McCormack K, Lin L, Iverson L E, Tanouye M A, Sigworth F J
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
Biophys J. 1992 Nov;63(5):1406-11. doi: 10.1016/S0006-3495(92)81703-4.
Shaker K+ channels are multimeric, probably tetrameric proteins. Substitution of a conserved leucine residue to valine (V2) at position 370 in the Drosophila Shaker 29-4 sequence results in large alterations in the voltage dependence of gating in the expressed channels. In order to determine the effects of this mutation in hybrid channels with a fixed stoichiometry of V2 and wild-type (WT) subunits we generated cDNA constructs of two linked-monomeric subunits similar to the tandem constructs previously reported by Isacoff, E. Y., Y. N. Jan, and L. Y. Jan. (1990. Nature (Lond.). 345:530-534). In addition, we constructed a tandem cDNA containing a wild-type subunit and a truncated nonfunctional subunit (Sh102) that suppresses channel expression. We report that the voltage-dependence of the channels produced with WT and V2 subunits varied significantly with the order of the subunits in the construct (WT-V2 or V2-WT), while the WT-Sh102 construct yielded currents that were much larger than expected. These results suggest that the tandem linkage of Shaker subunits does not guarantee the stoichiometry of the expressed channel proteins.
震荡器钾离子通道是多聚体蛋白，可能为四聚体。果蝇震荡器29 - 4序列中第370位的保守亮氨酸残基被缬氨酸取代（V2），导致所表达通道门控的电压依赖性发生巨大改变。为了确定这种突变在具有固定化学计量比的V2和野生型（WT）亚基的杂合通道中的影响，我们构建了两个类似伊萨科夫、Y. N. 扬和L. Y. 扬（1990年，《自然》（伦敦）。345:530 - 534）先前报道的串联构建体的两个相连单体亚基的cDNA构建体。此外，我们构建了一个包含野生型亚基和一个抑制通道表达的截短无功能亚基（Sh102）的串联cDNA。我们报道，由WT和V2亚基产生的通道的电压依赖性随构建体中亚基的顺序（WT - V2或V2 - WT）而显著变化，而WT - Sh102构建体产生的电流比预期大得多。这些结果表明，震荡器亚基的串联连接并不能保证所表达通道蛋白的化学计量比。
