INTRODUCTION

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 suggested 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

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 N_KT3 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 2-16. 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  Antibody

A BamHI-XhoI fragment encoding residues 17-354 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 17-354 to glutathione S-transferase. Purified fusion protein was obtained by standard procedures and injected into a goat at Bethyl Laboratories (Montgomery, TX).

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 Dulbecco's modified Eagle's medium-Ham's F-12 medium (Sigma) or Sf900 (Life Technologies, Inc.)(for Sf9 cells) for 1-2 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 _2a was performed as described by Linder et al. (4). ³H-Radiolabeled standards for myristate, palmitate, and stearate were purchased from American Radiolabeled Chemicals.

Membrane 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.

Cells 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 µM 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 Isoforms

An epitope common to the four known  subunit isoforms (1, 7, 8, 9, 10) was used to generate a generic  antiserum (_GEN). The _GEN antiserum was able to specifically recognize _1b (~72-75 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 ³H-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 cell-specific (data not shown).

Characterization of four  subunits from [³H]palmitate-labeled 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 _GEN antiserum was capable of recognizing all four known  subunit isoforms expressed in transfected tsA201 cells. Each lane contained 50 µg 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.

Characterization of the Acyl Moiety on _2a

Acylation 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 linkage consistent with palmitoylation.

Because palmitic acid can be naturally metabolized to myristic acid, reverse phase HPLC was used to identify the base-sensitive 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 2-16) 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 N-terminal 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.

Identification of sites of palmitoylation within the _2a subunit.

Effects of Palmitoylation on the Membrane Association and Solubility of _2a

To 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 wild-type _2a is expressed as a multiple series of bands of 68-72 kDa that were previously shown to arise from unidentified post-translational modifications (2). In contrast, the palmitoylation-deficient 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 either salt- or detergent-solubilized fractions of transfected cells metabolically labeled with [³H]palmitic acid (Fig. 3B, top). However, only _2a immunoprecipitated from the detergent-solubilized 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 membrane localization and solubility of _2a.

To 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 _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.

Electrophysiological analysis of channels containing wild-type or palmitoylation-deficient _2a subunits.

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.