| Budget Amount *help |
¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2002: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2001: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
Recently, epithelial calcium channels, ECaCl and ECaC2 (=CaT1), have been cloned as calcium selective epithelial channels.
(1) To investigate the role of ECaC2 in the kidney in vivo, the expression of ECaC2 mRNA and protein was examined. When mice were fed rich (or lack of) calcium in diet for one week, the low Ca diet led to hypocalcemia and increased expression of the channel. We next examined the influence of calcitonin, PTH and 1α, 25(OH)_2 vitamin D_3 on channel expression. Calcitonin, but not the other two hormones, increased the expression of ECaC2 protein.
(2) To elucidate the electrophysiologic differences based on the amino acid residues, we compared human and rodent ECaCl, and ECaC2 alignments, made mutants, and investigated their function in Xenopus and mammalian cells. The results indicate that the amino acid 579Q of ECaCl, corresponding to 587H of ECaC2, is of primary importance in the structure for the fast inactivation by intracellular Ca2+.
(3) The class of Ca2+ permeable cation channels is composed of large families with six transmembrane segments including TRP, VR, polycystin, ECaCs and melastatin (MLS). However, most of them are functionally silent and unexpressed in mammalian cells. An investigation of associated proteins brought us a possibility that blockade of calpain opens the silent channels. Using 1μM of blockers in whole cellular patch pipette fill we measured currents of CHO cells transfected by VR like 1 and 2, polycystin-2, or a melastatin-like new member (MLS3S). A significant conductance of every clone with characteristic rectification by blockers was demonstrated. The permeability to Ca2+ to them is similar to that reported. Western blot suggested that blockers did not affect an assembly of the protein but cleavage of the protein. Therefore, investigation of these families with the blockers may boost our knowledge of electrophysiologic function.
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