Abstract
The beta-subunits of Na,K-ATPase and H,K-ATPase have important functions in maturation and plasma membrane targeting of the catalytic alpha-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 beta-subunits resulted in considerable shifts of the voltage-dependent E1P/E2P distributions toward the E1P state as inferred from presteady-state current and voltage clamp fluorometric measurements of tetramethylrhodamine-6-maleimide-labeled 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 beta-(Y39W,Y43W) mutation but much more pronounced for the corresponding H,K-ATPase beta-(Y44W,Y48W) variant. Moreover in the Na,K-ATPase beta-(Y39W,Y43W) mutant, the apparent rate constant for reverse binding of extracellular Na+ and the subsequent E2P-E1P 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 E2P-E1P conversion was accelerated by the H,K-ATPase beta-(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 alpha-subunits, which are at interacting distance to the beta-tyrosines, resulted in similar E1 shifts, indicating their participation in stabilization of E2. Thus, interactions between selected residues within the transmembrane regions of alpha- and beta-subunits of P2C-type ATPases exert an E2-stabilizing effect, which is of particular importance for efficient H+ pumping by H,K-ATPase under in vivo conditions.
Highlights
The ubiquitous sodium pump and the closely related gastric proton pump are members of the P-type ATPase family that comprises more than 200 identified members [1, 2]
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 E1P/E2P distributions toward the E1P state as inferred from presteady-state current and voltage clamp fluorometric measurements of tetramethylrhodamine-6-maleimidelabeled ATPases
Even the observed repositioning of the transmembrane domain (TMD)2 itself could be responsible for the reported Kϩ effects in the N-terminally truncated -variant because mutations in the TMD have been shown to modify cation transport: tryptophan scanning mutagenesis in the TMD of the Na,K-ATPase -subunit revealed that the replacement of two tyrosines by tryptophan has distinct and additive consequences for the cation affinities of the holoenzyme [25]
Summary
Molecular Biology—The cDNAs of the sheep Na,K-ATPase 1-subunit, rat H,K-ATPase -subunit, rat gastric H,K-ATPase ␣-subunit, and a modified form of the sheep Na,K-ATPase ␣1subunit without extracellularly exposed cysteine residues (containing mutations C911S and C964A [31]) and with reduced ouabain sensitivity in the millimolar range (achieved by the mutations Q111R and N122D [32]) were subcloned into vector pTLN [33] as described previously [27, 28]. ATPase-expressing oocytes were preincubated for 15 min in a Rbϩ- and Kϩ-free solution (90 mM TMACl or NaCl, 20 mM tetraethylammonium chloride, 5 mM BaCl2, 5 mM NiCl2, 10 mM HEPES, pH 7.4) containing 100 M ouabain to ensure inhibition of the endogenous Na,K-ATPase and incubated for 15 min under temperature control in Rbϩ flux buffer at 21 °C Site-specific labeling was achieved by incubating oocytes in postloading buffer containing 5 M TMRM (Molecular Probes; stock solution, 5 mM in DMSO) for 5 min at room temperature in the dark followed by extensive washes in dye-free postloading buffer. Stationary currents of the Na,K-ATPase were measured upon a solution exchange from 0 to 10 mM Kϩ (10 mM KCl, 90 mM NaCl, 5 mM BaCl2, 5 mM NiCl2, 5 mM MOPS/Tris, pH 7.4, 10 M ouabain). The translocated charge Q was calculated from the integral of the fitted transient currents, and the resulting Q-V curves were approximated by a Boltzmanntype function,
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