Analysis of the 5 '-upstream region of mouse P/Q-type Ca2+ channel alpha(1A) subunit gene for expression in pancreatic islet beta cells using transgenic mice and HIT-T15 cells

The alpha-agatoxin-IVA-sensitive P/Q-type Ca2+ channel plays a role in insu lin release from the pancreatic islets of beta cells. To dissect the molecu lar mechanisms underlying beta cell expression of the P/Q-type channel, we characterized the 5'-upstream region of the mouse alpha(1A) subunit gene us ing transgenic mice and HIT insulinoma cells. The E. coli lacZ reporter gen e was expressed in pancreatic acini and islets in transgenic mice carrying the 6.3 kb or 3.0 kb of the 5'-upstream region, although those with 1.5 kb or 0.5 kb of the 5'-upstream region failed to show reporter expression on h istological examination. As the expression of alpha(1A) subunit gene could not be detected in acini using RT-PCR analysis, the reporter expression in acini might have been ectopic expression. When linked to the placental alka line phosphatase reporter gene to examine promoter activity for beta cell e xpression, the 6.3 kb and 3.0 kb fragment of the 5'-upstream region, but no t the smaller 1.5 kb fragment, were able to drive reporter gene expression in HIT cells. The sequence between 3.0 and 1.5 kb upstream of the start cod on enhanced thymidine kinase promoter activity in HIT cells, but not in fib roblast NIH3T3 cells. These results suggested that the beta cell-specific e lements of the alpha(1A) subunit gene are likely to be located in the dista l upstream region (-3021 to -1563) of the 5'-upstream sequence and that the 6.3 kb fragment of the 5'-upstream region alone might be a lack of a negat ive cis-regulatory element(s) to suppress the alpha(1A) subunit gene expres sion in acini.