Potassium and voltage dependence of the inorganic pyrophosphatase of intact vacuoles from Chenopodium rubrum.
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概要
The activity and the voltage dependence of the inorganic pyrophosphatase (PPase) was measured on intact vacuoles of Chenopodium rubrum cells using the patch-clamp technique. With K+ at the cytoplasmic side a negative current representing the forward mode of the pump was measured after addition of pyrophosphate (PPi). The pump was reversed and a positive current was detected after addition of orthophosphate (Pi) in the presence of K+ at the vacuolar side when a pH gradient across the tonoplast was applied. The PPase operates as a constant current source, because no voltage dependence was observed (-60 to 60 mV). The K+ dependence of the PPi-induced current was investigated by substitution of cytoplasmic K+ by other cations. The selectivity sequence was: K+ > or = Rb+ > NH4+ = Cs+ > Na+ > Li+ = choline+, and was independent of the membrane voltage and pHcyt. With Cs+ or Li+ in the bath and K+ inside the vacuole the PPi-induced current became voltage-dependent, and positive currents were observed even if the pump was geared to operate in the forward mode. We suggest a "tunneling' effect through a channel-like domain in the PPase molecule which, under defined electrochemical gradient conditions and in the presence of PPi, allows K+ ions to cross the energy barrier usually separating the cytoplasmic from the vacuolar face of the pump.