Functional Significance of E2 State Stabilization by Specific α/β-Subunit Interactions of Na,K- and H,K-ATPase

Abstract

The ␤-subunits of Na,K-ATPase and H,K-ATPase have important functions in maturation and plasma membrane targeting of the catalytic ␣-subunit but also modulate the transport activity of the holoenzymes. In this study, we show that tryptophan replacement of two highly conserved tyrosines in the transmembrane domain of both Na,K-and gastric H,K-ATPase ␤-subunits resulted in considerable shifts of the voltagedependent E 1 P/E 2 P distributions toward the E 1 P state as inferred from presteady-state current and voltage clamp fluorometric measurements of tetramethylrhodamine-6-maleimidelabeled ATPases. The shifts in conformational equilibria were accompanied by significant decreases in the apparent affinities for extracellular K ؉ that were moderate for the Na,K-ATPase ␤-(Y39W,Y43W) mutation but much more pronounced for the corresponding H,K-ATPase ␤-(Y44W,Y48W) variant. Moreover in the Na,K-ATPase ␤-(Y39W,Y43W) mutant, the apparent rate constant for reverse binding of extracellular Na ؉ and the subsequent E 2 P-E 1 P conversion, as determined from transient current kinetics, was significantly accelerated, resulting in enhanced Na ؉ competition for extracellular K ؉ binding especially at extremely negative potentials. Analogously the reverse binding of extracellular protons and subsequent E 2 P-E 1 P conversion was accelerated by the H,K-ATPase ␤-(Y44W,Y48W) mutation, and H ؉ secretion was strongly impaired. Remarkably tryptophan replacements of residues in the M7 segment of Na,K-and H,K-ATPase ␣-subunits, which are at interacting distance to the ␤-tyrosines, resulted in similar E 1 shifts, indicating their participation in stabilization of E 2 . Thus, interactions between selected residues within the transmembrane regions of ␣-and ␤-subunits of P 2C -type ATPases exert an E 2 -stabilizing effect, which is of particular importance for efficient H ؉ pumping by H,K-ATPase under in vivo conditions.

Cryo-EM structure of gastric H + ,K + -ATPase with a single occupied cation-binding site

“…4C). Together with previously reported findings regarding the E2 or E2P preference of H + ,K + -ATPase (19)(20)(21)(22)28), the proposed vectorial transport model (Fig. 4) describes how gastric H + ,K + -ATPase can generate the highly acidic condition in the gastric lumen.…”

Computation and Functional Studies Provide a Model for the Structure of the Zinc Transporter hZIP4

“…The supernatants were mixed and incubated with 50 l of NeutrAvidin resin at room temperature for 60 min with gentle shaking. The resin was washed four times with sol- (29), 50 oocytes were added to breaking buffer (20 mM HEPES, pH 7.4, 150 mM NaCl and 1 mM PMSF) and passed through a 25-gauge needle followed by 1 min of low speed centrifugation (100 ϫ g and 4°C). The supernatant was collected in a separate microcentrifuge tube and centrifuged (1 min at 100 ϫ g and 4°C).…”

Measuring Cation Transport by Na,K- and H,K-ATPase in <em>Xenopus</em> Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

“…To correlate the measured Rb + fluxes with the amount of H + ,K + -ATPase protein in the plasma membrane, we prepared plasma membrane (PM) fractions and total cellular membrane fractions (TM) from oocytes expressing the constructs from Figure 3A, and subjected the samples to SDS-PAGE (http://www.jove.com/video/758/electrophoretic-separation-of-proteins) 20 and Western blotting with antibodies directed against the H + ,K + -ATPase α-subunit. This plasma membrane purification procedure has first been described by Kamsteeg and Deen21 and, later, with some modifications in 4,22 . .…”

“…Using this technique, we were able to determine e.g. the apparent Rb + affinities of cation transport [4][5][6] , the influence of extra-and intracellular pH 7 and the effect of mutations of residues involved in cation coordination during transport 4,8 . An advantage of the technique is that ion fluxes can also be determined in combination with two-electrode voltage clamping of the oocytes, which on one hand assures accurate control of the membrane potential during transport and on the other hand allows to correlate ion flux with membrane current.…”