| Stomata are pores in the surfaces of a plant which allow the exchange of CO{dollar}sb2{dollar} and oxygen with the atmosphere, while also controlling the transpiration of water vapor. Guard cells open and close the stomatal pore in response to a variety of environmental signals, including light or darkness, CO{dollar}sb2{dollar} levels, and plant water status. Since potassium ion flux through channels in the guard cell membranes is of central importance in this process, there must be transduction pathways within the guard cell whereby the potassium channels are regulated in response to environmental and intrinsic signals. The goal of the research described in this thesis has been to investigate the mechanisms which regulate the inward-rectifying potassium (K{dollar}sp+sb{lcub}rm in{rcub}){dollar} channels in the plasma membrane of the guard cell, so as to gain further insight into this vital physiological function.; Previous studies had shown that abscisic acid, produced in response to drought, can induce an elevation of guard cell cytoplasmic calcium, which in turn can inhibit K{dollar}sp+sb{lcub}rm in{rcub}{dollar} channels. I carried out whole-cell patch clamp recordings on guard cell protoplasts from Vicia faba to further characterize this calcium effect. K{dollar}sp+sb{lcub}rm in{rcub}{dollar} currents were strongly inhibited when the free cytoplasmic calcium concentration was buffered to greater than 1 {dollar}mu{dollar}M, but this effect depended on the concentration and effectiveness of the chelator. Further, G protein modulators produced only a small amount of inhibition, indicating a much more limited and indirect role of G proteins than had previously been proposed.; A model of regulation of the channel by modulation of the phosphorylation state of the channel protein was explored using a cloned K{dollar}sp+sb{lcub}rm in{rcub}{dollar} channel. Site-directed mutations were introduced at several potential phosphorylation sites, then the mutant clones were expressed in oocytes and assayed electrophysiologically. One of these mutants had substantially reduced current amplitude and slower kinetics, consistent with a model that phosphorylation at this site is required for maximal opening or conduction of the channel. Further experiments will be required to directly demonstrate that phosphorylation of the channel protein is an actual regulatory mechanism in the native guard cell. |
