Dissecting movement of the transmembrane segments of non-gastric proton pump mutants with voltage-clamp fluorometry.

Abstract

The Na+,K+-ATPase (NKA) extrudes 3 Na+ and imports 2 K+ across the plasmalemma of every animal cell. The non-gastric H+,K+- ATPase (ngHKA) exports one H+ and imports one K+ in the apical membrane of several epithelia. Both P-type pumps have ∼65% identity and nearly identical catalytic cycles driven by binding of different ions to two major conformations (E1, high affinity for Na+ or H+ and E2 higher affinity for K+). Recently, we reported the structures of the wild type (WT) ngHKA and its electrogenic mutant K794A and the NKA-like K794S/A797P/W940/R949C (SPWC) mutant, in the K+-occluded E2 state. We also reported SPWC's AMPCP-bound E1 Cryo-EM structure, which is nearly identical to that of the E1(3Na+) NKA. We obtained the WT (Na+ and AlF-ADP) and K794A (Na+ and AMPPCP) structures. While the WT structure was identical to E2P state, the AMPPCP-bound K794A structure with (presumably) 2 Na+ bound has a mixed conformation. The P and N domains as well as cytoplasmic portion of transmembrane segment TM4 shows E1-like conformation, but the A domain, TM1-TM3 and the luminal portion of TM4 took an E2-like conformation. To evaluate displacements of the moving TM segments in all three constructs, we introduced a single Cys residue in the loops between TM1-TM2, TM3-4, or TM5-6, labeled them with tetramethylrhodamine maleimide (TMRM) and evaluated currents and fluorescence changes under voltage clamp fluorometry in Xenopus oocytes. Consistent with the stable E2 state in WT, a TMRM introduced in TM1-TM2 showed minimal voltage dependent changes in fluorescence, while the voltage dependence of the TM1-TM2 fluorescent signals observed with K794A and SPWC mutants were progressively closer to signals observed in NKA. Studies with TMRM introduced at TM3-4 and TM5-6 positions are underway. Funded by NSF MCB-2003251.