Sørensen J B, Cha A, Latorre R, Rosenman E, Bezanilla F
Department of Physiology, University of California, Los Angeles School of Medicine, Los Angeles, California 90095, USA.
J Gen Physiol. 2000 Feb;115(2):209-22. doi: 10.1085/jgp.115.2.209.
When attached outside the voltage-sensing S4 segment of the Shaker potassium channel, the fluorescent probe tetramethylrhodamine (TMRM) undergoes voltage-dependent fluorescence changes (DeltaF) due to differential interaction with a pH-titratable external protein-lined vestibule (Cha, A., and F. Bezanilla. 1998. J. Gen. Physiol. 112:391-408.). We attached TMRM at the same sites [corresponding to M356C and A359C in the wild-type (wt) channel] in a deletion mutant of Shaker where all but the five amino acids closest to S4 had been removed from the S3-S4 linker. In the deletion mutant, the maximal DeltaF/F seen was diminished 10-fold, and the DeltaF at M356C became pH independent, suggesting that the protein-lined vestibule is made up in large part by the S3-S4 linker. The residual DeltaF showed that the probe still interacted with two putative quenching groups near the S4 segment. One group was detected by M356C-TMRM (located outside of S3 in the deletion mutant) and reported on deactivation gating charge movement when applying hyperpolarizing voltage steps from a holding potential of 0 mV. During activating voltage steps from a holding potential of -90 mV, the fluorescence lagged considerably behind the movement of gating charge over a range of potentials. Another putative quenching group was seen by probes attached closer to the S4 and caused a DeltaF at extreme hyperpolarizations (more negative than -90 mV) only. A signal from the interaction with this group in the wt S3-S4 linker channel (at L361C) correlated with gating charge moving in the hyperpolarized part of the Q-V curve. Probe attached at A359C in the deletion mutant and at L361C in wt channel showed a biphasic DeltaF as the probe oscillated between the two groups, revealing that there is a transient state of the voltage sensor in between, where the probe has maximal fluorescence. We conclude that the voltage sensor undergoes two distinct conformational changes as seen from probes attached outside the S4 segment.
当荧光探针四甲基罗丹明（TMRM）连接在Shaker钾通道电压感应S4片段外部时，由于与一个可pH滴定的外部蛋白质内衬前庭存在差异相互作用，它会发生电压依赖性荧光变化（ΔF）（查，A.，和F.贝萨尼利亚。1998年。《普通生理学杂志》112:391 - 408）。我们将TMRM连接在Shaker的一个缺失突变体的相同位点[对应于野生型（wt）通道中的M356C和A359C]，在该突变体中，S3 - S4连接子中除了最靠近S4的五个氨基酸外的所有氨基酸都已被去除。在缺失突变体中，观察到的最大ΔF/F降低了10倍，并且M356C处的ΔF变得与pH无关，这表明蛋白质内衬前庭在很大程度上由S3 - S4连接子构成。残余的ΔF表明该探针仍与S4片段附近的两个假定淬灭基团相互作用。一个基团通过M356C - TMRM（位于缺失突变体的S3外部）检测到，并且在从0 mV的保持电位施加超极化电压阶跃时报告失活门控电荷移动情况。在从 - 90 mV的保持电位进行激活电压阶跃期间，在一系列电位范围内，荧光显著滞后于门控电荷的移动。另一个假定淬灭基团仅在连接更靠近S4的探针时在极端超极化（比 - 90 mV更负）时被观察到。来自野生型S3 - S4连接子通道（在L361C处）与该基团相互作用的信号与Q - V曲线超极化部分中的门控电荷移动相关。连接在缺失突变体的A359C处以及野生型通道的L361C处的探针显示出双相ΔF，因为探针在两个基团之间振荡，揭示了在两者之间存在电压传感器的一个瞬态，此时探针具有最大荧光。我们得出结论，从连接在S4片段外部的探针来看，电压传感器经历了两种不同的构象变化。
