Communication

Identification of Palmitoylation Sites within the L-type Calcium Channel 2a Subunit and Effects on Channel Function*
(Received for publication, August 5, 1996, and in revised form, August 30, 1996) Andy J. Chien, Kristen M. Carr, Roman E. Shirokov, Eduardo Rios, and M. Marlene Hosey From the Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611 and the Department of Physiology, Rush University, Chicago, Illinois 60612

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 43, Issue of October 25, pp. 2646526468, 1996 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

The hydrophilic 2a subunit of the L-type calcium channel was recently shown to be a membrane-localized, post-translationally modified protein (Chien, A. J., Zhao, X. L., Shirokov, R. E., Puri, T. S., Chang, C. F., Sun, D. D., Rios, E., and Hosey, M. M. (1995) J. Biol. Chem. 270, 30036 30044). In this study, we demonstrate that the rat 2a subunit was palmitoylated through a hydroxylamine-sensitive thioester linkage. Palmitoylation required a pair of cysteines in the N terminus, Cys3 and Cys4; mutation of these residues to serines resulted in mutant 2a subunits that were unable to incorporate palmitic acid. Interestingly, a palmitoylation-deficient 2a mutant still localized to membrane particulate fractions and was still able to target functional channel complexes to the plasma membrane similar to wild-type 2a. However, channels formed with a palmitoylationdeficient 2a subunit exhibited a dramatic decrease in ionic current per channel, indicating that although mutations eliminating palmitoylation did not affect channel targeting by the 2a subunit, they were important determinants of channel modulation by the 2a subunit. Three other known subunits that were analyzed were not palmitoylated, suggesting that palmitoylation could provide a basis for the regulation of L-type channels through modification of a specific isoform.

gested that the 2a subunit was post-translationally modified, resulting in an increase in apparent molecular mass from 68 kDa for the nascent protein to proteins of 70 72 kDa. Here we demonstrate that one modification of the 2a subunit involves palmitoylation, a post-translational modification that has been shown to facilitate the membrane localization of other hydrophilic proteins. Palmitoylation involves the addition of palmitic acid to cysteine residues through a thioester linkage (3, 4). This modification is thought to be dynamic due to the reversible nature of the thioester bond (3, 4). However, despite the increasing number of palmitoylation sites identified, there appears to be no consensus motif for predicting candidate cysteine residues, which may be acylated. The residues involved in palmitoylation of the 2a subunit were identified, and the functional roles of these amino acids were investigated in biochemical and electrophysiological studies using mutant proteins. Three other known subunits were also analyzed and found not to undergo palmitoylation.
EXPERIMENTAL PROCEDURES

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The L-type calcium channel 2a subunit is a highly hydrophilic protein with no predicted membrane-spanning regions (1). Nevertheless, we previously demonstrated that this protein was membrane-localized when expressed in human embryonic kidney tsA201 cells (2). In addition, pulse-chase analysis sug* This work was supported in part by National Institutes of Health Grants HL23306 (to M. M. H.) and AR43113 (to E. R.) and by grants from the American Heart Association of Metropolitan Chicago (to R. S.). 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. Supported by National Research Service Award Predoctoral Fellowship 1 F30 MH10770 from the National Institutes of Mental Health. Permanent address: A. A. Bogomoletz Inst. of Physiology, Ukrainian Academy of Sciences, Kiev 252024, Ukraine. To whom correspondence should be addressed: Northwestern University Medical School, Dept. of Molecular Pharmacology and Biological Chemistry S215, 303 E. Chicago Ave. Chicago, IL, 60611. Tel.: 312-5033692; Fax: 312-503-5349; E-mail: mhosey@nwu.edu.

Construction of Expression Vectors and Palmitoylation Mutants The cDNAs for the different subunit isoforms were the generous gift of Dr. Ed Perez-Reyes (Loyola University, Chicago IL). The rat 1, 2a, 3, and 4 subunits were each expressed in tsA201 cells (HEK293 cells transformed with SV40 large T antigen) using methods previously described (2). The NKT3 mutant was constructed by ligation of two PCR1 products synthesized with the following primers: 5 -AGCAACAGCTCGGTCAGG-3 (plus strand 1); 5 -GGATCCCATGAAGAGGAGGCAGG-3 (minus strand #1); 5 -GGATCCGCAGACTCCTACACCAGCC-3 (plus strand 2); and 5 -GCTTTGCTTGAGACTTTCCTCG-3 (minus strand 2). The plasmid pRBG- 2a (2) was used as a template. A BamHI site was inserted to encode for residues 17 and 18, resulting in a cDNA sequence encoding for the 2a subunit minus residues 216. The (C3S), (C4S), and (C3S/C4S) mutants were constructed with the megaprimer protocol using the following minus strand mutagenic primers: 5 -GGCGATGTACTAGTCCGCAGCTCTGCATGAAGAGGTGGC-3 (C3S); 5 -GGCGATGTACTAGTCCGCTGCACTGCATGAAGAGGTGGC-3 (C4S); and 5 -GGCGATGTACTAGTCCGCTGCTCTGCATGAAGAGGTGGC-3 (C3S/C4S). For all mutants, PCR fragments were then substituted as BglII-XhoI fragments into the pRBG 2a-KT3 plasmid (2). All mutations were confirmed by dideoxy sequencing. Generation and Characterization of the Generic AntibodyA BamHI-XhoI fragment encoding residues 17354 of 2a was generated by PCR and subcloned into the BamHI-XhoI sites of pGEX-4T3 (Pharmacia Biotech Inc.), resulting in an in-frame fusion of 2a residues 17354 to glutathione S-transferase. Purified fusion protein was obtained by standard procedures and injected into a goat at Bethyl Laboratories (Montgomery, TX). Metabolic Labeling of Mammalian tsA201 and Sf9 Insect Cells Transfection of tsA201 cells was performed in 100-mm tissue culture plates as described previously (2). Sf9 insect cells were cultured using standard techniques and infected with recombinant baculovirus containing the 2a cDNA (5). At 40 45 h post-transfection, medium was removed, and cells were incubated with 0.5 mCi/ml of [3H]palmitic acid (American Radiolabeled Chemicals, St. Louis, MO) in Dulbeccos modified Eagles medium-Hams F-12 medium (Sigma) or Sf900 (Life Technologies, Inc.)(for Sf9 cells) for 12 h at room temperature with gentle rocking. An aliquot of the medium was analyzed for 3H content at the start and end of the metabolic labeling. Incorporation of radioactivity into the cells was estimated between 70 and 98%. Palmitoylation experiments were performed a minimum of three times with identical results. HPLC analysis to determine the identity of the acyl group on 3 2a was performed as described by Linder et al. (4). H-Radiolabeled standards for myristate, palmitate, and stearate were purchased from
1 The abbreviations used are: PCR, polymerase chain reaction; HPLC, high performance liquid chromatography.

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American Radiolabeled Chemicals. Immunoprecipitations and SDS-Polyacrylamide Gel Electrophoresis AnalysisMembrane preparations, immunoprecipitations, and immunoblots were performed as described previously (2). For fluorography, acrylamide gels were stained, fixed, treated with Amplify (Amersham Corp.) or EN3HANCE (DuPont NEN), and then exposed to film (DuPont NEN) for 1 4 weeks. Whole-cell Patch-clamp AnalysisCells were analyzed by whole-cell patch-clamp using data acquisition and pulse generation protocols similar to those previously described (2). Voltage pulse duration was 300 ms. The pipette solution contained (in mM): 110 cesium aspartate, 20 CsCl, 10 EGTA, 10 HEPES, 5 Mg-ATP. The extracellular solution contained (in mM): 150 tetraethylammonium chloride, 10 CaCl2, 10 HEPES. To record intramembrane charge movement, ionic currents were blocked by the addition of 10 M GdCl3 in the bath (6). Holding potential was 90 mV. Asymmetric currents were obtained by subtracting control transients obtained by pulsing between 150 and 90 mV. Maximal amplitude of the ionic current and maximal intramembrane charge movement were related to cell capacitance in order to compare their densities.
RESULTS AND DISCUSSION

Palmitoylation of Different Subunit IsoformsAn epitope common to the four known subunit isoforms (1, 710) was used to generate a generic antiserum ( GEN). The GEN antiserum was able to specifically recognize 1b ( 7275 kDa), 2a (68 72 kDa), 3 ( 58 kDa), and 4 ( 58 kDa) in tsA201 cells transiently transfected with cDNAs for each isoform (Fig. 1A). In order to investigate the possibility that subunits may be palmitoylated, transiently transfected tsA201 cells expressing different isoforms were metabolically labeled with [3H]palmitic acid, immunoprecipitated with the GEN antiserum, and subsequently analyzed for acylation of proteins. The resulting fluorogram (Fig. 1B) indicated incorporation of 3 H-radiolabel in a 70-kDa protein in 2a cells (Fig. 1A, second lane), suggesting the addition of an acyl group. However, no specific radiolabeling of the 1b, 3, and 4 subunits was observed. The presence of the different subunits in the immunoprecipitates was confirmed by immunoblotting (data not shown). Experiments were performed three times with identical results. Acylation of the 2a protein also occurred in Sf9 insect cells, indicating that this phenomenon was not cellspecific (data not shown). Characterization of the Acyl Moiety on 2aAcylation with palmitic acid occurs most frequently through a thioester linkage, which is sensitive to base hydrolysis (3, 4). To assess the nature of the acylation linkage on 2a, immunoprecipitated 2a from metabolically labeled cells was split into two equal fractions and treated with either 1 M hydroxylamine (pH 7.5) or 1 M Tris-HCl (pH 7.5) and analyzed by subsequent SDS-polyacrylamide gel electrophoresis and fluorography (Fig. 1C). Immunoblotting was used to confirm that equal amounts of 2a protein were loaded in each lane (Fig. 1C, upper panel). Treatment with hydroxylamine led to a clear loss of 3H signal on the fluorogram (Fig. 1C, lower panel), indicating that the acyl moiety on the 2a protein was attached via a base-sensitive

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FIG. 1. Characterization of four subunits from [3H]palmitatelabeled cells. A, a linear model of the 2a protein depicts the two domains conserved among all subunits, as well as the epitope against which the GEN antiserum was generated. Immunoblots show that the subunit GEN antiserum was capable of recognizing all four known isoforms expressed in transfected tsA201 cells. Each lane contained 50 g of membrane particulate fractions. B, immunoprecipitates from

transfected cells expressing the different subunit isoforms that were metabolically labeled with [3H]palmitic acid. The electrophoretic migrations of each subunit, as determined by immunoblot, are indicated by arrows on the right. Numbers on the left indicate the migration of molecular mass standards. C, palmitoylated 2a from metabolically labeled cells was immunoprecipitated and assessed for sensitivity to either 1 M hydroxylamine (pH 7.5) or 1 M Tris-HCl (pH 7.5). Shown are the immunoblot (upper panel) and the corresponding 3H-fluorogram of the same blot (lower panel). D, base-hydrolyzed extracts from 2a were analyzed by reverse phase HPLC. The arrowheads on top indicate the migration of 3H-radiolabeled lipid standards: myristate (C14:0), palmitate (C16:0), and stearate (C18:0). Counts from the 2a base-hydrolyzed extract migrated as a single peak corresponding to the same elution fraction as the palmitic acid standard.

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FIG. 2. Identification of sites of palmitoylation within the 2a subunit. A, shown is a sequence alignment of 2a with other calcium channel subunits and palmitoylated proteins from the G protein family, the Src family of protein tyrosine kinases, and GAP43 (palmitoylated cysteine residues are in bold type). B, site-directed mutants at Cys3 and Cys4 were expressed in metabolically labeled transfected tsA cells. Both the immunoblot (lower panel) and the 3H-fluorogram (lower panel) are shown. 3H counts in each respective subunit are indicated at the bottom of the panel.

FIG. 3. Effects of palmitoylation on membrane localization and solubility of 2a. A, the palmitoylation-deficient mutants were assessed for localization to particulate (P) or supernatant (S) fractions as described previously (2). B, immunoprecipitates of fractions solubilized in either 1% digitonin or 0.4 M NaCl are indicated. Shown are the 3 H-fluorogram (lower panel) and the corresponding immunoblot (upper panel).

linkage consistent with palmitoylation. Because palmitic acid can be naturally metabolized to myristic acid, reverse phase HPLC was used to identify the basesensitive radioactive label. After labeling Sf9 cells expressing the 2a protein with [3H]palmitic acid, base hydrolysis and reverse phase HPLC were performed as described in Linder et al. (4). Fractions eluted off the column were assayed using scintillation counting. The base-hydrolyzed fraction eluted as a single peak, corresponding to the same elution profile as the palmitate standard. A presample run prior to loading of the base-hydrolyzed fraction onto the column gave counts undistinguishable from background. These results demonstrated that the 2a protein was acylated with palmitic acid through a base-sensitive linkage. Identification of Sites Important for Palmitoylation of 2a The addition of palmitic acid usually occurs through a thioester linkage on cysteine residues, although no consensus sequence exists for predicting the exact location of the modification. The N-terminal region of 2a contains a Met-Xaa-Cys motif found in several palmitoylated proteins (Fig. 2A; Ref. 3). To assess whether this region of the 2a protein was involved in palmitoylation, an N-terminal mutant was constructed that contained a 15-amino acid deletion (residues 216) of the unique region of the 2a N terminus (see Experimental Procedures). This mutant was not able to incorporate palmitic acid (data not shown), suggesting that palmitoylation of 2a required the Nterminal region of 2a. To further investigate the site or sites of palmitoylation, site-directed mutants were constructed in which cysteine residues 3 and 4 were mutated to serine residues. These mutants, 2a(C3S), 2a(C4S), and 2a(C3S/C4S), were expressed in tsA201 cells and analyzed for incorporation of palmitic acid. The results indicated that only the wild-type 2a was palmitoylated, whereas all three of the mutants did not

incorporate palmitic acid (Fig. 2B). A corresponding Western blot from the same experiment demonstrated the amount of protein in each immunoprecipitation (Fig. 2B). The immunoreactive bands were excised from the nitrocellulose and analyzed with liquid scintillation counting, which confirmed the results of the fluorogram; values obtained in this experiment are shown at the bottom of Fig. 2B. These results indicated that both Cys3 and Cys4 were necessary for palmitoylation of 2a. Effects of Palmitoylation on the Membrane Association and Solubility of 2aTo test for a potential role for palmitoylation in the membrane localization of the 2a subunit, transfected tsA201 cells expressing either the wild-type 2a or the palmitoylation-deficient mutants were fractionated by centrifugation into membrane particulate fractions and soluble cytosolic fractions as described previously (2). Both the palmitoylated 2a protein and the palmitoylation-deficient 2a mutants still localized to membrane particulate fractions (Fig. 3A). The wildtype 2a is expressed as a multiple series of bands of 68 72 kDa that were previously shown to arise from unidentified posttranslational modifications (2). In contrast, the palmitoylationdeficient mutants did not appear to convert to the higher molecular mass isoforms, as evidenced by the decreased immunoreactivity at 70 72 kDa (Fig. 3A). Rather, all the palmitoylation-deficient 2a subunits exhibited distinctly lower molecular masses (Fig. 3A). These result suggest that palmitoylation might be required for the post-translational modification to the larger size forms seen with the wild-type 2a protein. In addition, the palmitoylation-deficient mutants appeared to be more susceptible to proteolysis, because several smaller immunoreactive bands were seen for each mutant (Fig. 3A). Further studies were performed to assess contributions of palmitoylation to the interaction of the 2a subunit with the membrane. The 2a protein could be immunoprecipitated from

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FIG. 4. Electrophysiological analysis of channels containing wild-type or palmitoylation-deficient 2a subunits. A, traces of whole-cell calcium current (left) and whole-cell charge movement (right) are shown from representative cells expressing 1C alone and 1C with either 2a or the 2a(C3S/C4S) mutant. Note the difference in vertical scale for 1C charge movement measurements. B, results from several cells are plotted to allow comparison of current density (ICa) with charge movement (Qmax) in channels containing 1C and either 2a (q, n 34) or 2a(C3S/C4S) (, n 8) subunits. The inset is an enlargement of the boxed area. This inset contains data from cells expressing 1C alone (E, n 12), which had very low whole-cell calcium current as well as charge movement, which was at the limits of detection.

2a(C3S/C4S) (Fig. 4A, left panel). In contrast, charge movement was increased to similar values in either 1C/ 2a or 1C/ 2a(C3S/C4S) cells relative to 1C cells, which exhibited charge movement that was at the lower limits of detection (Fig. 4A, right panel). This latter result indicated that similar numbers of functional channels were present in the plasma membrane of cells expressing either the wild-type or mutant 2a subunits. Because previous studies have demonstrated a role for subunits to recruit functional channels to the membrane (2, 12, 13), the present results demonstrate that wild-type and palmitoylation-deficient 2a subunits are equivalent in this function. However, a plot of peak current amplitude versus charge movement demonstrated that the relationship of the amount of peak current to charge movement was dramatically decreased in channels with the 2a(C3S/C4S) mutants compared with channels with wild-type 2a (Fig. 4B), which indicated that less current was carried per functional channel in channels containing 2a(C3S/C4S). These results suggested that the lack of palmitoylation and/or the lack of further post-translational modification that could require palmitoylation may have resulted in a 2a subunit that was able to target channels to the membrane as described previously (2) but was altered in its ability to modify calcium channel currents. Although it is difficult to attribute these functional changes directly to the loss of palmitic acid, it is clear that Cys3 and Cys4 were important determinants of palmitoylation and 2a modulation of channel function. The molecular mechanisms underlying dynamic palmitoylation and de-palmitoylation remain unclear, although it has been suggested that agonists of certain receptors can stimulate the de-palmitoylation of proteins (14, 15). Intracellular enzymes involved in either the palmitoylation or de-palmitoylation of proteins have only recently been identified (16, 17). Continuing investigations on the pathways and enzymes involved in the regulation of palmitoylation and depalmitoylation should facilitate studies on the role of this post-translational modification with respect to channel function.

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AcknowledgmentsThe tsA201 cell lines were the generous gift of Dr. Richard Horn (Thomas Jefferson University, Philadelphia PA). We thank Tipu S. Puri for assistance with the Sf9 cell culture and Dr. Maureen Linder for helpful advice concerning the metabolic [3H]palmitate labeling and the reverse phase HPLC analysis.
REFERENCES
1. Perez-Reyes, E., Castellano, A., Kim, H. S., Bertrand, P., Baggstrom, E., Lacerda, A. E., Wei, X., and Birnbaumer, L. (1992) J. Biol. Chem. 267, 17921797 2. Chien, A. J., Zhao, X. L., Shirokov, R. E., Puri, T. S., Chang, C. F., Sun, D. D., Rios, E., and Hosey, M. M. (1995) J. Biol. Chem. 270, 30036 30044 3. Milligan, G., Parenti, M., and Magee, A. I. (1995) Trends Biochem. Sci. 20, 181186 4. Linder, M. E., Kleuss, C., and Mumby, S. M. (1995) Methods Enzymol. 250, 314 330 5. Gerhardstein, B., Puri, T., Zhao, X., and Hosey, M. (1996) Biophys. J. 70, 186 (abstr.) 6. Shirokov, R., Ferreira, G., Yi, J., Zhou, J., Chien, A., Hosey, M., and Rios E. (1996) Biophys. J. 70, 183 (abstr.) 7. Castellano, A., Wei, X., Birnbaumer, L., and Perez-Reyes, E. (1993) J. Biol. Chem. 268, 3450 3455 8. Castellano, A., Wei, X., Birnbaumer, L., and Perez-Reyes, E. (1993) J. Biol. Chem. 268, 12359 12366 9. Ruth, P., Rohrkasten, A., Biel, M., Bosse, E., Regulla, S., Meyer, H. E., Flockerzi, V., and Hofmann, F. (1989) Science 245, 11151118 10. Pragnell, M., Sakamoto, J., Jay, S. D., and Campbell, K. P. (1991) FEBS Lett. 291, 253258 11. Mikami, A., Imoto, K., Tanabe, T., Niidome, T., Mori, Y., Takeshima, H., Narumiya, S., and Numa, S. (1989) Nature 340, 230 233 12. Josephson, I. R., and Varadi, G. (1996) Biophys. J. 70, 12851293 13. Kamp, T. J., Perez-Garcia, M. T., and Marban, E. (1996) J. Physiol. 492, 89 96 14. Robinson, L. J., Busconi, L., and Michel, T. (1995) J. Biol. Chem. 270, 995998 15. Wedegaertner, P. B., and Bourne, H. R. (1994) Cell 77, 10631070 16. Berthiaume, L., and Resh, M. D. (1995) J. Biol. Chem. 270, 22399 22405 17. Camp, L. A., Verkruyse, L. A., Afendis, S. J., Slaughter, C. A., and Hofmann, S. L. (1995) J. Biol. Chem. 269, 2321223219

either salt- or detergent-solubilized fractions of transfected cells metabolically labeled with [3H]palmitic acid (Fig. 3B, top). However, only 2a immunoprecipitated from the detergentsolubilized fraction contained [3H]palmitate-labeled 2a (Fig. 3B, bottom). This result demonstrates that the palmitoylation of 2a modifies the nature of its association with the membrane, confirming our previous hypothesis, which predicted distinct salt- and detergent-soluble populations of the 2a subunit (2). Effects of Palmitoylation on Channel Targeting and Channel FunctionTo address potential roles of palmitoylation on channel function and/or protein multimerization, cells expressing both the cardiac 1C subunit (11) as well as either 2a or 2a(C3S/C4S) were analyzed. Both 2a and 2a(C3S/C4S) could be co-immunoprecipitated with 1C, indicating that the subunits could be co-expressed and directly interact (data not shown). However, electrophysiological analyses demonstrated striking differences in the biophysical properties of channels with the 2a(C3S/C4S) subunit (Fig. 4, A and B). Measurements of whole-cell calcium currents demonstrated that the expression of 2a increased currents relative to 1C alone; however, drastic reductions were seen in cells expressing 1C and