Computational Physiology Department, Simula Research Laboratory, Oslo, Norway; Institute of Informatics, University of Oslo, Oslo, Norway.
Neurophysiology Unit, GIGA Neurosciences, University of Liège, Liège, Belgium.
Biophys J. 2023 Apr 4;122(7):1143-1157. doi: 10.1016/j.bpj.2023.02.004. Epub 2023 Feb 9.
Small-conductance (SK) calcium-activated potassium channels are a promising treatment target in atrial fibrillation. However, the functional properties that differentiate SK inhibitors remain poorly understood. The objective of this study was to determine how two unrelated SK channel inhibitors, apamin and AP14145, impact SK channel function in excised inside-out single-channel recordings. Surprisingly, both apamin and AP14145 exert much of their inhibition by inducing a class of very-long-lived channel closures (apamin: τ = 11.8 ± 7.1 s, and AP14145: τ = 10.3 ± 7.2 s), which were never observed under control conditions. Both inhibitors also induced changes to the three closed and two open durations typical of normal SK channel gating. AP14145 shifted the open duration distribution to favor longer open durations, whereas apamin did not alter open-state kinetics. AP14145 also prolonged the two shortest channel closed durations (AP14145: τ = 3.50 ± 0.81 ms, and τ = 32.0 ± 6.76 ms versus control: τ = 1.59 ± 0.19 ms, and τ = 13.5 ± 1.17 ms), thus slowing overall gating kinetics within bursts of channel activity. In contrast, apamin accelerated intraburst gating kinetics by shortening the two shortest closed durations (τ = 0.75 ± 0.10 ms and τ = 5.08 ± 0.49 ms) and inducing periods of flickery activity. Finally, AP14145 introduced a unique form of inhibition by decreasing unitary current amplitude. SK channels exhibited two clearly distinguishable amplitudes (control: A = 0.76 ± 0.03 pA, and A = 0.54 ± 0.03 pA). AP14145 both reduced the fraction of patches exhibiting the higher amplitude (AP14145: 4/9 patches versus control: 16/16 patches) and reduced the mean low amplitude (0.38 ± 0.03 pA). Here, we have demonstrated that both inhibitors introduce very long channel closures but that each also exhibits unique effects on other components of SK gating kinetics and unitary current. The combination of these effects is likely to be critical for understanding the functional differences of each inhibitor in the context of cyclical Ca-dependent channel activation in vivo.
小电导钙激活钾通道 (SK) 是心房颤动有前途的治疗靶点。然而，区分 SK 抑制剂的功能特性仍知之甚少。本研究的目的是确定两种不相关的 SK 通道抑制剂，蜂毒肽和 AP14145，如何在分离的内外单通道记录中影响 SK 通道功能。令人惊讶的是，蜂毒肽和 AP14145 都通过诱导一类非常长寿命的通道关闭来发挥大部分抑制作用（蜂毒肽：τ = 11.8 ± 7.1 s，AP14145：τ = 10.3 ± 7.2 s），而在对照条件下从未观察到这些关闭。两种抑制剂还诱导了正常 SK 通道门控的三个关闭和两个开放持续时间的变化。AP14145 将开放持续时间分布移向更长的开放持续时间，而蜂毒肽则不改变开放状态动力学。AP14145 还延长了两个最短的通道关闭持续时间（AP14145：τ = 3.50 ± 0.81 ms，τ = 32.0 ± 6.76 ms，而对照：τ = 1.59 ± 0.19 ms，τ = 13.5 ± 1.17 ms），从而减缓了通道活动爆发期间的整体门控动力学。相比之下，蜂毒肽通过缩短两个最短的关闭持续时间（τ = 0.75 ± 0.10 ms 和 τ = 5.08 ± 0.49 ms）和诱导闪烁活动来加速爆发内的门控动力学。最后，AP14145 通过降低单位电流幅度引入了一种独特的抑制形式。SK 通道表现出两种明显可区分的幅度（对照：A = 0.76 ± 0.03 pA，A = 0.54 ± 0.03 pA）。AP14145 降低了显示较高幅度的斑块比例（AP14145：4/9 个斑块与对照：16/16 个斑块），并降低了平均低幅度（0.38 ± 0.03 pA）。在这里，我们已经证明，两种抑制剂都引入了非常长的通道关闭，但每种抑制剂也对 SK 门控动力学和单位电流的其他成分表现出独特的影响。这些影响的组合对于理解每种抑制剂在体内周期性 Ca 依赖性通道激活背景下的功能差异可能是至关重要的。
