Arginine substitution of a cysteine in transmembrane helix M8 converts Na ⁺ ,K ⁺ -ATPase to an electroneutral pump similar to H ⁺ ,K ⁺ -ATPase

Abstract

Na+,K+-ATPase and H+,K+-ATPase are electrogenic and nonelectrogenic ion pumps, respectively. The underlying structural basis for this difference has not been established, and it has not been revealed how the H+,K+-ATPase avoids binding of Na+ at the site corresponding to the Na+-specific site of the Na+,K+-ATPase (site III). In this study, we addressed these questions by using site-directed mutagenesis in combination with enzymatic, transport, and electrophysiological functional measurements. Replacement of the cysteine C932 in transmembrane helix M8 of Na+,K+-ATPase with arginine, present in the H+,K+-ATPase at the corresponding position, converted the normal 3Na+:2K+:1ATP stoichiometry of the Na+,K+-ATPase to electroneutral 2Na+:2K+:1ATP stoichiometry similar to the electroneutral transport mode of the H+,K+-ATPase. The electroneutral C932R mutant of the Na+,K+-ATPase retained a wild-type–like enzyme turnover rate for ATP hydrolysis and rate of cellular K+ uptake. Only a relatively minor reduction of apparent Na+ affinity for activation of phosphorylation from ATP was observed for C932R, whereas replacement of C932 with leucine or phenylalanine, the latter of a size comparable to arginine, led to spectacular reductions of apparent Na+ affinity without changing the electrogenicity. From these results, in combination with structural considerations, it appears that the guanidine+ group of the M8 arginine replaces Na+ at the third site, thus preventing Na+ binding there, although allowing Na+ to bind at the two other sites and become transported. Hence, in the H+,K+-ATPase, the ability of the M8 arginine to donate an internal cation binding at the third site is decisive for the electroneutral transport mode of this pump.

On the effect of hyperaldosteronism-inducing mutations in Na/K pumps

“…The mean parameters rendered from the fits to the individual experiments averaged in Fig. 8 D were V 1/2 = −38 ± 1 mV and slope factor kT/ez q = 32 ± 1 mV ( n = 7) for wild type, and V 1/2 = −109 ± 2 mV; kT/ez q = 54 ± 1 mV ( n = 6) for G99R, consistent with the mutant having an approximately eightfold decrease in overall Na + o apparent affinity; a 25-mV shift of the V 1/2 corresponds to a twofold change in external Na + o concentration ( Holmgren et al, 2000 ; Holmgren and Rakowski, 2006 ) or twofold change in overall external Na + affinity ( Li et al, 2005 ; Yaragatupalli et al, 2009 ; Meier et al, 2010 ; Vedovato and Gadsby, 2010 ; Holm et al, 2017 ).…”

Transient Electrical Currents Mediated by the Na+/K+-ATPase: A Tour from Basic Biophysics to Human Diseases

“…Similarly, Holm et al. ( 38 ) and Nielsen et al. ( 39 ) showed that changes in apparent affinity for external Na + estimated by biochemical data are comparable with those obtained by electrophysiological methods.…”

K+ binding and proton redistribution in the E2P state of the H+, K+-ATPase

“…1 ), which replaces a serine in the Na + , K + -ATPase 2 . A cysteine to arginine mutation in TM8 of the normally electrogenic Na + , K + -ATPase converts it to an electroneutral pump like HKA 14 , highlighting the importance of a positively charged residue in the binding site of HKA. K791 along with E820 are regarded as crucial to proton transfer and the selective K + binding in the E 2 P state of the HKA.…”

“…Unlike alkali cations, protons have to either piggyback on water molecules or will bind to the acidic amino acids thus “protonating” specific amino acids in the cation binding pocket. Such proton redistribution in the cation binding site of the closely related Na + , K + -ATPase has been extensively studied recently 8 , 14 , 23 – 28 because conformation-dependant protonation and deprotonation of acidic residues is deemed important for the release of Na + from the E 2 P.3Na + state, and is proposed to be critical in imparting cation selectivity (Na + versus K + ) to the Na + , K + -ATPase.…”