External pH effects on the depolarization-activated K channels in guard cell protoplasts of Vicia faba.

Abstract

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 stomata, respectively. During these aperture and pH changes, massive K § fluxes cross the cellular plasma membrane driving the osmotic turgor and volume changes of guard cells. Therefore, we examined the effect of extracellular pH on the depolarizationactivated K channels (KD channels), which constitute the K § efflux pathway, in the plasma membrane of Viciafaba guard cell protuplasts. We used patch clamp, both in whole cells as well as in excised outside-out membrane patches.Approximately 500 KD channels, at least, could be activated by depolarization in one protoplast (density: ~0.6 ~m-2). Acidification from pH 8.1 to 4.4 decreased markedly the whole-cell conductance, GK, of the KD channels, shifted its voltage dependence, GK-EM, to the right 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 GK-EM shifts, the estimated average negative surface charge spacing near the KD channel is 39 /~. To quantify the effects of protons on the rates of transitions between the hypothesized conformational states of the channels, we fitted the experimental macroscopic steady state conductancevoltage relationship 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 properties via a decrease in localized, rather than homogeneous, surface charge sensed by the gating moieties. In terms of either the CO or CCO model, the protonation of a site with a pKa of 4.8 decreases the voltage-independent number of channels, N, that are available for activation by depolarization.

The Role of Potassium Channels in the Temperature Control of Stomatal Aperture

“…The E,,,s of the two current types were determined from tailcurrent experiments (Hodgkin and Huxley, 1952). The linearity of the instantaneous whole-cell IK-E, relationships, as well as of the unitary is-EM relationships, in the presence of 11 mMK+ in the externa1 solution (Ilan et al, 1994;and our unpublished data), justifies the use of Equation 1 for the calculation of G, of both K, and K, channels. At a11 E,s, G, reflects two factors:…”

“…Plants of Vicia faba were grown under controlled environmental conditions as described previously (Ilan et al, 1994). Growth temperature was kept at 22°C during the day and 17°C at night.…”

“…Experiments were performed in a voltage-clamp mode as described by Ilan et al (1994), and we measured currents elicited by preprogrammed depolarizing and hyperpolarizing voltage pulses applied at 20-or 13-s intervals, respectively. A11 E, values were corrected for liquid-junction potentials, measured using 3 M KC1-agar bridges, by -17 mV when the [K+l, was 11 mM and by -8 mV when [Kt], was 50 mM.…”

“…IK values were obtained by subtracting the leak current (the instantaneous current at the beginning of the voltage pulses, Ilan et al, 1994) from the current measured at the end of the voltage pulses. We calculated G, at various E,s, from the relation between IK and E, .…”

The Clickable Guard Cell, Version II: Interactive Model of Guard Cell Signal Transduction Mechanisms and Pathways

“…fying K + (K + in ) channels (Schroeder et al, 1987;Pilot et al, 2001) and activates outward-rectifying K + (K + out ) channels (Schroeder et al, 1987;Hosy et al, 2003), resulting in a net K + efflux from guard cells (Figure 1). In addition, during stomatal closure ABA causes an alkalization of the guard cell cytosol (Blatt and Armstrong, 1993) which directly enhances K + out channel activity (Blatt, 1992;Blatt and Armstrong, 1993;Ilan et al, 1994;Miedema and Assmann, 1996), but also down-regulates the transient R-type anion channels (Schulz-Lessdorf et al, 1996). The efflux of both anions and K + from guard cells via anion and K + out channels contributes to loss of guard cell turgor, which leads to stomatal closing (Schroeder and Hagiwara, 1989;Pei et al, 1997;Vahisalu et al, 2008).…”