MEMBRANE TARGETING OF L-TYPE CALCIUM CHANNELS - ROLE OF PALMITOYLATION IN THE SUBCELLULAR-LOCALIZATION OF THE BETA(2A) SUBUNIT

In this study, we report that palmitoylation was a critical determinan t of the subcellular localization of the rat beta(2a) subunit of volta ge-dependent calcium channels. Immunohistochemical staining of transfe cted cells revealed that a palmitoylation-deficient beta(2a) subunit e xhibited a diffuse intracellular staining pattern, in contrast to the plasma membrane distribution seen with the wild-type beta(2a) subunit. Unexpectedly, mutations in regions distal to the palmitoylation sites at Cys(3) and Cys(4) affected palmitoylation of the beta(2a) protein. Mutations in an src homology 3 motif of the beta(2a) subunit affected both palmitoylation and subcellular localization of the beta(2a) prot ein, A mutation in the beta interaction domain, which disrupted intera ctions between the expressed alpha(1) and beta subunits, also resulted in a decreased palmitoylation and diffuse intracellular localization of the beta(2a) protein. Studies of chimeric proteins revealed that th e 16-amino acid N terminus of the beta(2a) subunit was sufficient to c onfer palmitoylation to the nonpalmitoylated beta(1b) and beta(3) isof orms. However, palmitoylation of chimeric beta subunits was by itself insufficient to restore the plasma membrane localization observed with the wild-type beta(2a) protein. Treatment of transfected cells with b refeldin A increased the amount of palmitic acid incorporated in the b eta(2a) protein, suggesting that palmitoylation of beta(2a) occurs dur ing or shortly after protein synthesis. Two other beta(2) variants, th e rabbit beta(2a) and beta(2b), which lack the palmitoylation sties at Cys(3) and Cys(4), exhibited a diffuse intracellular staining pattern and were not palmitoylated.