Lucchesi K J, Moczydlowski E
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Gen Physiol. 1991 Jun;97(6):1295-319. doi: 10.1085/jgp.97.6.1295.
Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue basic peptide that is a representative member of a widely distributed class of serine protease inhibitors known as Kunitz inhibitors. BPTI is also homologous to dendrotoxin peptides from mamba snake venom that have been characterized as inhibitors of various types of voltage-dependent K+ channels. In this study we compared the effect of DTX-I, a dendrotoxin peptide, and BPTI on large conductance Ca(2+)-activated K+ channels from rat skeletal muscle using planar bilayer methodology. As previously found for DTX-I (1990. Neuron. 2:141-148), BPTI induces the appearance of distinct subconductance events when present on the internal side of maxi K(Ca) channels. The single channel kinetics of substate formation follow the predictions of reversible binding of the peptide to a single site or class of sites with a Kd of 4.6 microM at 0 mV and 50 mM symmetrical KCl. The apparent association rate of BPTI binding decreases approximately 1,000-fold per 10-fold increase in ionic strength, suggestive of a strong electrostatic interaction between the basic peptide and negative surface charge in the vicinity of the binding site. The equilibrium Kd for BPTI and DTX-I is also voltage dependent, decreasing e-fold per 30 mV of depolarization. The unitary subconductance current produced by BPTI binding exhibits strong inward rectification in the presence of symmetrical KCl, corresponding to 15% of open channel current at +60 mV and 70% of open state at -40 mV. In competition experiments, the internal pore-blocking ions, Ba2+ and TEA+, readily block the substate with the same affinity as that for blocking the normal open state. These results suggest that BPTI does not bind near the inner mouth of the channel so as to directly interfere with cation entry to the channel. Rather, the mechanism of substate production appears to involve a conformational change that affects the energetics of K+ permeation.
牛胰蛋白酶抑制剂（BPTI）是一种由58个氨基酸残基组成的碱性肽，是广泛分布的一类丝氨酸蛋白酶抑制剂（称为Kunitz抑制剂）的代表性成员。BPTI也与来自曼巴蛇毒的树突毒素肽同源，这些肽已被鉴定为各种类型电压依赖性钾通道的抑制剂。在本研究中，我们使用平面双层方法比较了树突毒素肽DTX-I和BPTI对大鼠骨骼肌大电导钙激活钾通道的影响。如先前对DTX-I的研究结果（1990年。《神经元》。2:141 - 148）所示，当BPTI存在于大电导钙激活钾通道的内侧时，会诱导出明显的亚电导事件。亚状态形成的单通道动力学遵循肽与单个位点或一类位点可逆结合的预测，在0 mV和50 mM对称KCl条件下，解离常数（Kd）为4.6 microM。BPTI结合的表观缔合速率每增加10倍离子强度大约降低1000倍，这表明碱性肽与结合位点附近的负表面电荷之间存在强烈的静电相互作用。BPTI和DTX-I的平衡解离常数也依赖于电压，每去极化30 mV降低e倍。在对称KCl存在的情况下，BPTI结合产生的单位亚电导电流表现出强烈的内向整流，在 +60 mV时相当于开放通道电流的15%，在 -40 mV时相当于开放状态的70%。在竞争实验中，内部孔道阻断离子Ba2+和TEA+能够以与阻断正常开放状态相同的亲和力轻易阻断亚状态。这些结果表明，BPTI并非在通道内口附近结合以直接干扰阳离子进入通道。相反，亚状态产生的机制似乎涉及一种影响钾离子渗透能量学的构象变化。
