A Steep Dependence of Inward-Rectifying Potassium Channels on Cytosolic Free Calcium Concentration Increase Evoked by Hyperpolarization in Guard Cells

Inactivation of inward-rectifying K(+) channels (I(K,in)) by a rise in cytosolic free [Ca(2+)] ([Ca(2+)](i)) is a key event leading to solute loss from guard cells and stomatal closure. However, [Ca(2+)](i) action on I(K,in) has never been quantified, nor are its origins well understood. We used membrane voltage to manipulate [Ca(2+)](i) (A. Grabov and M.R. Blatt [1998] Proc Natl Acad Sci USA 95: 4778–4783) while recording I(K,in) under a voltage clamp and [Ca(2+)](i) by Fura-2 fluorescence ratiophotometry. I(K,in) inactivation correlated positively with [Ca(2+)](i) and indicated a K(i) of 329 ± 31 nm with cooperative binding of four Ca(2+) ions per channel. I(K,in) was promoted by the Ca(2+) channel antagonists Gd(3+) and calcicludine, both of which suppressed the [Ca(2+)](i) rise, but the [Ca(2+)](i) rise was unaffected by the K(+) channel blocker Cs(+). We also found that ryanodine, an antagonist of intracellular Ca(2+) channels that mediate Ca(2+)-induced Ca(2+) release, blocked the [Ca(2+)](i) rise, and Mn(2+) quenching of Fura-2 fluorescence showed that membrane hyperpolarization triggered divalent release from intracellular stores. These and additional results point to a high signal gain in [Ca(2+)](i) control of I(K,in) and to roles for discrete Ca(2+) flux pathways in feedback control of the K(+) channels by membrane voltage.