Membrane targeting of L-type calcium channels. Role of palmitoylation in the subcellular localization of the β(2a) subunit

In this study, we report that palmitoylation was a critical determinant of the subcellular localozation of the rat β(2a) subunit of voltage- dependent calcium channels. Immunohistochemical staining of transfected cells revealed that a palmitoylation-defieient β(2a) subunit exhibited a diffuse intracellular staining pattern, in contrast to the plasma membrane distribution seen with the wild-type β(2a) subunit. Unexpectedly, mutations in regions distal to the palmitoylation sites at Cys³ and Cys⁴ affected palmitoylation of the β(2a) protein. Mutations in an src homology 3 motif of the β(2a) subunit affected both palmitoylation and subcellular localization of the β(2a) protein. A mutation in the β interaction domain, which disrupted interactions between the expressed α₁ and β subunits, also resulted in a decreased palmitoylation and diffuse intracellular localization of the β(2a) protein. Studies of chimeric proteins revealed that the 16- amino acid N terminus of the β(2a) subunit was sufficient to confer palmitoylation to the nonpalmitoylated β(1b) and β₃ isoforms. However, palmitoylation of chimeric β subunits was by itself insufficient to restore the plasma membrane localization observed with the wild-type β(2a) protein. Treatment of transfected cells with brefeldin A increased the amount of palmitic acid incorporated in the β(2a) protein, suggesting that palmitoylation of β(2a), occurs during or shortly after protein synthesis. Two other β₂ variants, the rabbit β(2a) and β(2b), which lack the palmitoylation sties at Cys³ and Cys⁴, exhibited a diffuse intracellular staining pattern and were not palmitoylated.