Blumenthal E M, Kaczmarek L K
Interdepartmental Neuroscience Program, Yale University School of Medicine, New Haven, Connecticut 06510.
J Neurosci. 1994 May;14(5 Pt 2):3097-105. doi: 10.1523/JNEUROSCI.14-05-03097.1994.
The minK protein induces a slowly activating voltage-dependent potassium current when expressed in Xenopus oocytes. In order to measure the levels of minK protein in the plasma membrane, we have modified the minK gene by inserting a 9 amino acid epitope into the N-terminal domain of the protein sequence. When intact live oocytes are injected with the modified minK RNA and subsequently incubated with an antibody to this epitope, specific binding is detected, indicating that the N-terminal domain is extracellular. We found that when oocytes are injected with amounts of minK mRNA up to 50 ng, the levels of protein at the surface are proportional to the amount of injected mRNA. In contrast, the amplitude of the minK current recorded in the oocytes saturates at 1 ng of injected mRNA. Although the amplitude of the currents is not altered by increasing mRNA levels above 1 ng, the kinetics of activation of the current differ in oocytes with high or low levels of minK RNA. In particular, activation is slower with higher levels of minK protein in the plasma membrane. Finally, we find that increasing intracellular cAMP levels, which increases the amplitude of minK currents, does not alter surface expression of the minK protein but produces a small increase in the rate of activation of the current. Our results support a model in which minK protein forms functional potassium channels by association with a factor endogenous to the oocyte.
当在非洲爪蟾卵母细胞中表达时,minK蛋白会诱导一种缓慢激活的电压依赖性钾电流。为了测量质膜中minK蛋白的水平,我们通过在蛋白序列的N端结构域插入一个9个氨基酸的表位对minK基因进行了修饰。当向完整的活卵母细胞注射修饰后的minK RNA,随后用针对该表位的抗体进行孵育时,可检测到特异性结合,这表明N端结构域位于细胞外。我们发现,当向卵母细胞注射高达50 ng的minK mRNA时,表面蛋白水平与注射的mRNA量成正比。相比之下,在卵母细胞中记录到的minK电流幅度在注射1 ng mRNA时达到饱和。尽管当mRNA水平高于1 ng时电流幅度不会改变,但minK RNA水平高或低的卵母细胞中电流的激活动力学有所不同。特别是,质膜中minK蛋白水平较高时激活较慢。最后,我们发现增加细胞内cAMP水平会增加minK电流的幅度,但不会改变minK蛋白的表面表达,而是使电流激活速率略有增加。我们的结果支持这样一种模型,即minK蛋白通过与卵母细胞内源性因子结合形成功能性钾通道。