| Research Abstract |
Recent investigations including ours have suggested that a mammalian homologue of transient receptor potential (TRP) protein, TRPC6, is, the most abundantly expressed TRP protein in vascular smooth muscle tissues and plays a pivotal role in vascular tone regulation as a receptor-operated Ca^<2+> entry channel activated during the vascular sympathetic nerve excitation. Based on these findings, we have launched on the development of a new prototypic antihypertensive drug targeting TRPC6 by means of natural toxin screening which specifically inhibits its channel activities. In the first step of this project, we screened commercially available toxins extracted from both terrestrial and marine organisms which reportedly inhibit several voltage-dependent and fast ligand-gated channels, but none of them were found to be effective. In the next step, we collected crude toxin extracts from several snakes and tried to purify them to a single fraction containing TRPC6 channel-specific inhibitory a
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ctivities, by repeating the purification processes of the crude toxins with Sephadex chromatography and Ion exchange chromatography. As the result, two small subfractions which show potent and dose-dependent inhibitory actions on TRPC3 and TRPC7 (two closely related homologues of TRPC6) but not on TRPC6, have been isolated. The estimated molecular weight of toxins contained in the two subfractions is 65,000 and <1000KDa, respectively. Exploration of the site of actions of these subfractions by use of chimaeric TRP channels have revealed that the transmembrane region is involved in this inhibition. TRPC3, 6 and 7 channels constitute a subfamily amongst which more than 80% amino acid identity is found, but exhibit differential sensitivities to Ca^<2+>, flufenamate and mechanical stress imposed on the cell membrane. These findings strongly suggest that subtle differences in amino acid sequence, especially near the ion conductive pore region, would greatly change the sensitivity of these channels to various physiological modulators as well as pharmacological agents. In the subsequent period of research, we will put forward the purification process of the effective subfractions to yield single inhibitory peptides with computer-aided structural analysis, and simultaneously the identification of the sites of their actions, whereby the indispensable database for the development of specific TRP channel inhibitors is obtained, which would eventually lead to the discovery of a new generation of antihypertensive drug. Less
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