Cox Robert H, Fromme Samantha
Program in Cardiovascular Disease, Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA, 19096, USA.
Cell Biochem Biophys. 2016 Dec;74(4):499-511. doi: 10.1007/s12013-016-0763-4. Epub 2016 Sep 16.
We have shown that three components contribute to functional voltage gated K (K ) currents in rat small mesenteric artery myocytes: (1) Kv1.2 plus Kv1.5 with Kvβ1.2 subunits, (2) Kv2.1 probably associated with Kv9.3 subunits, and (3) Kv7.4 subunits. To confirm and address subunit stoichiometry of the first two, we have compared the biophysical properties of K currents in small mesenteric artery myocytes with those of K subunits heterologously expressed in HEK293 cells using whole cell voltage clamp methods. Selective inhibitors of Kv1 (correolide, COR) and Kv2 (stromatoxin, ScTx) channels were used to separate these K current components. Conductance-voltage and steady state inactivation data along with time constants of activation, inactivation, and deactivation of native K components were generally well represented by those of Kv1.2-1.5-β1.2 and Kv2.1-9.3 channels. The slope of the steady state inactivation-voltage curve (availability slope) proved to be the most sensitive measure of accessory subunit presence. The availability slope curves exhibited a single peak for both native K components. Availability slope curves for Kv1.2-1.5-β1.2 and Kv2.1-9.3 channels expressed in human embryonic kidney cells also exhibited a single peak that shifted to more depolarized voltages with increasing accessory to α subunit transfection ratio. Availability slope curves for SxTc-insensitive currents were similar to those of Kv1.2-1.5 expressed with Kvβ1.2 at a 1:5 molar ratio while curves for COR-insensitive currents closely resembled those of Kv2.1 expressed with Kv9.3 at a 1:1 molar ratio. These results support the suggested K subunit combinations in small mesenteric artery, and further suggest that Kv1 α and Kvβ1.2 but not Kv2.1 and Kv9.3 subunits are present in a saturated (4:4) stoichiometry.
我们已经证明，有三种成分对大鼠肠系膜小动脉肌细胞中的功能性电压门控钾（K⁺）电流有贡献：（1）带有Kvβ1.2亚基的Kv1.2加Kv1.5，（2）可能与Kv9.3亚基相关的Kv2.1，以及（3）Kv7.4亚基。为了确认并研究前两种成分的亚基化学计量，我们使用全细胞电压钳方法，比较了肠系膜小动脉肌细胞中K⁺电流的生物物理特性与在HEK293细胞中异源表达的K⁺亚基的生物物理特性。使用Kv1（correolide，COR）和Kv2（stromatoxin，ScTx）通道的选择性抑制剂来分离这些K⁺电流成分。Kv1.2 - 1.5 - β1.2和Kv2.1 - 9.3通道的电导 - 电压、稳态失活数据以及天然K⁺成分的激活、失活和去激活时间常数，通常能很好地反映天然K⁺成分的数据。稳态失活 - 电压曲线的斜率（可用性斜率）被证明是辅助亚基存在的最敏感指标。两种天然K⁺成分的可用性斜率曲线都呈现出一个单峰。在人胚胎肾细胞中表达的Kv1.2 - 1.5 - β1.2和Kv2.1 - 9.3通道的可用性斜率曲线也呈现出一个单峰，并且随着辅助亚基与α亚基转染比例的增加，该峰向更去极化的电压移动。对ScTx不敏感电流的可用性斜率曲线类似于以1:5摩尔比与Kvβ1.2一起表达的Kv1.2 - 1.5的曲线，而对COR不敏感电流的曲线与以1:1摩尔比与Kv9.3一起表达的Kv2.1的曲线非常相似。这些结果支持了在肠系膜小动脉中所提出的K⁺亚基组合，并进一步表明Kv1α和Kvβ1.2亚基以饱和（4:4）化学计量存在，而Kv2.1和Kv9.3亚基并非如此。
