Hamilton D W, Hills A, Blatt M R
Laboratory of Plant Physiology and Biophysics, Imperial College at Wye, Kent TN25 5AH, Wye, UK.
FEBS Lett. 2001 Feb 23;491(1-2):99-103. doi: 10.1016/s0014-5793(01)02176-7.
Ca2+ channels at the plasma membrane of stomatal guard cells contribute to increases in cytosolic free [Ca2+] (Ca2+) that regulate K+ and Cl- channels for stomatal closure in higher-plant leaves. Under voltage clamp, the initial rate of increase in Ca2+ in guard cells is sensitive to the extracellular divalent concentration, suggesting a close interaction between the permeant ion and channel gating. To test this idea, we recorded single-channel currents across the Vicia guard cell plasma membrane using Ba2+ as a charge carrying ion. Unlike other Ca2+ channels characterised to date, these channels activate at hyperpolarising voltages. We found that the open probability (P(o)) increased strongly with external Ba2+ concentration, consistent with a 4-fold cooperative action of Ba2+ in which its binding promoted channel opening in the steady state. Dwell time analyses indicated the presence of a single open state and at least three closed states of the channel, and showed that both hyperpolarising voltage and external Ba2+ concentration prolonged channel residence in the open state. Remarkably, increasing Ba2+ concentration also enhanced the sensitivity of the open channel to membrane voltage. We propose that Ba2+ binds at external sites distinct from the permeation pathway and that divalent binding directly influences the voltage gate.
气孔保卫细胞质膜上的Ca2+通道有助于提高胞质游离Ca2+浓度（[Ca2+]i），从而调节高等植物叶片气孔关闭时的K+和Cl-通道。在电压钳制下，保卫细胞中[Ca2+]i的初始增加速率对细胞外二价离子浓度敏感，这表明渗透离子与通道门控之间存在密切相互作用。为了验证这一观点，我们以Ba2+作为载流离子，记录了蚕豆保卫细胞质膜上的单通道电流。与迄今已鉴定的其他Ca2+通道不同，这些通道在超极化电压下激活。我们发现，开放概率（P(o)）随外部Ba2+浓度的增加而强烈增加，这与Ba2+的4倍协同作用一致，即其结合促进了通道在稳态下的开放。驻留时间分析表明，该通道存在单一开放状态和至少三种关闭状态，并表明超极化电压和外部Ba2+浓度均延长了通道在开放状态的停留时间。值得注意的是，增加Ba2+浓度也增强了开放通道对膜电压的敏感性。我们提出，Ba2+结合在与渗透途径不同的外部位点，二价离子结合直接影响电压门控。
