TPK1 Is a Vacuolar Ion Channel Different from the Slow-Vacuolar Cation Channel

摘要

TPK1 (formerly KCO1) is the founding member of the family of two-pore domain K⁺ channels in Arabidopsis (Arabidopsis thaliana), which originally was described following expression in Sf9 insect cells as a Ca²⁺- and voltage-dependent outwardly rectifying plasma membrane K⁺ channel. In plants, this channel has been shown by green fluorescent protein fusion to localize to the vacuolar membrane, which led to speculations that the TPK1 gene product would be a component of the nonselective, Ca²⁺ and voltage-dependent slow-vacuolar (SV) cation channel found in many plants species. Using yeast (Saccharomyces cerevisiae) as an expression system for TPK1, we show functional expression of the channel in the vacuolar membrane. In isolated vacuoles of yeast yvc1 disruption mutants, the TPK1 gene product shows ion channel activity with some characteristics very similar to the SV-type channel. The open channel conductance of TPK1 in symmetrically 100 mm KCl is slightly asymmetric with roughly 40 pS at positive membrane voltages and 75 pS at negative voltages. Similar to the SV-type channel, TPK1 is activated by cytosolic Ca²⁺, requiring micromolar concentration for activation. However, in contrast to the SV-type channel, TPK1 exhibits strong selectivity for K⁺ over Na⁺, and its activity turned out to be independent of the membrane voltage over the range of ±80 mV. Our data clearly demonstrate that TPK1 is a voltage-independent, Ca²⁺-activated, K⁺-selective ion channel in the vacuolar membrane that does not mediate SV-type ionic currents.