Research representative: Prof. ITO, Yushi
(Graduate School of Medical Sciences)
|ü@We have found nifedipine-insensitive (NI), rapidly inactivating, voltage-dependent Ca2+ channels with unique biophysical and pharmacological properties in the terminal branches of guinea pig mesenteric artery, by using a whole-cell mode of the patch-clamp technique. The fraction of the current (NI-ICa) carried in these channels appeared to increase markedly along the lower branches of mesenteric artery, amounting to almost 100% of ICa in the periphery. With 5 mmol/L Ba2+ as the charge carrier, NI-ICa was activated with a threshold of -50 mV, peaked at -10 mV, and was half-activated at -11 mV and inactivated at -52 mV, generating a potential range of constant activation near the resting membrane potential. The NI-ICa was rundown resistant, not subject to Ca2+-dependent inactivation, and the channel exhibited pore properties typical of high voltage-activated Ca2+ channels: Ba2+ was ~ 2-fold more permeable than Ca2+, and Cd2+ was a better blocker than Ni2+ (IC50, 6 and 68 â╩mol/L, respectively). Relatively specific blockers for N- and P/Q-type Ca2+-channels such as âÍ-conotoxins GVIA and MVIIC (each 1 â╩mol/L) and w-agatoxin IVA (1 â╩mol/L) were ineffective at inhibiting this NI-ICa channel, whereas nimodipine blocked the current (~ 75% with 1 mmol/L: IC50: 107 â╩mol/L). Although these properties are reminiscent of R-type Ca2+ channels, expression of the â┐1E mRNA was not detected with reverse transcriptase-polymerase chain reaction. This indicates the predominant presence of NI, high voltage-activated Ca2+ channels with novel properties, which may be abundantly expressed in peripheral small arterioles and contribute to their tone regulation.
ü@If the NI-ICa is indeed carried via a novel Ca2+ channel subtype, structural analysis of the channel compared with HVA and LVA families is likely to reveal nonconserved gating regions that can be exploited for rational drug design for the treatment of cardiovascular diseases.
ü@In order to get more insight into the molecular nature of NI-ICa channel and facilitate the understanding of their molecular entity, we investigated the pharmacological profile of NI-ICa in further detail, in comparison with those of R- and T-type Ca2+ channels, which resemble NI-ICa channels. For this purpose, we employed cell lines stably expressing â┐1E and â┐1G. We chose â┐1G rather than other T-type VDCC isoforms as it has been characterized and found to be expressed in some vascular tissues, we also used myocytes dissociated from the rat rather than guinea-pig mesenteric artery to record NI-ICa channels, since far greater genetic information is available for the rat than the guinea-pig. To validate this choice, in the first part of the study, we confirmed that NI-ICa almost identical to guinea-pig is present in the rat mesenteric terminal artery. Surprisingly, the results of this study have shown that despite clear biophysical differences, the rat NI-ICa channels exhibit very similar sensitivities to many organic compounds including mibefradil compared with T-type Ca2+ channels.