Membrane targeting of L-type calcium channels -: Role of palmitoylation in the subcellular localization of the β2a subunit

In this study, we report that palmitoylation was a critical determinant of the subcellular localization of the rat beta(2a) subunit of voltage-dependent calcium channels. Immunohistochemical staining of transfected cells revealed that a palmitoylation-deficient beta(2a) subunit exhibited 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) protein, A mutation in the beta interaction domain, which disrupted interactions 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 the 16-amino acid N terminus of the beta(2a) subunit was sufficient to confer palmitoylation to the nonpalmitoylated beta(1b) and beta(3) isoforms. 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 brefeldin A increased the amount of palmitic acid incorporated in the beta(2a) protein, suggesting that palmitoylation of beta(2a) occurs during or shortly after protein synthesis. Two other beta(2) variants, the 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.