Moss G W, Marshall J, Moczydlowski E
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066, USA.
J Gen Physiol. 1996 Dec;108(6):473-84. doi: 10.1085/jgp.108.6.473.
Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue protein with three disulfide bonds that belongs to the Kunitz family of serine proteinase inhibitors. BPTI is an extremely potent inhibitor of trypsin, but it also specifically binds to various active and inactive serine proteinase homologs with KD values that range over eight orders of magnitude. We previously described an interaction of BPTI at an intracellular site that results in the production of discrete subconductance events in large conductance Ca2+ activated K+ channels (Moss, G.W.J., and E. Moczydlowski. 1996, J. Gen. Physiol, 107:47-68). In this paper, we summarize a variety of accumulated evidence which suggests that BPTI binds to a site on the KCa channel protein that structurally resembles a serine proteinase. One line of evidence includes the finding that the complex of BPTI and trypsin, in which the inhibitory loop of BPTI is masked by interaction with trypsin, is completely ineffective in the production of substate events in the KCa channel. To further investigate this notion, we performed a sequence analysis of the alpha-subunit of cloned slowpoke KCa channels from Drosophila and mammals. This analysis suggests that a region of approximately 250 residues near the COOH terminus of the KCa channel is homologous to members of the serine proteinase family, but is catalytically inactive because of various substitutions of key catalytic residues. The sequence analysis also predicts the location of a Ca(2+)-binding loop that is found in many serine proteinase enzymes. We hypothesize that this COOH-terminal domain of the slowpoke KCa channel adopts the characteristic double-barrel fold of serine proteinases, is involved in Ca(2+)-activation of the channel, and may also bind other intracellular components that regulate KCa channel activity.
牛胰蛋白酶抑制剂（BPTI）是一种含有58个氨基酸残基且具有三个二硫键的蛋白质，属于丝氨酸蛋白酶抑制剂的库尼茨家族。BPTI是一种极强的胰蛋白酶抑制剂，但它也能特异性地结合各种活性和非活性丝氨酸蛋白酶同源物，其解离常数（KD）值跨越八个数量级。我们之前描述过BPTI在细胞内位点的一种相互作用，这种相互作用会导致大电导Ca2+激活K+通道产生离散的亚电导事件（莫斯，G.W.J.，和E.莫齐德洛夫斯基。1996年，《普通生理学杂志》，107:47 - 68）。在本文中，我们总结了各种积累的证据，这些证据表明BPTI与KCa通道蛋白上一个在结构上类似于丝氨酸蛋白酶的位点结合。一条证据线索包括这样一个发现：BPTI与胰蛋白酶的复合物，其中BPTI的抑制环因与胰蛋白酶相互作用而被掩盖，在KCa通道中产生亚状态事件时完全无效。为了进一步研究这一概念，我们对来自果蝇和哺乳动物的克隆慢poke KCa通道的α亚基进行了序列分析。该分析表明，KCa通道COOH末端附近约250个残基的区域与丝氨酸蛋白酶家族成员同源，但由于关键催化残基的各种取代而无催化活性。序列分析还预测了许多丝氨酸蛋白酶中存在的一个Ca(2 +)结合环的位置。我们假设慢poke KCa通道的这个COOH末端结构域采用丝氨酸蛋白酶特有的双桶折叠结构，参与通道的Ca(2 +)激活，并且还可能结合其他调节KCa通道活性的细胞内成分。
