| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2001: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
In mouse C127 cells, we found a new swelling-activated Gd-sensitive anionic current, which had no outward rectification, and inactivated at voltages greater than ±25 mV. Single-channels of 300-400 pS in on cell and inside-out patches displayed inactivation pattern similar to whole-cell currents. The channels had an ATP-binding site in the middle of the pore and were ATP-conductive with P_<ATP>/P_<Cl> of 0.09. The pharmacological profile of single-channel activity was same to that found in swelling-induced ATP release. Thus, it is concluded that the volume- and voltage-dependent ATP-conductive large-conductance anion channel (VDACL) serves as a conductive pathway for the swelling-induced ATP release in C127i cells.
Arachidonic acid (AA) down-regulated both VDACL currents and swelling-induced ATP release in physiological concentration range with K_d of 4-6 μM. This effect was direct and not mediated by downstream metabolic products. A membrane-impermeable analog arachidonyl coenzyme A was
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effective only from the cytosolic side suggesting that the binding site is localized intracellularly. ATP^4-currents were also reversibly inhibited by AA. Thus, we conclude that swelling-induced ATP release and its putative pathway, VDACL anion channel, are under a negative control by intracellular arachidonic acid signaling in mammary C 127 cells.
We have attempted to identify VDACL channel in two different ways. (1) Since electrophysiologically, VDACL - channel resembles the mitochondrial voltage-dependent anion channel (VDAC), we have tested the hypothesis that VDACL corresponds to plasmalemmal (pl-) subtype of VDAC. We detected the mRNA expression of pl-VDACL which was identical to the reported pl-VDAC sequence. However, we failed to reproduce VDACL channels phenotype by heterologous expression. Next, we tested the effects specific antagonists of mitochondrial ATP/ADP translocator, atracyloside and bongcrekic acid on ATP release and found no consistent effect, therefore, this transporter is unlikely to be a molecular candidate for VDACL channel. Less
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