PandP2003

Research representative: Prof. ITO, Yushi
(Graduate School of Medical Sciences)

　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-I_Ca) carried in these channels appeared to increase markedly along the lower branches of mesenteric artery, amounting to almost 100% of I_Ca in the periphery. With 5 mmol/L Ba²⁺ as the charge carrier, NI-I_Ca 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-I_Ca 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 μmol/L, respectively). Relatively specific blockers for N- and P/Q-type Ca²⁺-channels such as ω-conotoxins GVIA and MVIIC (each 1 μmol/L) and w-agatoxin IVA (1 μmol/L) were ineffective at inhibiting this NI-I_Ca channel, whereas nimodipine blocked the current (~ 75% with 1 mmol/L: IC₅₀: 107 μmol/L). Although these properties are reminiscent of R-type Ca²⁺ channels, expression of the α_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.

　If the NI-I_Ca 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.

　In order to get more insight into the molecular nature of NI-I_Ca channel and facilitate the understanding of their molecular entity, we investigated the pharmacological profile of NI-I_Ca in further detail, in comparison with those of R- and T-type Ca²⁺ channels, which resemble NI-I_Ca channels. For this purpose, we employed cell lines stably expressing α_1E and α_1G. We chose α_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-I_Ca 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-I_Ca channels exhibit very similar sensitivities to many organic compounds including mibefradil compared with T-type Ca²⁺ channels.