Relative Movement Of The α-Subunit's First And Last External Loops Throughout Na/K-Pump Cycle

Abstract

Structural models of the Na/K-pump α-subunit show that conserved residues D121 (loop 1-2, pig-α1 numbering) and R972 (loop 9-10) are 5-7 A apart in E2, and more than 12 A away in E1. To check whether these residues reach conformation-dependent atomic proximity, the substitutions D121C and R972C were introduced (individually and concurrently) into the ouabain-insensitive C104Y-Xenopus-α1. Each mutant was coexpressed with Xenopus-β3 in Xenopus oocytes and their function tested in Na+-loaded oocytes under two-electrode voltage clamp. K+o-activated Na/K-pump currents were observed in oocytes injected with D121C or R972C, but not in those injected with D121C-R972C, unless the eggs were exposed to TCEP (10-50 mM, ∼20 min), consistent with the presence of a pump-inhibiting disulfide.To identify the conformation locked by the disulfide, we used palytoxin to transform Na/K-pumps into channels. Palytoxin-induced currents (IPTX) in outside-out patches from oocytes expressing D121C-R972C, bathed in Na+ solutions, were insensitive to MTSET+ application. Patch exposure to DTT restored MTSET+-sensitivity (∼65% IPTX reduction) without affecting IPTX amplitude. Palytoxin stabilizes an E2P-like structure; thus, the lack of DTT effect on IPTX suggest that cross-linking between D121C and R972C occurs in E2P, with the external cation pathway open. Moreover, pump inhibition by spontaneous disulfide formation indicates that conformational mobility between these residues is required for the E2 to E1 transition.The slow component of ouabain-sensitive transient charge movement in 125 mM Nao+ was measured in Na+-loaded oocytes expressing these mutants. The center of the equilibrium distribution of charge (voltage of equal occupancy of E1 and E2) for R972C (V1/2= -34±8 mV) was identical to that of the C104Y-α1 template (V1/2= -35±3 mV). Therefore, it appears that a D121-R972 salt bridge is not necessary for E2P stabilization among Na+-occupied states.