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pH dependence of the Slc4a11-mediated H+ conductance is influenced by intracellular lysine residues and modified by disease-linked mutations.
American Journal of Physiology-Cell Physiology ( IF 5.0 ) Pub Date : 2020-07-28 , DOI: 10.1152/ajpcell.00128.2020
Bianca N Quade 1 , Aniko Marshall 1 , Mark D Parker 1, 2, 3
Affiliation  

SLC4A11 is the only member of the SLC4 family that transports protons rather than bicarbonate. SLC4A11 is expressed in corneal endothelial cells, and its mutation causes corneal endothelial dystrophy, although the mechanism of pathogenesis is unknown. We previously demonstrated that the magnitude of the H+ conductance (Gm) mediated by SLC4A11 is increased by rises in intracellular as well as extracellular pH (pHi and pHe). To better understand this feature and whether it is altered in disease, we studied the pH dependence of wild-type and mutant mouse Slc4a11 expressed in Xenopus oocytes. Using voltage-clamp circuitry in conjunction with a H+-selective microelectrode and a microinjector loaded with NaHCO3, we caused incremental rises in oocyte pHi and measured the effect on Gm. We find that the rise of Gm has a steeper pHi dependence at pHe =8.50 than at pHe =7.50. Data gathered at pHe =8.50 can be fit to the Hill equation enabling the calculation of a pK value that reports pHi dependence. We find that mutation of lysine residues that are close to the first transmembrane span (TM1) causes an alkaline shift in pK. Furthermore, two corneal-dystrophy-causing mutations close to the extracellular end of TM1, E399K and T401K (E368K and T370K in mouse), cause an acidic shift in pK, while a third mutation in the fourth intracellular loop, R804H (R774H in mouse), causes an alkaline shift in pK. This is the first description of determinants of SLC4A11 pH dependence and the first indication that a shift in pH dependence could modify disease expressivity in some cases of corneal dystrophy.

中文翻译:

Slc4a11 介导的 H+ 电导的 pH 依赖性受细胞内赖氨酸残基的影响,并受疾病相关突变的修饰。

SLC4A11 是 SLC4 家族中唯一运输质子而不是碳酸氢盐的成员。SLC4A11在角膜内皮细胞中表达,其突变导致角膜内皮营养不良,但发病机制尚不清楚。我们之前已经证明,由 SLC4A11 介导的 H +电导 ( G m ) 的大小随着细胞内和细胞外 pH 值(pH i和 pH e)的升高而增加。为了更好地了解这一特征以及它是否在疾病中发生改变,我们研究了在非洲爪蟾卵母细胞中表达的野生型和突变型小鼠 Slc4a11 的 pH 依赖性。将电压钳电路与 H +结合使用选择性微电极和装有 NaHCO 3的显微注射器,我们引起卵母细胞 pH i 的增量上升并测量对G m的影响。我们发现G m的上升在 pH e  = 8.50 时比在 pH e  = 7.50 时具有更陡峭的 pH i依赖性。在 pH e  = 8.50 下收集的数据可以拟合 Hill 方程,从而能够计算报告 pH i依赖性的 ap K值。我们发现接近第一跨膜跨度 (TM1) 的赖氨酸残基的突变导致 p K的碱性位移. 此外,靠近 TM1、E399K 和 T401K(小鼠中的 E368K 和 T370K)细胞外末端的两个导致角膜营养不良的突变导致 p K的酸性变化,而第四个细胞内环中的第三个突变 R804H(小鼠中的 R774H) mouse),导致 p K发生碱性变化。这是对 SLC4A11 pH 依赖性决定因素的首次描述,并且首次表明 pH 依赖性的转变可能会改变某些角膜营养不良病例的疾病表现。
更新日期:2020-08-20
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