Wang G K, Simon R, Wang S Y
Department of Anesthesia, Harvard Medical School, Boston, Massachusetts.
J Gen Physiol. 1991 Nov;98(5):1005-24. doi: 10.1085/jgp.98.5.1005.
Quaternary ammonium (QA) blockers are well-known structural probes for studying the permeation pathway of voltage-gated K+ channels. In this study we have examined the effects of a series of n-alkyl-trimethylammonium compounds (Cn-QA) on batrachotoxin (BTX)-activated Na+ channels from skeletal muscle incorporated into planar lipid bilayers. We found that these amphipathic QA compounds (Cn-QA where n = 10-18) block single Na+ channels preferentially from the internal side with equilibrium dissociation constants (KD) in the submicromolar to micromolar range. External application of amphipathic QA compounds is far less effective, by a factor of greater than 200. The block can be described by a QA molecule binding to a single site in the Na+ channel permeation pathway. QA binding affinity is dependent on transmembrane voltage with an effective valence (delta) of approximately 0.5. QA dwell times (given as mean closed times, tau c) increase as a function of n-alkyl chain length, ranging from approximately 13 ms for C10-QA to 500 ms for C18-QA at +50 mV. The results imply that there is a large hydrophobic region within the Na+ channel pore which accepts up to 18 methylene groups of the Cn-QA cation. This hydrophobic domain may be of clinical significance since it also interacts with local anesthetics such as cocaine and mepivacaine. Finally, like BTX-activated Na+ channels in bilayers, unmodified Na+ channels in GH3 cells are also susceptible to QA block. Amphipathic QA cations elicit both tonic and use-dependent inhibitions of normal Na+ currents in a manner similar to that of local anesthetic cocaine. We conclude that amphipathic QA compounds are valuable structural probes to study the permeation pathway of both normal and BTX-activated Na+ channels.
季铵（QA）阻滞剂是研究电压门控钾通道渗透途径的著名结构探针。在本研究中，我们检测了一系列正烷基三甲基铵化合物（Cn-QA）对掺入平面脂质双分子层的骨骼肌中由蟾毒素（BTX）激活的钠通道的影响。我们发现，这些两亲性QA化合物（n = 10 - 18时的Cn-QA）优先从内侧阻断单个钠通道，平衡解离常数（KD）在亚微摩尔至微摩尔范围内。两亲性QA化合物的外部应用效果要差得多，相差超过200倍。这种阻断可以用一个QA分子结合到钠通道渗透途径中的单个位点来描述。QA结合亲和力取决于跨膜电压，有效价（δ）约为0.5。QA停留时间（以平均关闭时间τc表示）随着正烷基链长度的增加而增加，在+50 mV时，范围从C10-QA的约13毫秒到C18-QA的500毫秒。结果表明，钠通道孔内有一个大的疏水区域，可容纳Cn-QA阳离子的多达18个亚甲基基团。这个疏水结构域可能具有临床意义，因为它也与可卡因和美吡卡因等局部麻醉剂相互作用。最后，与双层膜中BTX激活的钠通道一样，GH3细胞中的未修饰钠通道也易受QA阻断。两亲性QA阳离子以类似于局部麻醉剂可卡因的方式引起正常钠电流的强直和使用依赖性抑制。我们得出结论，两亲性QA化合物是研究正常和BTX激活的钠通道渗透途径的有价值的结构探针。
