Na+/K+ Pump Ion Binding Site Interactions Regulate the Proton Leak Pathway

Abstract

The Na+/K+ pump (NKA) is a membrane bound transporter located in animal cells which transports three Na+ ions out for two K+ ions into the cell for every one ATP molecule hydrolyzed. Two out-of-three ion-binding sites within the protein reciprocally bind two K+ or two Na+ ions (shared sites), while the remaining site exclusively binds Na+. Without these two ions present the NKA passively transports H+, likely through the Na-exclusive site. In order to understand the proton transport mechanism voltage-clamp was used to study the effect of tetrapropylammonium (TPA) and Beryllium Fluoride (BeFx) on inward proton current (IH) transport by NKA expressed in Xenopus oocytes. TPA (a shared site blocker) had a dual effect on IH; between pH 7.6-6.0 TPA partially inhibited IH, and at pH 5.0 (where an inhibitory effect on the steady state IH is present), TPA partially activated IH. When the phosphate analogue BeFx was injected into oocytes, stabilizing an externally open conformation of the ion binding sites, K+ still inhibited inward current at pH 7.6. At pH 5.0 K+ activated IH resembling the effect of TPA on fully functional NKA. These results illustrate the complex interaction between the occupancy of shared sites and proton permeation. Blocking access at the shared sites with TPA activated IH by relieving the inhibition normally observed at high [H+]. Partial inhibition by TPA at pH 5.0 may reflect incomplete block of H+ access or a reduced rate of the conformational changes needed for H+ translocation. After BeFx inhibition NKA was still capable of interacting with K+. The mixed occupancy of K+ and H+ at low pH activated the leak, likely relieving the H+ inhibition due to K+ occlusion. Supported by NIH GM 061583 to CG and NSF MCB-1243842 to PA.