The electrophysiological characterization of plant calcium channels and their role in calcium signaling

grantor: University of TorontoThe identification and characterization of Ca$\sp{2+}$ channels in the vacuolar and the plasma membrane of higher plants was accomplished through the use of conventional patch clamp techniques. Intact vacuoles from sugar beet cells were isolated and used to identify Ca$\sp{2+}$ currents at the whole vacuole level and outside-out patches of membrane were used for single channel analysis. Permeability and selectivity studies strongly suggested that one of the channels identified in the vacuolar membrane could efficiently and selectively release Ca$\sp{2+}$ from the vacuole resulting in increased levels of cytosolic Ca$\sp{2+}.$ A second channel co-residing with the vacuolar-release channel is the SV (slow-vacuolar type) channel. Although this channel is active at non-physiological potentials, the Ca$\sp{2+}$ permeability of the SV channel suggests that it could be involved in the sequestration of Ca$\sp{2+}$ following an increase in cytosolic Ca$\sp{2+}$ levels. The proposed role of the SV channel in Ca$\sp{2+}$-induced Ca$\sp{2+}$-release is also discussed. Stimulus-induced fluctuations in cytosolic Ca$\sp{2+}$ are mediated by Ca$\sp{2+}$ channels located in both the vacuolar and plasma membrane. The role of a plasma membrane Ca$\sp{2+}$ channel in stimulus-response coupling and the mechanisms of channel regulation were investigated by exposing protoplasts isolated from tomato cell near-isogenic for the resistance gene Cf5 to elicitor preparations containing the avr5 gene from race 4 (IF$\sb4)$ of the fungus Cladosporium fulvum. The stimulation of Ca$\sp{2+}$ channels by IF$\sb4,$ was up-regulated by phosphorylation mediated by the action of a G protein. The concerted action of Ca$\sp{2+}$ channels in the vacuolar and plasma membrane in maintaining Ca$\sp{2-}$ homeostasis and the mechanisms by which plant cells generate and perceive a stimulus-specific Ca$\sp{2+}$ signal is discussed.Ph.D