Department of Physiology & Membrane Biology, University of California, Davis, Davis, CA.
Neurobiology Course, Marine Biological Laboratory, Woods Hole, MA.
J Gen Physiol. 2019 Mar 4;151(3):292-315. doi: 10.1085/jgp.201812213. Epub 2018 Nov 5.
Allosteric ligands modulate protein activity by altering the energy landscape of conformational space in ligand-protein complexes. Here we investigate how ligand binding to a K channel's voltage sensor allosterically modulates opening of its K-conductive pore. The tarantula venom peptide guangxitoxin-1E (GxTx) binds to the voltage sensors of the rat voltage-gated K (Kv) channel Kv2.1 and acts as a partial inverse agonist. When bound to GxTx, Kv2.1 activates more slowly, deactivates more rapidly, and requires more positive voltage to reach the same K-conductance as the unbound channel. Further, activation kinetics are more sigmoidal, indicating that multiple conformational changes coupled to opening are modulated. Single-channel current amplitudes reveal that each channel opens to full conductance when GxTx is bound. Inhibition of Kv2.1 channels by GxTx results from decreased open probability due to increased occurrence of long-lived closed states; the time constant of the final pore opening step itself is not impacted by GxTx. When intracellular potential is less than 0 mV, GxTx traps the gating charges on Kv2.1's voltage sensors in their most intracellular position. Gating charges translocate at positive voltages, however, indicating that GxTx stabilizes the most intracellular conformation of the voltage sensors (their resting conformation). Kinetic modeling suggests a modulatory mechanism: GxTx reduces the probability of voltage sensors activating, giving the pore opening step less frequent opportunities to occur. This mechanism results in K-conductance activation kinetics that are voltage-dependent, even if pore opening (the rate-limiting step) has no inherent voltage dependence. We conclude that GxTx stabilizes voltage sensors in a resting conformation, and inhibits K currents by limiting opportunities for the channel pore to open, but has little, if any, direct effect on the microscopic kinetics of pore opening. The impact of GxTx on channel gating suggests that Kv2.1's pore opening step does not involve movement of its voltage sensors.
变构配体通过改变配体-蛋白质复合物构象空间的能量景观来调节蛋白质活性。在这里，我们研究了配体与 K 通道电压传感器的结合如何变构调节其 K 导电孔的打开。狼蛛毒液肽 guangxitoxin-1E（GxTx）与大鼠电压门控 K（Kv）通道 Kv2.1 的电压传感器结合，并作为部分反向激动剂。当与 GxTx 结合时，Kv2.1 的激活速度更慢，失活速度更快，并且需要更正的电压才能达到与未结合通道相同的 K 电导。此外，激活动力学更呈 S 形，表明与打开相关的多个构象变化受到调节。单通道电流幅度表明，当 GxTx 结合时，每个通道都能打开至全电导。GxTx 对 Kv2.1 通道的抑制作用是由于长寿命关闭状态的发生增加导致开放概率降低所致；GxTx 本身不会影响最后孔打开步骤的时间常数。当细胞内电势小于 0 mV 时，GxTx 将 Kv2.1 电压传感器的门控电荷捕获在其最细胞内位置。然而，当施加正电压时，门控电荷会发生位移，这表明 GxTx 稳定了电压传感器的最细胞内构象（其静息构象）。动力学模型表明了一种调节机制：GxTx 降低了电压传感器激活的概率，使孔打开步骤发生的机会减少。这种机制导致 K 电导激活动力学对电压有依赖性，即使孔打开（限速步骤）本身没有固有电压依赖性。我们得出结论，GxTx 将电压传感器稳定在静息构象中，并通过限制通道孔打开的机会来抑制 K 电流，但对孔打开的微观动力学几乎没有直接影响。GxTx 对通道门控的影响表明，Kv2.1 的孔打开步骤不涉及其电压传感器的移动。
