Biological significance of palytoxin-sensitive ion channel associated with NaィイD1+ィエD1,KィイD1+ィエD1-ATPase molecule

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1) Electophysiological properties of palytoxin (PTX)-induced channel current in vascular smooth muscle cells and megakaryocytes were investigated using a patch clamp technique. PTX-induced channel in both cell types was permeable to monovalent cations but not to divalent cations. Ouabain, an inhibitor of Na,K-ATPase, suppressed the current, suggesting that PTX-sensitive channel is associated with Na,K-ATPase. A current dependent on Na pump was observed in the presence of PTX. This suggests that the Na pump is still operable after induction of the channel by PTX.2) PTX increased cytosolic CaィイD12+ィエD1 ([CaィイD12+ィエD1]ィイD2iィエD2) in vascular smooth muscle cells. Analysis of [CaィイD12+ィエD1]ィイD2iィエD2 mobilization revealed that one mechanism for the increase is activation of L-type CaィイD12+ィエD1 channels as a result of depolarization. Another mechanism is inhibition of CaィイD12+ィエD1 extrusion through NaィイD1+ィエD1-CaィイD12+ィエD1 exchanger since PTX increased [NaィイD1+ィエD1]ィイD2iィエD2 and caused depolar … More ization.3) To explore whether catalytic subunit of Na,K-ATPase is responsible for induction of PTX-sensitive channel, we transfected yeast cells, which do not have endogenous Na,K-ATPase, with wild type Na,K-ATPase (NNN) gene or its chimeric gene (NCN), in which the catalytic subunit was replaced with that of endoplasmic reticulum Ca-ATPase, and observed the effect of PTX on KィイD1+ィエD1 efflux. PTX increased KィイD1+ィエD1 efflux from NNN- and NCN-expressed cells but not from non-transformed cells. Ouabain inhibited the efflux. When ouabain-resistant NNN and NCN were transfected to yeast, PTX also increased KィイD1+ィエD1 efflux. KィイD1+ィエD1 efflux in these cells was insensitive to ouabain. Na azide and Na orthovanadate, which inhibit Na,K-ATPase, depressed the effect of PTX in an cDNA-transfected cells.These data suggest that PTX-sensitive channel is originally a pore to transport NaィイD1+ィエD1 and KィイD1+ィエD1 in the enzyme and the site of channel is far from the catalytic subunit. Induction of the channel by PTX seems to depend on a conformation state of the enzyme. Less