Oh Y, Smith P R, Bradford A L, Keeton D, Benos D J
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294.
Am J Physiol. 1993 Jul;265(1 Pt 1):C85-91. doi: 10.1152/ajpcell.1993.265.1.C85.
To determine the mechanism by which vasopressin increases apical membrane Na+ entry, we evaluated whether or not this hormone could recruit Na+ channels from a subapical membrane pool using specific polyclonal antibodies raised against high amiloride affinity bovine renal papillary Na+ channels. We also studied the effect of protein kinase A (PKA)-mediated phosphorylation on single-channel activity of highly purified Na+ channels incorporated into planar lipid bilayer membranes. PKA induced a significant increase in open-channel probability (Po) with no change in single-channel conductance. As shown previously and reconfirmed in the present work, PKA catalyzed the phosphorylation of a single subunit of this Na+ channel protein, namely, a 300-kDa polypeptide. On the other hand, protein kinase C, in combination with diacylglycerol, Ca2+, and phosphatidylserine, phosphorylated both the 130- and 55-kDa subunits of this purified Na+ channel, with a concomitant decrease in Po of both untreated and previously PKA-treated channels. We also found, in expression studies conducted in confluent monolayers of amphibian renal A6 cells, that vasopressin did not induce the apical insertion of new channel proteins. These observations support the hypothesis that vasopressin increases the apical Na+ permeability by activating Na+ channels already resident in the apical membrane by a direct phosphorylation mechanism rather than by cytoplasmic recruitment of latent Na+ channels.
为了确定血管加压素增加顶端膜Na⁺内流的机制，我们使用针对高阿米洛利亲和力的牛肾乳头Na⁺通道产生的特异性多克隆抗体，评估了该激素是否能从顶端下膜池募集Na⁺通道。我们还研究了蛋白激酶A（PKA）介导的磷酸化对掺入平面脂质双分子层膜中的高度纯化的Na⁺通道单通道活性的影响。PKA使开放通道概率（Po）显著增加，而单通道电导没有变化。如先前所示并在本研究中再次证实，PKA催化该Na⁺通道蛋白的单个亚基即300 kDa多肽的磷酸化。另一方面，蛋白激酶C与二酰基甘油、Ca²⁺和磷脂酰丝氨酸结合，使该纯化的Na⁺通道的130 kDa和55 kDa亚基均发生磷酸化，同时未处理和先前经PKA处理的通道的Po均降低。我们还发现，在两栖类肾A6细胞汇合单层进行的表达研究中，血管加压素不会诱导新通道蛋白的顶端插入。这些观察结果支持以下假设：血管加压素通过直接磷酸化机制激活已经存在于顶端膜中的Na⁺通道，而不是通过潜在Na⁺通道的胞质募集来增加顶端Na⁺通透性。
