EXTERNAL PH EFFECTS ON THE DEPOLARIZATION-ACTIVATED K-CHANNELS IN GUARD-CELL PROTOPLASTS OF VICIA-FABA

Previous studies reveal that the pH of the apoplastic solution in the guard cell walls may vary between 7.2 and 5.1 in closed and open stoma ta, respectively. During these aperture and pH changes, massive K+ flu xes cross the cellular plasma membrane driving the osmotic turgor and volume changes of guard cells. Therefore, we examined the effect of ex tracellular pH on the depolarization-activated K channels (K-D channel s), which constitute the K+ efffux pathway, in the plasma membrane of Vicia faba guard cell protoplasts. We used patch clamp, both in whole cells as well as in excised outside-out membrane patches. Approximatel y 500 K-D channels, at least, could be activated by depolarization in one protoplast (density: similar to 0.6 mu m-2). Acidification from pH 8.1 to 4.4 decreased markedly the whole-cell conductance, G(K), of th e K-D channels, shifted its voltage dependence, G(K)-E(M), to the righ t on the voltage axis, slowed the rate of activation and increased the rate of deactivation, whereas the single channel conductance was not affected significantly. Based on the G(K)-E(M) shifts, the estimated a verage negative surface charge spacing near the K-D channel is 39 Angs trom. To quantify the effects of protons on the rates of transitions b etween the hypothesized conformational states of the channels, we fitt ed the experimental macroscopic steady state conductance-voltage relat ionship and the voltage dependence of time constants of activation and deactivation, simultaneously, with a sequential three-state model CCO . In terms of this model, protonation affects the voltage-dependent pr operties via a decrease in localized, rather than homogeneous, surface charge sensed by the gating moieties. In terms of either the CO or CC O model, the protonation of a site with a pK(a) of 4.8 decreases the v oltage-independent number of channels, N, that are available for activ ation by depolarization.