ABA stimulation of outward K+ current (IK,out) in Vicia faba guard cells has been correlated with a rise in cytosolic pH (pHi). However, the underlying mechanism by which IK,out is affected by pHi has remained unknown. Here, we demonstrate that pHi regulates outward K+ current in isolated membrane patches from Vicia faba guard cells. The stimulatory effect of alkalinizing pHi was voltage insensitive and independent of the two free calcium levels tested, 50 nM and 1 microM. The single-channel conductance was only slightly affected by pHi. Based on single-channel measurements, the kinetics of time-activated whole-cell current, and the analysis of current noise in whole-cell recordings, we conclude that alkaline pHi enhances the magnitude of IK,out by increasing the number of channels available for activation. The fact that the pHi effect is seen in excised patches indicates that signal transduction pathways involved in the regulation of IK,out by pHi, and by implication, components of hormonal signal transduction pathways that are downstream of pHi, are membrane-delimited.
Arabidopsis thaliana has become a powerful tool in genetics and molecular biology. In order to use Arabidopsis as a model system for electrophysiological studies on plant cells, a detailed characterization of the transporters present in the plasma and vacuolar membranes of this species is required. We used the patch-clamp technique to study ion channels in the plasma membrane of Arabidopsis mesophyll cells. The most prominent conductance in these cells was a K(+)-selective, voltage-dependent, outwardly-rectifying channel (IK,out). In the whole-cell configuration, IK,out was observed in 100% of the cells assayed. In contrast, inward current was observed in less than 50% of the cells which were bathed in 100 mM K+, and was totally absent from cells bathed in 10 mM K+. The activation kinetics of IK,out were modulated by the external K+ concentration with a faster activation at low external K+. Tail-current analysis revealed that in addition to K+, IK,out is also permeable to Ca2+ and Ba2+. Externally applied Ba2+ also caused a voltage-dependent decrease in current magnitude, indicating that IK,out is also partially blocked by this classic K+ channel blocker. Single channels studied in outside-out patches showed Ca2+ and Ba2+ sensitivity, voltage dependence and time activation similar to that of IK,out in the whole-cell configuration. Given their permeability to Ca2+, these channels may function as an avenue for Ca2+ influx as well as K+ efflux, both of which may affect photosynthesis.
In vivo studies with leaf cells of aquatic plant species such as Elodea nuttallii revealed the proton permeability and conductance of the plasma membrane to be strongly pH dependent. The question was posed if similar pH dependent permeability changes also occur in isolated plasma membrane vesicles. Here we report the use of acridine orange to quantify passive proton fluxes. Right-side out vesicles were exposed to pH jumps. From the decay of the applied DeltapH the proton fluxes and proton permeability coefficients (PH+) were calculated. As in the intact Elodea plasma membrane, the proton permeability of the vesicle membrane is pH sensitive, an effect of internal pH as well as external pH on PH+ was observed. Under near symmetric conditions, i.e., zero electrical potential and zero DeltapH, PH+ increased from 65 x 10(-8) at pH 8.5 to 10(-1) m/sec at pH 11 and the conductance from 13 x 10(-6) to 30 x 10(-4) S/m2. At a constant pHi of 8 and a pHo going from 8.5 to 11, PH+ increased more than tenfold from 2 to 26 x 10(-6) m/sec. The calculated values of PH+ were several orders of magnitude lower than those obtained from studies on intact leaves. Apparently, in plasma membrane purified vesicles the transport system responsible for the observed high proton permeability in vivo is either (partly) inactive or lost during the procedure of vesicle preparation. The residue proton permeability is in agreement with values found for liposome or planar lipid bilayer membranes, suggesting that it reflects an intrinsic permeability of the phospholipid bilayer to protons. Possible implications of these findings for transport studies on similar vesicle systems are discussed.
Although there is consensus that the slow vacuolar or SV channel is a Ca2+ release channel, the underlying mechanism of operation is still controversial. The main reason is that the voltage sensitivity of SV gating seems to exclude activation at hyperpolarized (physiological) membrane potentials. Inspired by a study of Gambale et al. (1993) and supported by simulation studies presented here, we interpreted SV activation and deactivation kinetics in terms of a cyclic state diagram originally applied to animal cation-selective channels. A cyclic state diagram allows two pathways of activation operating in opposite directions. One pathway represents the frequently observed slow activation at moderate depolarization (< 130 mV). With the open state (O) next to the closed state initially occupied (C1), direct transitions from C1 to O can account for the fast activation observed at higher depolarized potentials (> 130 mV). We hypothesize that similar state transitions directly to O may also occur during hyperpolarization. The implication of this proposed mechanism is that SV accomplishes its physiological role during hyperpolarization-evoked deactivation. Despite their rare occurrence and possibly short duration, these opening events may last long enough to substantially raise the local cytosolic free Ca2+ level at the channel mouth by as much as 600 nM/ms. Because under in vivo conditions the Ca2- flux is inwardly directed, the mechanism presented here revives the notion that the SV channel can be subject to calcium-induced calcium release.
Application of patch clamp techniques to higher-plant cells has been subject to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wall. Because the wall is an integral component of plant cells, and because cell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts of Vicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell remains intact within its tissue environment. "Laser-assisted" patch clamping reveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch clamp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production of individual plant protoplasts isolated from their tissue environment by enzymatic digestion of the cell wall. Given the large conductances of the channels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and electrical signalling in guard cells.
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