Grabov A, Blatt MR
Laboratory of Plant Physiology and Biophysics, University of London, Wye College, Wye, Kent TN25 5AH, United Kingdom.
Plant Physiol. 1999 Jan;119(1):277-88. doi: 10.1104/pp.119.1.277.
Inactivation of inward-rectifying K+ channels (IK,in) by a rise in cytosolic free [Ca2+] ([Ca2+]i) is a key event leading to solute loss from guard cells and stomatal closure. However, [Ca2+]i action on IK,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 [1998] Proc Natl Acad Sci USA 95: 4778-4783) while recording IK,in under a voltage clamp and [Ca2+]i by Fura-2 fluorescence ratiophotometry. IK,in inactivation correlated positively with [Ca2+]i and indicated a Ki of 329 +/- 31 nM with cooperative binding of four Ca2+ ions per channel. IK,in was promoted by the Ca2+ channel antagonists Gd3+ and calcicludine, both of which suppressed the [Ca2+]i rise, but the [Ca2+]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 IK,in and to roles for discrete Ca2+ flux pathways in feedback control of the K+ channels by membrane voltage.
胞质游离[Ca2+]（[Ca2+]i）升高导致内向整流钾通道（IK,in）失活，是保卫细胞溶质流失和气孔关闭的关键事件。然而，[Ca2+]i对IK,in的作用从未被量化，其来源也尚未完全了解。我们利用膜电压来操控[Ca2+]i（A. Grabov和M.R. Blatt [1998] Proc Natl Acad Sci USA 95: 4778 - 4783），同时在电压钳制下记录IK,in，并通过Fura - 2荧光比率光度法测量[Ca2+]i。IK,in失活与[Ca2+]i呈正相关，表明每个通道结合四个Ca2+离子时的解离常数Ki为329±31 nM。Ca2+通道拮抗剂钆（Gd3+）和氨氯地平（calcicludine）可促进IK,in失活，二者均抑制[Ca2+]i升高，但[Ca2+]i升高不受K+通道阻滞剂铯（Cs+）的影响。我们还发现，ryanodine（介导Ca2+诱导Ca2+释放的细胞内Ca2+通道拮抗剂）可阻断[Ca2+]i升高，Fura - 2荧光的Mn2+淬灭表明膜超极化触发了细胞内钙库的二价阳离子释放。这些以及其他结果表明，[Ca2+]i对IK,in的控制具有高信号增益，且离散的Ca2+通量途径在膜电压对K+通道的反馈控制中发挥作用。
