cDNA cloning of the new nigedipine-insenstive voltage-dependent Ca^2+ channels in the peripheral resistant artery..

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We attempted the cDNA cloning of nifedipine-insensitive voltage-dependent Ca^<2+> channels (VDCC) which predominantly distribute in the peripheral mesenteric arteries and exhibit unique properties distinct from those of hitherto-known VDCCs. (1)We first performed a RT-PCR detection of so far known VDCC isoforms, especially of T -type, in rat aorta, mesenteric artery, cerebral artery and brain. While the transcripts of α1G isoform were almost equally amplified from all tissues examined, those of α1H were variable depending on the region of arteries, and all was entirely undetectable. (2)We then constructed a cDNA library from about 3000 segments dissected from the peripheral regions of rat mesenteric artery. By using this library as a template, the presence of both α1G and α1H was confirmed by PCR amplification. (3)We next attempted to amplify the splice variants of α1H isoform from this library. For this purpose, the full length of the wild-type α1H was divided into 6 regions with some … More base overlaps, for each of which specific primer pairs were designed. PCR amplification was performed with these primers, and DNA fragments corresponding to four middle regions excluding the N-terminal (which includes the transcription initiation site)' and C-terminal ends were obtained. Direct sequencing of these DNA fragments revealed several important mutations therein. (4)We are now performing a sequence comparison between the four DNA fragments and the splice variants of α1H isoform recently cloned from human uterine smooth muscle, to find any significant similarities. In parallel with this, the electrophysiological properties of each human α1H splice variant expressed in HEK293 cells are now being thoroughly investigated, especially with respect to the threshold potential for activation and inactivation. We have already found, in collaboration with others, that some α1H splice variants (those having defects in the III-IV linker region) evaluated in the Xenopus oocyte system show appreciable depolarization shifts of activation potential. We will also focus on a possible modulatory effect of calmodulin-dependent kinase II -mediated phosphorylation on α1H channel gating, since this would be a mechanism by which the activation threshold is dynamically regulated in in situ by its phosphorylated state and may perhaps explain the unique gating properties of NICCs. Less