Braun G, Lengyel M, Enyedi P, Czirják G
Department of Physiology, Semmelweis University, Budapest, Hungary.
Br J Pharmacol. 2015 Apr;172(7):1728-38. doi: 10.1111/bph.13019. Epub 2015 Jan 8.
Pharmacological separation of the background potassium currents of closely related K2P channels is a challenging problem. We previously demonstrated that ruthenium red (RR) inhibits TASK-3 (K2 P 9.1), but not TASK-1 (K2 P 3.1) channels. RR has been extensively used to distinguish between TASK currents in native cells. In the present study, we systematically investigate the RR sensitivity of a more comprehensive set of K2 P channels.
K(+) currents were measured by two-electrode voltage clamp in Xenopus oocytes and by whole-cell patch clamp in mouse dorsal root ganglion (DRG) neurons.
RR differentiates between two closely related members of the TREK subfamily. TREK-2 (K2 P 10.1) proved to be highly sensitive to RR (IC50 = 0.2 μM), whereas TREK-1 (K2 P 2.1) was not affected by the compound. We identified aspartate 135 (D135) as the target of the inhibitor in mouse TREK-2c. D135 lines the wall of the extracellular ion pathway (EIP), a tunnel structure through the extracellular cap characteristic for K2 P channels. TREK-1 contains isoleucine in the corresponding position. The mutation of this isoleucine (I110D) rendered TREK-1 sensitive to RR. The third member of the TREK subfamily, TRAAK (K2 P 4.1) was more potently inhibited by ruthenium violet, a contaminant in some RR preparations, than by RR. DRG neurons predominantly express TREK-2 and RR-resistant TREK-1 and TRESK (K2 P 18.1) background K(+) channels. We detected the RR-sensitive leak K(+) current component in DRG neurons.
We propose that RR may be useful for distinguishing TREK-2 (K2P 10.1) from TREK-1 (K2P 2.1) and other RR-resistant K2 P channels in native cells.
对密切相关的K2P通道的背景钾电流进行药理学分离是一个具有挑战性的问题。我们之前证明了钌红（RR）抑制TASK - 3（K2P 9.1）通道，但不抑制TASK - 1（K2P 3.1）通道。RR已被广泛用于区分天然细胞中的TASK电流。在本研究中，我们系统地研究了更全面的一组K2P通道对RR的敏感性。
通过在非洲爪蟾卵母细胞中采用双电极电压钳以及在小鼠背根神经节（DRG）神经元中采用全细胞膜片钳来测量K⁺电流。
RR可区分TREK亚家族的两个密切相关成员。结果表明，TREK - 2（K2P 10.1）对RR高度敏感（IC50 = 0.2 μM），而TREK - 1（K2P 2.1）不受该化合物影响。我们确定天冬氨酸135（D135）是小鼠TREK - 2c中抑制剂的作用靶点。D135位于细胞外离子通道（EIP）壁上，EIP是一种贯穿K2P通道细胞外帽的隧道结构。TREK - 1在相应位置含有异亮氨酸。将该异亮氨酸突变为天冬氨酸（I110D）使TREK - 1对RR敏感。TREK亚家族中的第三个成员TRAAK（K2P 4.1）对钌紫（某些RR制剂中的一种污染物）的抑制作用比RR更强。DRG神经元主要表达TREK - 2以及对RR有抗性的TREK - 1和TRESK（K2P 18.1）背景K⁺通道。我们在DRG神经元中检测到了对RR敏感的泄漏K⁺电流成分。
我们认为RR可能有助于在天然细胞中区分TREK - 2（K2P 10.1）与TREK - 1（K2P 2.1）以及其他对RR有抗性的K2P通道。
