Gating and conductance in an outward-rectifying K+ channel from the plasma membrane of Saccharomyces cerevisiae

Abstract

The plasma membrane of the yeast Saccharomyces cerevisiae has been investigated by patch-clamp techniques, focusing upon the most conspicuous ion channel in that membrane, a K(+)-selective channel. In simple observations on inside-out patches, the channel is predominantly closed at negative membrane voltages, but opens upon polarization towards positive voltages, typically displaying long flickery openings of several hundred milliseconds, separated by long gaps (G). Elevating cytoplasmic calcium shortens the gaps but also introduces brief blocks (B, closures of 2-3 msec duration). On the assumption that the flickery open intervals constitute bursts of very brief openings and closings, below the time resolution of the recording system, analysis via the beta distribution revealed typical closed durations (interrupts, I) near 0.3 msec, and similar open durations. Overall behavior of the channel is most simply described by a kinetic model with a single open state (O), and three parallel closed states with significantly different lifetimes: long (G), short (B) and very short (I). Detailed kinetic analysis of the three open/closed transitions, particularly with varied membrane voltage and cytoplasmic calcium concentration, yielded the following stability constants for channel closure: K1 = 3.3 x e-zu in which u = eVm/kT is the reduced membrane voltage, and z is the charge number; KG = 1.9 x 10(-4) ([Ca2+].ezu)-1; and KB = 2.7 x 10(3)([Ca2+].ezu)2. Because of the antagonistic effects of both membrane voltage (Vm) and cytoplasmic calcium concentration ([Ca2+]cyt) on channel opening from the B state, compared with openings from the G state, plots of net open probability (Po) vs. either Vm or [Ca2+] are bell-shaped, approaching unity at low calcium (microM) and high voltage (+150 mV), and approaching 0.25 at high calcium (10 mM) and zero voltage. Current-voltage curves of the open channel are sigmoid vs. membrane voltage, saturating at large positive or large negative voltages; but time-averaged currents, along the rising limb of Po (in the range 0 to +150 mV, for 10 microM [Ca2+]) make this channel a strong outward rectifier. The overall properties of the channel suggest that it functions in balancing charge movements during secondary active transport in Saccharomyces.