Uebele V N, England S K, Chaudhary A, Tamkun M M, Snyders D J
Department of Pharmacology, Vanderbilt University, School of Medicine, Nashville, Tennessee 37232, USA.
J Biol Chem. 1996 Feb 2;271(5):2406-12. doi: 10.1074/jbc.271.5.2406.
The voltage-sensitive currents observed following hKv1.5 alpha subunit expression in HEK 293 and mouse L-cells differ in the kinetics and voltage dependence of activation and slow inactivation. Molecular cloning, immunopurification, and Western blot analysis demonstrated that an endogenous L-cell Kv beta 2.1 subunit assembled with transfected hKv 1.5 protein. In contrast, both mRNA and protein analysis failed to detect a beta subunit in the HEK 293 cells, suggesting that functional differences observed between these two systems are due to endogenous L-cell Kv beta 2.1 expression. In the absence of Kv beta 2.1, midpoints for activation and inactivation of hKv1.5 in HEK 293 cells were -0.2 +/- 2.0 and -9.6 +/- 1.8 mV, respectively. In the presence of Kv beta 2.1 these values were -14.1 +/- 1.8 and -22.1 +/- 3.7 mV, respectively. The beta subunit also caused a 1.5-fold increase in the extent of slow inactivation at 50 mV, thus completely reconstituting the L-cell current phenotype in the HEK 293 cells. These results indicate that 1) the Kv beta 2.1 subunit can alter Kv1.5 alpha subunit function, 2) beta subunits are not required for alpha subunit expression, and 3) endogenous beta subunits are expressed in heterologous expression systems used to study K+ channel function.
在HEK 293细胞和小鼠L细胞中表达hKv1.5α亚基后观察到的电压敏感性电流，在激活和缓慢失活的动力学及电压依赖性方面存在差异。分子克隆、免疫纯化和蛋白质印迹分析表明，内源性L细胞Kvβ2.1亚基与转染的hKv 1.5蛋白组装在一起。相比之下，mRNA和蛋白质分析均未能在HEK 293细胞中检测到β亚基，这表明在这两个系统中观察到的功能差异是由于内源性L细胞Kvβ2.1的表达所致。在没有Kvβ2.1的情况下，HEK 293细胞中hKv1.5激活和失活的中点分别为-0.2±2.0 mV和-9.6±1.8 mV。在有Kvβ2.1存在的情况下，这些值分别为-14.1±1.8 mV和-22.1±3.7 mV。β亚基还使50 mV时缓慢失活的程度增加了1.5倍，从而在HEK 293细胞中完全重建了L细胞电流表型。这些结果表明：（1）Kvβ2.1亚基可改变Kv1.5α亚基的功能；（2）α亚基的表达不需要β亚基；（3）内源性β亚基在用于研究钾通道功能的异源表达系统中表达。
