title: Two-electrode voltage-clamp analysis of Na,K-ATPase asparagine 776 mutants.
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.
authors.affiliation: Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, Frankfurt am Main, Germany. J.Koenderink@ncmls.kun.nl
authors.forename: Jan B
authors.initials: JB
authors.lastname: Koenderink
authors.forename: Sven
authors.initials: S
authors.lastname: Geibel
authors.forename: Eva
authors.initials: E
authors.lastname: Grabsch
authors.forename: Jan Joep H H M
authors.initials: JJ
authors.lastname: De Pont
authors.forename: Ernst
authors.initials: E
authors.lastname: Bamberg
authors.forename: Thomas
authors.initials: T
authors.lastname: Friedrich
mesh_terms: D000409:Alanine; D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine; D001224:Aspartic Acid; D005260:Female; D005973:Glutamine; D007700:Kinetics; D009865:Oocytes / Q000502:physiology*; D018408:Patch-Clamp Techniques; D021122:Protein Subunits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; D005973:Glutamine; D001224:Aspartic Acid; D001216:Asparagine; D000254:Sodium-Potassium-Exchanging ATPase; D000409:Alanine