Large-conductance Ca-activated K channels (BK channels) are formed by Slo1 subunits as a homotetramer. Besides Ca, other divalent cations, such as Cd, also activate BK channels when applied intracellularly by shifting the conductance-voltage relation to more negative voltages. However, we found that if the inside-out patch containing BK channels was treated with solution containing reducing agents such as dithiothreitol (DTT), then subsequent Cd application completely inhibited BK currents. The DTT-dependent Cd inhibition could be reversed by treating the patch with solutions containing HO, suggesting that a redox reaction regulates the Cd inhibition of BK channels. Similar DTT-dependent Cd inhibition was also observed in a mutant BK channel, Core-MT, in which the cytosolic domain of the channel is deleted, and in the proton-activated Slo3 channels but not observed in the voltage-gated Shaker K channels. A possible mechanism for the DTT-dependent Cd inhibition is that DTT treatment breaks one or more disulfide bonds between cysteine pairs in the BK channel protein and the freed thiol groups coordinate with Cd to form an ion bridge that blocks the channel or locks the channel at the closed state. However, surprisingly, none of the mutations of all cysteine residues in Slo1 affect the DTT-dependent Cd inhibition. These results are puzzling, with an apparent contradiction: on one hand, a redox reaction seems to regulate Cd inhibition of the channel, but on the other hand, no cysteine residue in the Slo1 subunit seems to be involved in such inhibition.

中文翻译：

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BK 型 Ca2+ 激活的 K+ 通道的氧化还原依赖性 Cd2+ 抑制

大电导 Ca 激活 K 通道（BK 通道）由 Slo1 亚基形成同源四聚体。除了 Ca 之外，其他二价阳离子（例如 Cd）在细胞内应用时也会通过将电导-电压关系转变为更负的电压来激活 BK 通道。然而，我们发现，如果用含有二硫苏糖醇 (DTT) 等还原剂的溶液处理含有 BK 通道的由内而外贴片，则随后的 Cd 应用会完全抑制 BK 电流。通过用含有 H2O 的溶液处理贴片可以逆转 DTT 依赖性 Cd 抑制，这表明氧化还原反应调节 BK 通道的 Cd 抑制。在突变 BK 通道 Core-MT（其中通道的胞质结构域被删除）和质子激活 Slo3 通道中也观察到类似的 DTT 依赖性 Cd 抑制，但在电压门控 Shaker K 通道中未观察到。DTT 依赖性 Cd 抑制的一种可能机制是 DTT 处理破坏 BK 通道蛋白中半胱氨酸对之间的一个或多个二硫键，并且游离的硫醇基团与 Cd 配合形成离子桥，从而阻断通道或将通道锁定在关闭状态。然而，令人惊讶的是，Slo1 中所有半胱氨酸残基的突变均不影响 DTT 依赖性 Cd 抑制。这些结果令人费解，存在明显的矛盾：一方面，氧化还原反应似乎调节 Cd 对通道的抑制，但另一方面，Slo1 亚基中似乎没有半胱氨酸残基参与这种抑制。