation of figures is greatly appreciated. We thank T. Balla for PH domain coding vector; F. Hofmann for plasmids encoding the channel accessory subunits; A. Sorkin for the EGF receptor-coding plasmid; M. R. Montminy for YKIDN and KIXCN constructs; and S. Sollott for valuable suggestions.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: NIA, National Institutes of Health, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8343; Fax: 410-558-8318; E-mail: soldatovn@grc.nia.nih.gov.

Published, JBC Papers in Press, December 6, 2002, DOI 10.1074/jbc.M211254200

    ABBREVIATIONS

The abbreviations used are: CaM, calmodulin; FRET, fluorescence resonance energy transfer; CREB, cAMP-responsive element-binding protein; ECFP, enhanced cyan fluorescent proteins; EYFP, enhanced yellow fluorescent protein; nt, nucleotide; PH, pleckstrin homology; EGF, epidermal growth factor; ACh, acetylcholine; M1AChR, muscarinic acetylcholine receptor..

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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