Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University, Xuzhou 221004, People's Republic of China.
Jiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology, Xuzhou Medical University, Xuzhou 221004, People's Republic of China.
J Neurosci. 2023 Apr 12;43(15):2665-2681. doi: 10.1523/JNEUROSCI.1365-22.2023. Epub 2023 Mar 10.
The Slack channel (KCNT1, Slo2.2) is a sodium-activated and chloride-activated potassium channel that regulates heart rate and maintains the normal excitability of the nervous system. Despite intense interest in the sodium gating mechanism, a comprehensive investigation to identify the sodium-sensitive and chloride-sensitive sites has been missing. In the present study, we identified two potential sodium-binding sites in the C-terminal domain of the rat Slack channel by conducting electrophysical recordings and systematic mutagenesis of cytosolic acidic residues in the rat Slack channel C terminus. In particular, by taking advantage of the M335A mutant, which results in the opening of the Slack channel in the absence of cytosolic sodium, we found that among the 92 screened negatively charged amino acids, E373 mutants could completely remove sodium sensitivity of the Slack channel. In contrast, several other mutants showed dramatic decreases in sodium sensitivity but did not abolish it altogether. Furthermore, molecular dynamics (MD) simulations performed at the hundreds of nanoseconds timescale revealed one or two sodium ions at the E373 position or an acidic pocket composed of several negatively charged residues. Moreover, the MD simulations predicted possible chloride interaction sites. By screening predicted positively charged residues, we identified R379 as a chloride interaction site. Thus, we conclude that the E373 site and the D863/E865 pocket are two potential sodium-sensitive sites, while R379 is a chloride interaction site in the Slack channel. The research presented here identified two distinct sodium and one chloride interaction sites located in the intracellular C-terminal domain of the Slack (Slo2.2, KCNT1) channel. Identification of the sites responsible for the sodium and chloride activation of the Slack channel sets its gating property apart from other potassium channels in the BK channel family. This finding sets the stage for future functional and pharmacological studies of this channel.
Slack 通道（KCNT1、Slo2.2）是一种钠激活和氯激活钾通道，可调节心率并维持神经系统的正常兴奋性。尽管人们对钠门控机制非常感兴趣，但对确定钠敏和氯敏位点的全面研究仍付之阙如。在本研究中，我们通过对大鼠 Slack 通道胞质酸性残基进行电生理记录和系统突变，在大鼠 Slack 通道 C 末端鉴定出两个潜在的钠结合位点。特别是，利用 M335A 突变体，该突变体导致 Slack 通道在没有胞质钠的情况下开放，我们发现在筛选的 92 个带负电荷的氨基酸中，E373 突变体可完全消除 Slack 通道的钠敏性。相比之下，其他几个突变体虽然显著降低了钠敏性，但并未完全消除。此外，在数百纳秒时间尺度上进行的分子动力学（MD）模拟显示，E373 位置处有一个或两个钠离子，或者由几个带负电荷的残基组成的酸性口袋。此外，MD 模拟预测了可能的氯相互作用位点。通过筛选预测的正电荷残基，我们确定 R379 为氯相互作用位点。因此，我们得出结论，E373 位点和 D863/E865 口袋是两个潜在的钠敏位点，而 R379 是 Slack 通道中的氯相互作用位点。本研究鉴定了位于 Slack（Slo2.2、KCNT1）通道胞内 C 末端的两个独特的钠和一个氯相互作用位点。鉴定出负责 Slack 通道钠和氯激活的位点使其门控特性与 BK 通道家族中的其他钾通道不同。这一发现为该通道的未来功能和药理学研究奠定了基础。
