钠钾ATP酶天冬酰胺776突变体的双电极电压钳分析

Two-electrode voltage-clamp analysis of Na,K-ATPase asparagine 776 mutants.

作者信息

Koenderink Jan B, Geibel Sven, Grabsch Eva, De Pont Jan Joep H H M, Bamberg Ernst, Friedrich Thomas

机构信息

Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, Frankfurt am Main, Germany.

出版信息

Ann N Y Acad Sci. 2003 Apr;986:150-4. doi: 10.1111/j.1749-6632.2003.tb07152.x.

DOI:10.1111/j.1749-6632.2003.tb07152.x

PMID:12763788

Abstract

Steady-state and pre-steady-state currents of Asn(776) mutants of Na,K-ATPase are presented. The stationary current generated by N776Q strongly depends on the membrane potential, but has a negative slope, opposite to that of the wild-type enzyme. The apparent rate constant of the reaction sequence E(1)P(Na(+)) <--> E(2)P + Na(+) of this mutant is rather independent of the membrane potential and is at resting and depolarizing membrane potential higher than that of the wild-type enzyme. Thus, the voltage-dependent increase of the rate coefficient of the wild type that is associated with extracellular Na(+) rebinding is almost absent in the N776Q mutant. These findings indicate that dislocating the carboxamide group of Asn(776) decreases the affinity of sodium at its extracellular binding site.

摘要

本文展示了钠钾ATP酶天冬酰胺（776）突变体的稳态电流和稳态前电流。由N776Q产生的稳态电流强烈依赖于膜电位，但具有负斜率，这与野生型酶相反。该突变体反应序列E(1)P(Na(+)) <--> E(2)P + Na(+)的表观速率常数相当独立于膜电位，并且在静息和去极化膜电位下均高于野生型酶。因此，与细胞外钠离子重新结合相关的野生型速率系数的电压依赖性增加在N776Q突变体中几乎不存在。这些发现表明，天冬酰胺（776）的羧酰胺基团移位会降低钠离子在其细胞外结合位点的亲和力。