感应电荷突变对 KV7.2 通道失活的影响

IF 3.3 2区 医学 Q1 PHYSIOLOGY Journal of General Physiology Pub Date : 2024-03-04 Epub Date: 2024-01-18 DOI:10.1085/jgp.202213284
Baharak Mehrdel, Carlos A Villalba-Galea
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引用次数: 0

摘要

KV7 家族的钾选择性电压门控通道是许多细胞类型中电兴奋性的关键调节器。移除人类 KV7.2 最外层的假定感应电荷(R198)会使其激活电压依赖性转向更负的电位。这表明,去除电压感应结构域第四(S4)段 "顶部 "的电荷有助于激活。在此,我们假设恢复该电荷将使激活恢复到正常电压范围。我们在 KV7.2 中引入了突变 R198H,并设想用质子滴定引入的组氨酸将恢复感应电荷。正如预测的那样,突变体的激活电压依赖性随外部 pH 值(pHEXT)的变化而变化,而野生型(WT)通道的激活电压依赖性变化不大。另一方面,R198H 突变体的失活动力学对 pHEXT 变化非常敏感,在 pHEXT 为 6 时很容易失活,而在 pHEXT 为 8 时失活则变得缓慢。相比之下,KV7.2 WT 的失活动力学随 pHEXT 的变化不大。这表明残基 198 的电荷对去活至关重要。然而,令人吃惊的是,突变体 R198Q--一种非滴定突变--也显示出较高的 pHEXT 敏感活性。因此,我们得出结论:不是 198 位的电荷,而是通道胞外面的质子化状态调节了开放通道的稳定性,并且残基 198 的电荷是电压传感器有效停用通道所必需的,从而克服了高 pHEXT 的稳定作用。
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Effect of a sensing charge mutation on the deactivation of KV7.2 channels.

Potassium-selective, voltage-gated channels of the KV7 family are critical regulators of electrical excitability in many cell types. Removing the outermost putative sensing charge (R198) of the human KV7.2 shifts its activation voltage dependence toward more negative potentials. This suggests that removing a charge "at the top" of the fourth (S4) segment of the voltage-sensing domain facilitates activation. Here, we hypothesized that restoring that charge would bring back the activation to its normal voltage range. We introduced the mutation R198H in KV7.2 with the idea that titrating the introduced histidine with protons would reinstate the sensing charge. As predicted, the mutant's activation voltage dependence changed as a function of the external pH (pHEXT) while modest changes in the activation voltage dependence were observed with the wild-type (WT) channel. On the other hand, the deactivation kinetics of the R198H mutant was remarkably sensitive to pHEXT changes, readily deactivating at pHEXT 6, while becoming slower to deactivate at pHEXT 8. In contrast, the KV7.2 WT displayed modest changes in the deactivation kinetics as a function of pHEXT. This suggested that the charge of residue 198 was critical for deactivation. However, in a surprising turn, the mutant R198Q-a non-titratable mutation-also displayed a high pHEXT sensitivity activity. We thus concluded that rather than the charge at position 198, the protonation status of the channel's extracellular face modulates the open channel stabilization and that the charge of residue 198 is required for the voltage sensor to effectively deactivate the channel, overcoming the stabilizing effect of high pHEXT.

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来源期刊
CiteScore
6.00
自引率
10.50%
发文量
88
审稿时长
6-12 weeks
期刊介绍: General physiology is the study of biological mechanisms through analytical investigations, which decipher the molecular and cellular mechanisms underlying biological function at all levels of organization. The mission of Journal of General Physiology (JGP) is to publish mechanistic and quantitative molecular and cellular physiology of the highest quality, to provide a best-in-class author experience, and to nurture future generations of independent researchers. The major emphasis is on physiological problems at the cellular and molecular level.
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