The N terminus of the Cardiac L-type Ca Channel a1C Subunit THE INITIAL SEGMENT IS UBIQUITOUS AND CRUCIAL FOR PROTEIN KINASE C MODULATION, BUT IS NOT DIRECTLY PHOSPHORYLATED*

The first 46 amino acids (aa) of the N terminus of the rabbit heart (RH) L-type cardiac Ca channel a1C subunit are crucial for the stimulating action of protein kinase C (PKC) and also hinder channel gating (Shistik, E., Ivanina, T., Blumenstein, Y., and Dascal, N. (1998) J. Biol. Chem. 273, 17901–17909). The mechanism of PKC action and the location of the PKC target site are not known. Moreover, uncertainties in the genomic sequence of the N-terminal region of a1C leave open the question of the presence of RH-type N terminus in Ltype channels in mammalian tissues. Here, we demonstrate the presence of a1C protein containing an RH-type initial N-terminal segment in rat heart and brain by using a newly prepared polyclonal antibody. Using deletion mutants of a1C expressed in Xenopus oocytes, we further narrowed down the part of the N terminus crucial for both inhibitory gating and for PKC effect to the first 20 amino acid residues, and we identify the first 5 aa as an important determinant of PKC action and of N-terminal effect on gating. The absence of serines and threonines in the first 5 aa and the absence of phosphorylation by PKC of a glutathione S-transferase-fusion protein containing the initial segment suggest that the effect of PKC does not arise through a direct phosphorylation of this segment. We propose that PKC acts by attenuating the inhibitory action of the N terminus via phosphorylation of a remote site, in the channel or in an auxiliary protein, that interacts with the initial segment of the N terminus.

The first 46 amino acids (aa) of the N terminus of the rabbit heart (RH) L-type cardiac Ca 2؉ channel ␣ 1C subunit are crucial for the stimulating action of protein kinase C (PKC) and also hinder channel gating ( Here, we demonstrate the presence of ␣ 1C protein containing an RH-type initial N-terminal segment in rat heart and brain by using a newly prepared polyclonal antibody. Using deletion mutants of ␣ 1C expressed in Xenopus oocytes, we further narrowed down the part of the N terminus crucial for both inhibitory gating and for PKC effect to the first 20 amino acid residues, and we identify the first 5 aa as an important determinant of PKC action and of N-terminal effect on gating. The absence of serines and threonines in the first 5 aa and the absence of phosphorylation by PKC of a glutathione S-transferase-fusion protein containing the initial segment suggest that the effect of PKC does not arise through a direct phosphorylation of this segment. We propose that PKC acts by attenuating the inhibitory action of the N terminus via phosphorylation of a remote site, in the channel or in an auxiliary protein, that interacts with the initial segment of the N terminus. Voltage-dependent L-type Ca 2ϩ channels regulate contraction of cardiac and smooth muscle and excitability and gene expression in the brain (2)(3)(4). They consist of three subunits: ␣ 1 (main, pore-forming subunit), ␤, and ␣ 2 /␦. The ␣ 1 subunits in the heart, smooth muscle, and brain are products of the ␣ 1C gene (5). The existence of several cDNA isoforms and the genomic sequence of the ␣ 1C DNA suggest the presence of splice variants of RNA and thus of several isoforms of the ␣ 1C protein (6 -8), but the actual composition of ␣ 1C protein isoforms in tissues is still poorly characterized.
The ␣ 1C subunit appears to be the main target for modulation by protein kinases A and C (PKA and PKC, 1 respectively), although ␤ is also a substrate (9). Both kinases increase the activity of the channel (10 -12). PKC has been proposed to mediate the enhancement of L-type Ca 2ϩ channels by intracellular ATP (13), angiotensin II (14), glucocorticoids (15), PACAP (16), and arginine-vasopressin (17). After the initial enhancement by PKC-activating phorbol esters, the Ca 2ϩ current is often decreased (18,19), but it is not clear whether the inhibition is phosphorylation-related (20,21). The dual effect of PKC activators is fully reconstituted in Xenopus oocytes expressing ␣ 1C , with or without ␣ 2 /␦ and/or ␤; the presence of ␤ attenuates the enhancing action of PKC (21,22). In the nerve cells, either stimulation (23)(24)(25)(26) or inhibition (27)(28)(29) of L-type channels by PKC has been reported.
The ␣ 1 subunit is composed of four homologous membranespanning internal domains, each with six transmembrane ␣-helixes and a pore-forming reentrant P loop (30). C and N termini and linkers between domains I-II, II-III, and III-IV are cytosolic. The initial 46 aa of the N terminus of rabbit heart (RH) ␣ 1C are crucial for PKC modulation (1). The cytosolic N-terminal part of RH ␣ 1C is 154 aa long. Deletion of the first 40 aa or more causes a 5-10-fold increase in the current via RH-type Ca 2ϩ channels expressed in Xenopus oocytes (1,31). This is a result of a change in channel gating because the truncation causes an increase in open probability, without increasing the amount of ␣ 1C protein in the plasma membrane (1). These and additional findings led us to propose that the N terminus of ␣ 1C acts as an inhibitory gate, and its removal enhances channel activation; PKC increases the current by attenuating the inhibitory action of the N terminus (1). It is not known whether PKC phosphorylates the N terminus.
Despite the importance of the first 46 aa of the RH-type N terminus, its presence in L-type channel proteins in vivo remains uncertain. The only other cDNA of ␣ 1C containing a stretch encoding this protein sequence is that cloned from rat aorta and heart (6). ␣ 1C cDNAs cloned from rabbit lung, human heart, and rat brain (7, 32-34) do not encode this stretch (see Fig. 2A). It has been proposed that these variations correspond to splice variants of the ␣ 1C gene (7), but even this is not certain. The structure of the genomic DNA of human ␣ 1C has not been fully resolved in this region; none of the known exons correspond to the RH-type N terminus (8). In contrast, a recent study that utilized an RNase protection assay showed that RNA of the RH-type initial segment is predominant in human heart (35). These discrepancies make it important to clarify whether L-type Ca 2ϩ channel isoforms with the RH-type N terminus are common in mammalian tissues.
Here we demonstrate the abundance of the RH-type N terminus in rat heart and brain and map the segment critical for PKC modulation and for inhibition of gating to the very beginning of the N terminus. Our results strongly suggest that PKC effect is not mediated by phosphorylation of this initial segment.
The cDNA constructs of the GST fusion proteins of the whole N terminus (N 1-154 ) and of the loop I-II were described previously (1). The cDNAs for N 47-154 and N 87-154 (encoding the corresponding ␣ 1C segments) were made by a PCR procedure and inserted into EcoRI and NotI sites of pGEX-4T-1 (Amersham Pharmacia Biotech) and thus linked in-frame to GST. N 1-46(S44A) , with serine 44 replaced by alanine, and a cDNA for N(neuronal) 1-124 , encoding aa 1-124 of rat brain rbCII ␣ 1C isoform (7), were inserted into EcoRI and XhoI sites of pGEX-4T-1. All PCR products were sequenced at the Tel Aviv University Sequencing Facility. Fusion proteins were generated by transformation into Escherichia coli strain BL-21 (Stratagene) followed by induction with 1 mM isopropyl-1-thio-b-D-galactopyranoside and affinity purification with GST beads (Amersham Pharmacia Biotech) and elution with 15 or 20 mM reduced glutathione. In the preparation of N 1-46(S44A) , protease inhibitors were used: aprotinin (10 g/ml), benzamidine (5 mM), Pefabloc SC (0.2 mM), and EDTA (1 mM). N 1-46(S44A) was additionally dialyzed to 0.1 M ammonium acetate buffer, pH 7.0, aliquoted, and lyophilized. Materials and enzymes for molecular biology were purchased from Roche Molecular Biochemicals, Promega, or MBI Fermentas.
Oocytes and Electrophysiology-Xenopus laevis frogs were maintained and dissected as described (37). Oocytes were injected with equal amounts (by weight) of the mRNAs of ␣ 1C or its mutants, of ␣ 2 /␦, and, in the experiment shown in Fig. 2F, of ␤2A (2.5 ng for electrophysiological, 5 ng for biochemical experiments) and incubated for 3-5 days at 20 -22°C in NDE96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 2.5 mM Na-pyruvate, 50 g/ml gentamycin, 5 mM HEPES, pH 7.5). Whole cell currents were recorded using the Gene Clamp 500 amplifier (Axon Instruments, Foster City, CA) using the two-electrode voltage clamp technique in a solution containing 40 mM Ba(OH) 2 , 50 mM NaOH, 2 mM KOH, and 5 mM HEPES, titrated to pH 7.5 with methanesulfonic acid (37). Stimulation, data acquisition, and analysis were performed using pCLAMP software (Axon Instruments). Ba 2ϩ currents were measured by a 200-ms step to 20 mV from a holding potential of Ϫ80 mV. To study the effect of PMA (10 nM; Sigma), the voltage pulses were delivered every 10 -20 s (see Ref. 1 for details of PMA use).
Antibodies-Card-I and Card-C antibodies were kindly provided by M. M. Hosey (Northwestern University, Chicago, IL) (38). A new antibody (Card-N) was raised against the GST fusion protein N 1-46(S44A). Two New Zealand female rabbits were immunized with 0.3 mg of N 1-46(S44A) in complete Freund's adjuvant, and reimmunized monthly with 0.2 mg of N 1-46(S44A) in incomplete Freund's adjuvant. Blood samples were taken 10 days after the immunization. The specific antibodies in the sera were detected by enzyme-linked immunosorbent assay on immobilized N 1-46(S44A) , in the presence of excess of soluble GST (20 g/ml). The reacting sera were chosen for antibody purification. Immobilized N 1-46S44A , GST, and E. coli lysate were prepared using Affi-Gel 10 (Bio-Rad) according to the recommendations of the manufacturer. Crude IgG fraction was prepared from the serum by 50% saturation (NH 4 ) 2 SO 4 precipitation and dialyzed in 100 mM Tris-HCl, pH 8.0. To eliminate anti-GST antibodies, the IgG fraction was incubated with GST beads (Affi-Gel 10, Bio-Rad) overnight at 4°C. The bound material was eluted with 4.5 M MgCl 2 . The procedure was repeated with the unbound material several times until no antibodies were eluted from GST beads. To eliminate antibodies against possible contaminating bacterial proteins, the same procedure was performed with immobilized heat-shocked E. coli lysate proteins. The unbound material was applied to N 1-46S44A -GST beads (Affi-Gel 10, Bio-Rad), incubated 2 h at room temperature or overnight at 4°C, and eluted with 3.5 M MgCl 2 . The eluted antibodies were dialyzed against 10 mM Tris-HCl, pH 8.0, and then against phosphate-buffered saline containing 0.025% NaN 3 .
Preparation of Tissues and Western Blot Analysis-Tissues from 3-week old Wistar rats were frozen in liquid N 2 , crushed while frozen, and homogenized on ice in a buffer (0.32 M sucrose, 1 mM EDTA, 50 mM Tris) containing the Roche Molecular Biochemicals protease inhibitor mixture. Crude membranes were prepared by centrifugations at 4°C (two times at 1100 ϫ g for 10 min to remove the debris and the nuclei, and then for 1 h at 100,000 ϫ g) and resuspension of the pellet in the above buffer. The amount of protein was determined by the Bradford assay. The membranes were stored in aliquots at Ϫ80°C. Protein samples (35 g per lane) were separated on 6% polyacrylamide-SDS gels and transferred to nitrocellulose membranes for Western blotting with the various antisera at the following dilutions: Card-C, 1:1000; Card-I, 1:375; Card-N, 1:750 or 1:1000. The filters were visualized using the SuperSignal Substrate kit (Pierce).

RESULTS AND DISCUSSION
The RH-type N Terminus Is Present in Rat Heart and Brain-A polyclonal antibody (Card-N) directed against a GST fusion protein of the first 46 aa of the RH-type ␣ 1C N terminus was raised in rabbit (since a GST-fusion construct containing the WT 46 aa of RH ␣ 1C appeared to degrade in the course of bacterial synthesis, Card-N was actually raised against a more stable fusion protein, N 1-46(S44A) , in which serine 44 was mutated to alanine). Card-N was compared with two previously characterized antibodies, Card-I (against residues 812-929 of the II-III domain linker) and Card-C (against residues 2156 -2169 in the end of the C terminus) (37,38). Card-N immunoprecipitated the WT RH ␣ 1C protein expressed in Xenopus oocytes and metabolically labeled with [ 35 S]methionine/cysteine (Fig. 1A, lane 2), but not the truncated mutant missing the first 46 aa, ␣ 1C ⌬N 2-46 (Fig. 1A, lane 1). Card-I and Card-C immunoprecipitated both the WT ␣ 1C and ␣ 1C ⌬N 2-46 (Fig. 1, A  and B); the level of expression of the full-length ␣ 1C was higher than that of ␣ 1C ⌬N 2-46 (Fig. 1B), as reported previously (1). Card-C precipitated the WT channel more efficiently than Card-N (Fig. 1A). No 35 S-labeled ␣ 1C was detected in oocytes that were not injected with RNA by any of the antibodies (Fig.  1, A and B). The fact that bands of the same size of WT ␣ 1C are detected by antibodies directed to the extreme N and C termini and a mid-portion of the channel supports the notion (37) that the oocyte expresses the whole-length protein not truncated at any of its termini. Notably, under the conditions used here, the WT ␣ 1C protein runs on SDS-polyacrylamide gels as an ϳ207-kDa band, as reported previously in the oocytes (1,37). Because the calculated molecular mass of this protein is ϳ242 kDa (36), the error (underestimate) of its size is about 35 kDa. The underestimation may result from the established fact that hydrophobic proteins tend to run on SDS-polyacrylamide gels faster than standard, water-soluble molecular mass markers (40).
Western blots of rat ventricle membranes with Card-N revealed a major band at ϳ207 kDa and a minor band at ϳ160 kDa; labeling of both bands was completely suppressed in the presence of the N 1-46(S44A) GST-fusion protein against which the antibody was raised (Fig. 1C). This result confirms the specificity of Card-N and demonstrates, for the first time on the protein level, the presence of an RH-type initial segment in the N terminus of ␣ 1C in the rat heart.
Western blots of rat heart (ventricle), brain, and liver were done with all three antibodies (Fig. 1D). Card-C antibody detected an ϳ207-kDa band in the ventricle. A higher, ϳ240-kDa band was observed in all tissues. However, we cannot discard the possibility that this labeling is nonspecific because this band was not detected by the other two antibodies. The Card-I and Card-N antibodies detected the ϳ207-kDa band in ventricle and in the brain, although labeling with Card-I was weak. This may be because of sequence divergence between the rabbit cardiac ␣ 1C and isoforms of rat brain channel that show variability in the loop II-III (41). With Card-N, the intensity of this band varied in different blots, and in some instances it was even stronger in the brain than in the ventricle (data not shown). Because brain ␣ 1C was not detected by Card-C but detected by Card-N, the very end of the C terminus in a majority of brain ␣ 1C protein may be missing or different from the cardiac one. An additional band at ϳ160 kDa was detected in the liver by Card-I and Card-N but not by Card-C; Card-N detected a similar band in the ventricle. These results support the notion (42) that only a small percentage of the L-type channel in the heart is truncated at the end of its C terminus (see also Ref. 43). The actual size of the truncated protein is probably higher than 160 kDa because of the underestimation of the size by SDS-polyacrylamide gel electrophoresis in our conditions. Because ␣ 1C RNA has been found in whole liver but not in hepatocytes (44), the ϳ160-kDa protein may be present in nonhepatocyte tissues, e.g. in the blood vessels.
The main conclusion of this part of the study is that ␣ 1C protein containing the RH-type N terminus is present in rat heart and brain, and the L-type Ca 2ϩ current in these tissues can be expected to be stimulated by PKC. The variability of PKC effects on neuronal L-type channels (23)(24)(25)(26)(27)(28)(29) may result from the presence of different isoforms of ␣ 1C in different neuronal cell types.
The First Five Amino Acid Residues of ␣ 1C Are Critical for PKC Modulation-Deletion of the first 46 aa residues of the ␣ 1C , which are unique to the RH-type N terminus ( Fig. 2A), increases the Ca 2ϩ channel current and also eliminates the PKC-induced augmentation (1). The effects of deletions shorter than 40 aa have not been studied (1,45). To narrow down the segment crucial for PKC effect, we have prepared three additional deletion mutants: ␣ 1C ⌬N 2-5 , ␣ 1C ⌬N 2-20 , and ␣ 1C ⌬N 2-25 , lacking aa 2-5, 2-20, and 2-25, accordingly. All truncated channels produced whole-cell currents 3-10-fold larger than WT in Ͼ5 oocyte batches. For a quantitative comparison, RNAs of WT and of four deletion mutants were prepared on the same day, in parallel, and injected (together with ␣ 2 /␦ subunit) into oocytes of the same donor. Fig. 2B shows that deletion of the first 20 aa was sufficient to cause a maximal current increase, similar to that produced by the deletion of 46 or 139 aa. Deletion of aa 2-5 also significantly (p Ͻ 0.01) increased the current but less well than of 20 or more aa. Because coexpression of the ␤2A subunit increased WT currents better than those of Nterminal truncation mutants ␣ 1C ⌬N 2-46 and ␣ 1C ⌬N 2-139 , we proposed that part of ␤2A-induced enhancement is because of an allosteric hindrance of the N-terminal inhibition of gating (1). Fig. 2F shows that the enhancement of peak currents of ␣ 1C ⌬N 2-5 and ␣ 1C ⌬N 2-20 caused by coexpression of ␤2A is also weaker than that of the WT channels; this was observed at all voltages. Thus, the first 20 aa are crucial for the inhibitory effect of the N terminus on L-type channel gating and on its interaction with ␤2A, and the first 5 aa constitute an important component. Fig. 2 shows representative current traces (Fig. 2C) and time course of the effect of the phorbol ester PMA (Fig. 2D) in oocytes expressing WT, ⌬N 2-5 , or ⌬N 2-20 ␣ 1C (with the ␣ 2 /␦ subunit). Two oocyte batches in which the WT channel showed high sensitivity to PMA, and the Ca 2ϩ channel current was increased 1.6 -4-fold (summarized in Fig. 2E), have been used in these experiments. The removal of aa 2-5 reduced the PMA effect by more than 90%; the remaining increase (13 Ϯ 4.4%; mean Ϯ S.E.) was small but statistically significant (p Ͻ 0.02). Channels lacking the first 20 aa were insensitive to PMA (6.4 Ϯ 8.1%). Thus, the first 5 aa are very important, and the first 20 aa are crucial for the PKC effect.
PKC Does Not Phosphorylate the Segment Crucial for Its Physiological Effect-Based on effects of PKC activators and inhibitors, the effect of PMA is expected to result from a PKCcatalyzed phosphorylation (21,22). Because none of the first 5 aa of ␣ 1C are serines or threonines ( Fig. 2A), it is not possible that PKC directly phosphorylates this segment. None of the residues in the first 20 aa is a consensus PKC site, but a cryptic site (T 10 or S 18 ) might be a target for PKC. Therefore, we have examined in vitro phosphorylation by purified PKC of GSTfusion proteins of segments of the N terminus: N 1-46(S44A) ,  3 and 6) from oocytes of one donor injected with the corresponding RNAs, but not from uninjected oocytes (lanes 1 and 4). In panels A and B, ␣ 1C was coexpressed with ␣ 2 /␦. In each lane, immunoprecipitates from 5 oocytes were loaded. C, Western blot of rat ventricular membranes with the Card-N antibody in the absence (lane 2) or presence (lane 1) of the GST-fusion protein N 1-46(S44A) . 10 ml of the antibody (dilution 1:1000) were incubated overnight at 4°C with 80 g of N 1-46(S44A) , then additional 80 g were added, and the antibody/GST fusion protein mixture (lane 1), or 10 ml of the antibody without N 1-46(S44A) (lane 2), were incubated with the nitrocellulose membranes for 2 h at room temperature. D, detecting the Ca 2ϩ channel ␣ 1C subunit isoforms in ventricle, brain, and liver by immunoblots with Card-N, Card-I, and Card-C. N 1-154 (the whole N terminus), N 47-154 , and N 84 -154 . As controls, we used GST and the GST fusion proteins of the loop I-II of RH ␣ 1C and of the N terminus of the rat brain ␣ 1C , N(neuronal) 1-124 . Fig. 3 shows that N 1-154 and N 47-154 were strongly phosphorylated; weaker signals were observed in N 87-154 and in N(neuronal) 1-124 . GST alone, N 1-46(S44A) , and loop I-II were not phosphorylated. Thus, the first 20 aa, contained within the GST fusion protein N 1-46(S44A) , are not phosphorylated under these conditions, whereas other parts of the N terminus are. The physiological significance of the phosphorylation of these distal parts of the N terminus is unclear at present.
In summary, our results demonstrate the presence of ␣ 1C protein isoform(s) containing an RH-type N terminus in rat heart (ventricle) and brain. Further, we have demonstrated that the initial 20 amino acids are crucial both for the inhibitory gating by the N terminus, the allosteric interaction of the N terminus with the ␤ subunit, and for the PKC effect. Removal of the first 5 aa already strongly hampers the inhibitory function of the N terminus and almost fully abolishes the PKC effect. The correlation between the location of residues crucial for these two functions supports the hypothesis (1) that PKC exerts its stimulating action by attenuating the inhibition im-posed on the channel by the N terminus.
Determination of the mechanism of PKC action is a challenge for the future. Our data suggest that the effect of PKC is not attained by a direct phosphorylation of the initial 20 aa of FIG. 3. In vitro phosphorylation of ␣ 1C fusion proteins by purified PKC. GST-fusion proteins of the indicated constructs were phosphorylated as described under "Experimental Procedures." The products were resolved by SDS-polyacrylamide gel electrophoresis, followed by autoradiography. FIG. 2. The importance of the initial N-terminal segment of ␣ 1C in modulation of the channel activity and in mediation of the PKC effect. A, alignment of protein sequences predicted from cDNA clones from rabbit heart (36), rat heart (6), and rat brain rbC-II (7). Identical amino acids are shown by dashes; empty spaces are gaps introduced for optimal sequence alignment. B, comparison of Ca 2ϩ channel currents (carried by Ba 2ϩ , I Ba ) in groups of oocytes of one donor injected with RNAs of the indicated constructs of ␣ 1C , together with the RNA of ␣ 2 /␦. 8 to 9 oocytes were tested in each group. Currents were normalized to the mean I Ba measured in the WT group. The increase in I Ba compared with control was significant in all groups (p Ͻ 0.05 or better, ANOVA multiple comparison test followed by Dunn's test). C, representative current traces in oocytes of one batch expressing ␣ 1C WT or ␣ 1C ⌬N 2-5 (with ␣ 2 /␦) before and 10 min after the addition of 10 nM PMA. D, time course of PMA effect on I Ba in representative oocytes of the same donor, expressing ␣ 2 /␦ and the indicated construct of ␣ 1C . In each cell, the stability of the current was verified for at least 3 min before the addition of PMA (not shown). PMA addition is shown as t ϭ 0. E, a summary of the effect of PMA on channels containing ␣ 2 /␦ and either ␣ 1C WT, ␣ 1C ⌬N 2-5 , or ␣ 1C ⌬N 2-20 . Two batches of oocytes, 11 to 12 oocytes in each group. The effect of PMA is presented as percent increase of I Ba in each cell 10 min after the addition of PMA. Asterisks indicate statistically significant differences (p Ͻ 0.02 or better). F, the effect of coexpression of ␤2A subunit on channels containing ␣ 2 /␦ and either ␣ 1C WT, ␣ 1C ⌬N 2-5 , or ␣ 1C ⌬N 2-20 (at ϩ10 mV). the N terminus. This conclusion is supported both by the amino acid composition of this segment, especially of the first 5 aa crucial for PKC action, and by the absence of phosphorylation of the GST fusion protein containing the initial segment. What can be the mechanism of PKC action? There are at least two possibilities. PKC may phosphorylate a site at ␣ 1C which is remote from the initial N-terminal segment but interacts with it directly or allosterically. If such interaction is permissive for N-terminal effect on gating (inhibition), phosphorylation by PKC may weaken the inhibition. Another possibility is that PKC phosphorylates an auxiliary protein, yet unidentified, which either aids the N-terminal inhibition (and the phosphorylation obstructs this effect), or attenuates the N-terminal inhibition when phosphorylated by PKC.