Distinct effects of Q925 mutation on intracellular and extracellular Na+ and K+ binding to the Na+, K+-ATPase

Abstract

Three Na+ sites are defined in the Na+-bound crystal structure of Na+, K+-ATPase. Sites I and II overlap with two K+ sites in the K+-bound structure, whereas site III is unique and Na+ specific. A glutamine in transmembrane helix M8 (Q925) appears from the crystal structures to coordinate Na+ at site III, but does not contribute to K+ coordination at sites I and II. Here we address the functional role of Q925 in the various conformational states of Na+, K+-ATPase by examining the mutants Q925A/G/E/N/L/I/Y. We characterized these mutants both enzymatically and electrophysiologically, thereby revealing their Na+ and K+ binding properties. Remarkably, Q925 substitutions had minor effects on Na+ binding from the intracellular side of the membrane – in fact, mutations Q925A and Q925G increased the apparent Na+ affinity – but caused dramatic reductions of the binding of K+ as well as Na+ from the extracellular side of the membrane. These results provide insight into the changes taking place in the Na+-binding sites, when they are transformed from intracellular- to extracellular-facing orientation in relation to the ion translocation process, and demonstrate the interaction between sites III and I and a possible gating function of Q925 in the release of Na+ at the extracellular side.

Structure and function of H+/K+ pump mutants reveal Na+/K+ pump mechanisms

“…The inhibitory phase of Na + ,K + -ATPase activity at high K + concentrations (Fig. 5f , IC 50,K+ = 96 mM) resembles those reported in mutants that disrupt Na + affinity 26 , 27 , 33 , 34 , and probably reflects a combination of increased K + affinity and reduced affinity for the competing Na + for the pump in the E1 conformation. In contrast to the high Na + -independent activities observed in other ngHKA constructs (Fig.…”

“…Gln923 and Asp926 contribute Na + -coordinating side chains to site III in the NKA 8 , 25 – 27 and are conserved in HKA pumps (Gln942 and Asp945, respectively, in ngHKA). Between these two residues, facing the other side of the TM8 helix, NKA has tryptophan (Trp924) while both HKAs have an isoleucine (Ile943 in ngHKA).…”

“…4b, c ) and resembles currents observed in certain NKA mutants that reduce apparent affinity for Na + (refs. 27 , 29 , 30 ). SWC’s non-sigmoidal Q–V curve (Fig.…”

“…This leak has a complex dependence on extracellular Na + , being inhibited at physiological concentrations, but activated at Na + concentrations ≤5 mM 43 . Therefore, provided the proton permeation pathway through site III is maintained, various NKA mutations that reduce affinity for Na + frequently reduce the ion’s leak-inhibitory effect and show enhanced leak currents in the presence of physiological Na + concentrations 27 , 29 , 30 . However, mutations that directly interrupt the H + pathway at site III block the leak 44 , 45 .…”

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

“…For example, C-terminal deletions substantially shifted the charge distribution of the slow component to more negative voltages, indicating a reduction of the apparent affinity for external Na þ (37). Similarly, Holm et al (38) and Nielsen et al (39) showed that changes in apparent affinity for external Na þ estimated by biochemical data are comparable with those obtained by electrophysiological methods. Further, the combination of both approaches provides strength to the specific structural inferences in these studies.…”