Braun A P, Heist E K, Schulman H
Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
J Physiol. 2000 Sep 15;527 Pt 3(Pt 3):479-92. doi: 10.1111/j.1469-7793.2000.00479.x.
The large conductance, calcium-sensitive K+ channel (BKCa channel) is a voltage-activated ion channel in which direct calcium binding shifts gating to more negative cellular membrane potentials. We hypothesized that the calcium-binding domain of BKCa channels may mimic the role played by calmodulin (CaM) in the activation of calcium-CaM-dependent enzymes, in which a tonic inhibitory constraint is removed on CaM binding. To examine such a hypothesis, we used peptides from the autoregulatory domains of CaM kinase II (CK291-317) and cNOS (the constitutive nitric oxide synthase; cNOS725-747) as probes for the calcium-dependent activation of murine BKCa channels transiently expressed in HEK 293 cells. We found that these CaM-binding peptides produced potent, time-dependent inhibition of mammalian BKCa channel current following voltage-dependent activation. Inhibition was observed in both the presence and the absence of cytosolic free calcium. Similar application of CK291-31 had no effect on either the amplitude or kinetics of voltage-dependent, macroscopic currents recorded from rabbit smooth muscle Kv1.5 potassium channels transiently expressed in HEK 293 cells. Cytosolic application of both CK291-317 and tetraethylammonium (TEA) produced an additive and non-competitive block of BKCa current. This finding suggests that the peptide-binding site is distinct (e.g. outside the pore region of the channel) from that of TEA. Our results are thus consistent with a model in which the BKCa channel's voltage-dependent gating process is under an intramolecular constraint that is relieved upon calcium binding. The intrinsic calcium sensor of the channel may thus interact with an inhibitory domain present in the BKCa channel, and by doing so, remove an inhibitory 'constraint' that permits voltage-dependent gating to occur at more negative potentials.
大电导钙敏感钾通道（BKCa通道）是一种电压激活离子通道，其中直接的钙结合将门控转移到更负的细胞膜电位。我们推测，BKCa通道的钙结合结构域可能模拟钙调蛋白（CaM）在激活钙-CaM依赖性酶中所起的作用，即在CaM结合时消除了一种紧张性抑制约束。为了检验这一假设，我们使用了来自CaM激酶II（CK291-317）和组成型一氧化氮合酶（cNOS；cNOS725-747）自调控结构域的肽作为探针，用于检测在HEK 293细胞中瞬时表达的小鼠BKCa通道的钙依赖性激活。我们发现，这些CaM结合肽在电压依赖性激活后对哺乳动物BKCa通道电流产生了强大的、时间依赖性的抑制作用。在有和没有胞质游离钙的情况下均观察到抑制作用。类似地应用CK291-31对在HEK 293细胞中瞬时表达的兔平滑肌Kv1.5钾通道记录的电压依赖性宏观电流的幅度或动力学均无影响。胞质应用CK291-317和四乙铵（TEA）对BKCa电流产生了相加性和非竞争性阻断。这一发现表明，肽结合位点与TEA的结合位点不同（例如在通道孔区域之外）。因此，我们的结果与一个模型一致，即BKCa通道的电压依赖性门控过程受到分子内约束，钙结合后该约束得以解除。通道的内在钙传感器可能因此与BKCa通道中存在的抑制结构域相互作用，并通过这样做消除一种抑制“约束”，从而允许电压依赖性门控在更负的电位下发生。
