A single amino acid mutation results in a rapid inactivation of epithelial calcium channels.
Epithelial Ca(2+) channel (ECaC1 and 2 = CAT1) molecules are characterized by properties including inward rectification and Ca(2+)-dependent fast and slow inactivation. To elucidate the electrophysiological differences based on the amino acid residues, we compared human and rodent ECaC1, and ECaC2 alignments, made mutants, and investigated their function in Xenopus and mammalian cells. Expression of the ECaC1 mutant Q579H and a H587Q mutation in ECaC2 in Xenopus oocytes resulted in a possible change in the rate of fast decay. Currents of H587C and H587N were not detected, and the H587R diminished the rate of rapid decay. Treatment of the oocytes with BAPTA magnified the amplitude of the current and abolished the decay. The expressions of mutants, therefore, implied that H587 in ECaC2 is a position related to the mechanism of the rapid decay rather than the magnitude of the current or the slow decay. Decay measurements were carefully performed in mammalian cells by tight-seal patch clamping. The rapid decay was exaggerated in H587C and H587N mutants but was undetectable in the H587R mutant. The results indicate that the amino acid 579Q of ECaC1, corresponding to 587H of ECaC2, is of primary importance in the structure for the fast inactivation by intracellular Ca(2+).
Suzuki
Makoto
M
Department of Pharmacology, Jichi Medical School, 3311-1, Yakushiji, Minamikawachi, Tochigi 329-04, Japan. macsuz@jichi.ac.jp
Ohki
Gaku
G
Ishibashi
Kenichi
K
Imai
Masashi
M
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D015220:Calcium Channels / Q000235:genetics* / Q000502:physiology*; D006224:Cricetinae; D004553:Electric Conductivity; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D050916:TRPV Cation Channels; D014162:Transfection; D014981:Xenopus
D015220:Calcium Channels; D050916:TRPV Cation Channels; C492239:TRPV5 protein, human; C120776:TRPV6 channel