Fan J S, Jiang M, Dun W, McDonald T V, Tseng G N
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0641, USA.
Biophys J. 1999 Jun;76(6):3128-40. doi: 10.1016/S0006-3495(99)77464-3.
The fast-inactivation process in the hERG channel can be affected by mutations in the pore or S6 domain, similar to the C-type inactivation in the Shaker channel. However, differences in the kinetics and voltage dependence of inactivation between these two channels suggest that different structural determinants may be involved. To explore this possibility, we mutated a serine in the outer mouth region of hERG (S631) to residues of different physicochemical properties and compared the resulting changes in the channel's inactivation process with those resulting from mutations of an equivalent position in the Shaker channel (T449). The most dramatic differences are seen when this position is occupied by a charged residue: S631K and S631E disrupted C-type inactivation in hERG, whereas T449K and T449E facilitate C-type inactivation in Shaker. S631K and S631E also disrupted the K selectivity of hERG pore, a change not seen in T449K or T449E of Shaker. To further study why there are such differences, we replaced S631 with cysteine. This allowed us to manipulate the properties of thiol groups at position 631 and correlate side-chain properties here with changes in channel function. S631C behaved like the wild-type channel when the thiol groups were in the reduced state. Oxidizing thiol groups with H2O2 or modifying them with MTSET or MTSES disrupted C-type inactivation and K selectivity, similar to the phenotype of S631K and S631E. The same thiol-modifying maneuvers did not affect the wild-type channel function. Our results suggest differences in the outer mouth structure between hERG and Shaker, and we propose a "molecular spring" hypothesis to explain these differences.
hERG通道中的快速失活过程可能会受到孔道或S6结构域突变的影响，这与Shaker通道中的C型失活类似。然而，这两种通道在失活动力学和电压依赖性方面的差异表明，可能涉及不同的结构决定因素。为了探究这种可能性，我们将hERG外口区域的一个丝氨酸（S631）突变为具有不同物理化学性质的残基，并将通道失活过程中产生的变化与Shaker通道中相同位置的突变（T449）所产生的变化进行比较。当这个位置被带电荷的残基占据时，会出现最显著的差异：S631K和S631E破坏了hERG中的C型失活，而T449K和T449E则促进了Shaker中的C型失活。S631K和S631E还破坏了hERG孔道的K+选择性，而Shaker的T449K或T449E则未出现这种变化。为了进一步研究为何存在这些差异，我们将S631替换为半胱氨酸。这使我们能够操控631位硫醇基团的性质，并将此处的侧链性质与通道功能的变化联系起来。当硫醇基团处于还原状态时，S631C的行为类似于野生型通道。用H2O2氧化硫醇基团或用MTSET或MTSES对其进行修饰会破坏C型失活和K+选择性，这与S631K和S631E的表型相似。相同的硫醇修饰操作不会影响野生型通道的功能。我们的结果表明hERG和Shaker在外口结构上存在差异，并且我们提出了一个“分子弹簧”假说来解释这些差异。
