Department of Biophysics, Center for Molecular Biomedicine, Friedrich Schiller University of Jena and Jena University Hospital, Jena D-07745, Germany.
J Gen Physiol. 2012 Apr;139(4):305-19. doi: 10.1085/jgp.201110720.
Scorpion β toxins, peptides of ∼70 residues, specifically target voltage-gated sodium (Na(V)) channels to cause use-dependent subthreshold channel openings via a voltage-sensor trapping mechanism. This excitatory action is often overlaid by a not yet understood depressant mode in which Na(V) channel activity is inhibited. Here, we analyzed these two modes of gating modification by β-toxin Tz1 from Tityus zulianus on heterologously expressed Na(V)1.4 and Na(V)1.5 channels using the whole cell patch-clamp method. Tz1 facilitated the opening of Na(V)1.4 in a use-dependent manner and inhibited channel opening with a reversed use dependence. In contrast, the opening of Na(V)1.5 was exclusively inhibited without noticeable use dependence. Using chimeras of Na(V)1.4 and Na(V)1.5 channels, we demonstrated that gating modification by Tz1 depends on the specific structure of the voltage sensor in domain 2. Although residue G658 in Na(V)1.4 promotes the use-dependent transitions between Tz1 modification phenotypes, the equivalent residue in Na(V)1.5, N803, abolishes them. Gating charge neutralizations in the Na(V)1.4 domain 2 voltage sensor identified arginine residues at positions 663 and 669 as crucial for the outward and inward movement of this sensor, respectively. Our data support a model in which Tz1 can stabilize two conformations of the domain 2 voltage sensor: a preactivated outward position leading to Na(V) channels that open at subthreshold potentials, and a deactivated inward position preventing channels from opening. The results are best explained by a two-state voltage-sensor trapping model in that bound scorpion β toxin slows the activation as well as the deactivation kinetics of the voltage sensor in domain 2.
蝎β毒素是一种约 70 个残基的肽，它特异性地靶向电压门控钠 (Na(V)) 通道，通过电压传感器捕获机制引起电压依赖性亚阈通道开放。这种兴奋作用常常被一种尚未被理解的抑制模式所覆盖，其中 Na(V) 通道活性被抑制。在这里，我们使用全细胞膜片钳法分析了来自 Tityus zulianus 的β-毒素 Tz1 对异源表达的 Na(V)1.4 和 Na(V)1.5 通道的这两种门控修饰模式。Tz1 以电压依赖性方式促进 Na(V)1.4 的开放，并以反转的使用依赖性抑制通道开放。相比之下，Na(V)1.5 的开放则完全被抑制，没有明显的使用依赖性。使用 Na(V)1.4 和 Na(V)1.5 通道的嵌合体，我们证明了 Tz1 的门控修饰取决于域 2 中的电压传感器的特定结构。尽管 Na(V)1.4 中的残基 G658 促进了 Tz1 修饰表型之间的电压依赖性转变，但 Na(V)1.5 中的等效残基 N803 则消除了这些转变。Na(V)1.4 域 2 电压传感器的门控电荷中和鉴定出位置 663 和 669 的精氨酸残基分别是该传感器向外和向内运动的关键。我们的数据支持了一种模型，即 Tz1 可以稳定域 2 电压传感器的两种构象：一个预激活的向外位置导致 Na(V) 通道在亚阈电位下开放，以及一个失活的向内位置阻止通道开放。结果最好用二态电压传感器捕获模型来解释，即结合的蝎β毒素会减缓域 2 电压传感器的激活和失活动力学。
