Inhibition of Na,K ATPase partial reactions by organic amines

Abstract

The Na,K‐ATPase (NKA) transports 3 Na ions out of the cell and 2 K ions into the cell for every one ATP molecule hydrolyzed. Two of the three ion‐binding sites within the protein reciprocally bind two K or two Na ions (shared sites), while the other site exclusively binds Na). Three modes of transport can be studied with two electrode voltage clamp (TEVC) in Xenopus oocytes to evaluate NKA function and its partial reactions involving external ion binding: 1) outward pump current (IP) induced by forward NKA cycling when extracellular K is added, 2) transient charge movement (QNa) induced by voltage‐dependent binding and release of Na, rate limited by the NKA conformational change and 3) uncoupled proton import at negative voltages without external Na or K the pump transports H down their electrochemical gradient (IH). Tetrapropylammonium (TPA) and ethylenediamine (EDA), are known competitive inhibitors of extracellular K binding and their mechanisms of action are not fully understood (particularly for EDA). We used TEVC to evaluate the effect of TPA and EDA on IP, QNa and IH. TPA acted as a competitive inhibitor of K binding by reducing its apparent affinity for IP activation in a voltage‐independent manner IC50 of 8.78 ± 0.12 mM. EDA also acted as a K competitor, but its IC50 changed from 8.96 ± 0.48 mM at −100 mV to 15.92 ± 1.84 mM a 0 mV. When evaluating QNa, the position of the charge voltage curve (Q‐V), parameterized by the curve's center voltage (V1/2) related to the apparent affinity for external Na (more negative V1/2 indicating a reduction of Na affinity and viceversa), was −54.39 ± 1.65 mV in the absence of amines, −120.88 ± 2.15 mV in the presence of 20 mM TPA and −10.49 ± 6.13 mV in the presence of 15 mM EDA. While TPA did not significantly affect the total charge moved (related to the number of pumps performing the Na binding reactions) EDA reduced it by 53.6 ± 0.3%. This indicates that TPA acts as a prototypical competitive inhibitor of external Na reducing Na apparent affinity, but EDA does not. Finally, TPA inhibited IH (~70% maximal inhibition) at both pH 7.6 and pH 6.0, while EDA had no effect on IH at pH 7.6, but increased IH (by 2 fold) at pH 6.0 at −180 mV. Our results are consistent with the larger TPA acting as a large extracellular access‐channel blocker therefore competing with Na, K and H without reaching the ion‐binding sites, while EDA reaches the shared sites domain, probably becoming occluded there, therefore making NKAs unavailable to induce QNa. The complex effects of EDA on IH may reflect the complex nature of its occlusion there and are still under evaluation.Support or Funding InformationWork supported by NIH GM 061583 to CG and NSF MCB‐1243842 to PA.