Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada.
Am J Physiol Cell Physiol. 2013 Aug 15;305(4):C468-78. doi: 10.1152/ajpcell.00147.2013. Epub 2013 Jun 12.
The human ether-a-go-go related (hERG) potassium channel has unusual functional characteristics in that the rates of channel activation and deactivation are much slower than inactivation, which is attributed to specific structural elements within the NH2 terminus and the S1-S4 voltage-sensing domains (VSD). Although the charged residues in the VSD have been extensively modified and mutated as a result, the role and importance of specific hydrophobic residues in the S4 has been much less explored in studies of hERG gating. We found that charged, but not neutral or hydrophobic, amino acid substitution of isoleucine 521 at the outer end of the S4 transmembrane domain resulted in channels activating at much more negative voltages associated with a marked hyperpolarization of the conductance-voltage (G-V) relationship. The contributions of different physicochemical properties to this effect were probed by chemical modification of channels substituted with cysteine at position I521. When positively charged reagents including tetramethyl-rhodamine-5-maleimide (TMRM), 1-(2-maleimidylethyl)-4-[5-(4-methoxyphenyl)oxazol-2-yl] pyridinium methane-sulfonate (PyMPO), [2-(trimethylammonium)ethyl] methanethiosulfonate chloride (MTSET), and 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) were bound to the cysteine, I521C channels activated at more negative membrane potentials. To examine the contributions to hERG gating of other residues at the outer end of S4 (520-528), we performed a cysteine scan combined with MTSET modification. Only L520C, along with I521C, shows a substantial hyperpolarizing shift of the G-V relationship upon MTSET modification. The data indicate that the neutral, hydrophobic residue I521 at the extracellular end of S4 is critical for stabilizing the closed conformation of the hERG channel relative to the open state and by comparison with Shaker supports the alignment of hERG I521 with Shaker L361.
人 Ether-a-go-go 相关（hERG）钾通道具有不同寻常的功能特性，其通道激活和失活的速率远慢于失活，这归因于 NH2 末端和 S1-S4 电压感应结构域（VSD）内的特定结构元素。尽管 VSD 中的带电残基已被广泛修饰和突变，但在 hERG 门控研究中，S4 中特定疏水性残基的作用和重要性却鲜为人知。我们发现，S4 跨膜结构域外端的异亮氨酸 521 的带电（而非中性或疏水性）氨基酸取代会导致通道在更负的电压下激活，与电导-电压（G-V）关系的明显超极化有关。通过对位置 I521 处用半胱氨酸取代的通道进行化学修饰，研究了不同物理化学性质对这种效应的贡献。当带正电荷的试剂（包括四甲基罗丹明-5-马来酰亚胺（TMRM）、1-（2-马来酰亚胺基乙基）-4-[5-（4-甲氧基苯基）恶唑-2-基]吡啶𬭩甲烷磺酸盐（PyMPO）、[2-（三甲基铵）乙基]甲硫基磺酸盐氯化物（MTSET）和 2-氨基乙基甲硫基磺酸盐氢溴酸盐（MTSEA）与半胱氨酸结合时，I521C 通道在更负的膜电位下激活。为了研究 S4 外端（520-528）的其他残基对 hERG 门控的贡献，我们进行了半胱氨酸扫描结合 MTSET 修饰。只有 L520C 和 I521C 在 MTSET 修饰后会使 G-V 关系发生明显的超极化移位。数据表明，S4 细胞外端的中性疏水性残基 I521 对于稳定 hERG 通道的关闭构象相对于开放状态非常重要，并且与 Shaker 相比，支持 hERG I521 与 Shaker L361 的对齐。
