A steep dependence of inward-rectifying potassium channels on cytosolic free calcium concentration increase evoked by hyperpolarization in guard cells

Abstract

Inactivation of inward-rectifying K+ channels (I(κ,in)) by a rise in cytosolic free [Ca2+[ ([Ca2+l(i)) is a key event leading to solute loss from guard cells and stomatal closure. However, [Ca2+l(i) action on l(κ,in) has never been quantified, nor are its origins well understood. We used membrane voltage to manipulate [Ca2+](i) (A. Grabov and M.R. Blatt [19981 Proc Natl Acad Sci USA 95: 4778-4783) while recording I(κ,in) under a voltage clamp and [Ca2+](i) by Fura-2 fluorescence ratiophotometry. I(κ,in) inactivation correlated positively with [Ca2+l(i) and indicated a K(i) of 329 ± 31 nM with cooperative binding of four Ca2+ ions per channel. I(κ,in) was promoted by the Ca2+ channel antagonists Gd3+ and calcicludine, both of which suppressed the [Ca2+](i) rise, but the [Ca2+l(i) rise was unaffected by the K+ channel blocker Cs+. We also found that ryanodine, an antagonist of intracellular Ca2+ channels that mediate Ca2-induced Ca2+ release, blocked the [Ca2+](i) rise, and Mn2+ 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 [Ca2+](i) control of I(κ,in) and to roles for discrete Ca2+ flux pathways in feedback control of the K+ channels by membrane voltage.