Selective open-channel block of Shaker (Kv1) potassium channels by s-nitrosodithiothreitol (SNDTT).
Selective open-channel block of Shaker (Kv1) potassium channels by s-nitrosodithiothreitol (SNDTT).
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S-硝基硫代硫醇（SNDTT）的振动振摇摇床（KV1）钾通道的选择性开放通道块。
DOI：
10.1085/jgp.118.1.113
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发表时间：
2001-07
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大的季铵（QA）离子通过以1：1的化学计量比在水腔中结合来阻断电压门控K+（Kv）通道，所述水腔仅在通道打开时暴露于细胞质。S-亚硝基二硫苏糖醇（SNDTT; ONSCH 2CH（OH）CH（OH）CH 2SNO）在Kv通道中产生定性相似的“开放通道阻滞”，尽管结构截然不同。SNDTT很小，电中性，并且不是很疏水。在全细胞电压钳位鱿鱼巨纤维叶神经元中，浴应用SNDTT导致可逆的时间依赖性Kv通道阻滞，但不是Na+或Ca 2+通道。在HEK 293细胞中表达的灭活去除的ShakerB（ShB Δ）Kv1通道也被类似地阻断，并用于进一步研究SNDTT的作用。剂量-反应数据与两个SNDTT分子顺序结合到单个通道的方案一致，其中第一个分子的结合足以产生阻断。第二SNDTT分子结合的解离常数（Kd 2 = 0.14 mM）低于第一分子结合的解离常数（Kd 1 = 0.67 mM），表明协同性。半阻断浓度（K 1/2）为10.2 mM。这种电中性化合物的稳态阻断具有电压依赖性（约-0.3 e0），其幅度与QA离子的报告相似，但方向相反。SNDTT上的两个亚硝酰基（每个硫原子上一个）都需要阻断，但不涉及这些反应性基团转移到通道半胱氨酸残基。SNDTT经历缓慢的分子内反应（τ = 770 s），其中这些NO基团被释放，导致SNDTT效应的自发逆转。与内部四乙基铵的竞争表明浴应用SNDTT穿过细胞膜在内部位点起作用，最有可能在通道腔内。最后，SNDTT对Kv1通道具有显著的选择性。当在HEK 293细胞中单独表达时，大鼠Kv1.1 - 1.6显示出显著的SNDTT时间依赖性阻滞，而Kv2.1、3.1b或4.2未观察到这种效应。
Large quaternary ammonium (QA) ions block voltage-gated K+ (Kv) channels by binding with a 1:1 stoichiometry in an aqueous cavity that is exposed to the cytoplasm only when channels are open. S-nitrosodithiothreitol (SNDTT; ONSCH2CH(OH)CH(OH)CH2SNO) produces qualitatively similar “open-channel block” in Kv channels despite a radically different structure. SNDTT is small, electrically neutral, and not very hydrophobic. In whole-cell voltage-clamped squid giant fiber lobe neurons, bath-applied SNDTT causes reversible time-dependent block of Kv channels, but not Na+ or Ca2+ channels. Inactivation-removed ShakerB (ShBΔ) Kv1 channels expressed in HEK 293 cells are similarly blocked and were used to study further the action of SNDTT. Dose–response data are consistent with a scheme in which two SNDTT molecules bind sequentially to a single channel, with binding of the first being sufficient to produce block. The dissociation constant for the binding of the second SNDTT molecule (K d2 = 0.14 mM) is lower than that of the first molecule (K d1 = 0.67 mM), indicating cooperativity. The half-blocking concentration (K 1/2) is ∼0.2 mM. Steady-state block by this electrically neutral compound has a voltage dependence (about −0.3 e0) similar in magnitude but opposite in directionality to that reported for QA ions. Both nitrosyl groups on SNDTT (one on each sulfur atom) are required for block, but transfer of these reactive groups to channel cysteine residues is not involved. SNDTT undergoes a slow intramolecular reaction (τ ≈ 770 s) in which these NO groups are liberated, leading to spontaneous reversal of the SNDTT effect. Competition with internal tetraethylammonium indicates that bath-applied SNDTT crosses the cell membrane to act at an internal site, most likely within the channel cavity. Finally, SNDTT is remarkably selective for Kv1 channels. When individually expressed in HEK 293 cells, rat Kv1.1–1.6 display profound time-dependent block by SNDTT, an effect not seen for Kv2.1, 3.1b, or 4.2.