CLONING OF A NOVEL ALPHA-1-SUBUNIT OF THE VOLTAGE-DEPENDENT CALCIUM-CHANNEL FROM THE BETA-CELL

To study the molecular regulation of voltage-dependent Ca 2+ channels (VDCCs) in the beta-cell, we have cloned a cDNA for the alpha1-subunit from a hamster insulin-secreting cell line (HIT-T15). The cDNA (HCa3a) encodes a 1610-amino acid protein with four repeating membrane domains and an overall structure characteristic of other alpha1-subunits. Although the cDNA shows a high degree of sequence homology (97%) with a rat brain alpha1-subunit (RBalpha1), the C-terminal 15 amino acids of HCa3a share no similarity with any cloned alpha1 protein. High stringency Northern blot analysis revealed a single transcript of approximately 8.6 kilobases in HIT cells and hamster pancreas. A similarly sized species was detected in hamster brain, heart, and skeletal muscle. Using polymerase chain reaction and a primer set unique to HCa3a, this alpha1 isoform was found to be expressed in islet cell lines derived from rat, mouse, and hamster. The HIT cell alpha1-subunit is also expressed in discrete regions of the rat central nervous system, including the cortex, cerebellum, hypothalamus, and brain stem. The expression of two alpha1 isoforms (HCa3a and cardiac) in the HIT cell underscores the possible complexity of VDCCs in the regulation of beta-cell signal transduction. With its widespread tissue distribution, HCa3a does not conform to the current classification system used for L-type VDCCs; this suggests that an alternative system of classification is required.