Ouabain affinity determining residues lie close to the Na/K pump ion pathway

Abstract

The Na/K pump establishes essential ion concentration gradients across animal cell membranes. Cardiotonic steroids, such as ouabain, are specific inhibitors of the Na/K pump. We exploited the marine toxin, palytoxin, to probe both the ion translocation pathway through the Na/K pump and the site of its interaction with ouabain. Palytoxin uncouples the pump's gates, which normally open strictly alternately, thus allowing both gates to sometimes be open, so transforming the pump into an ion channel. Palytoxin therefore permits electrophysiological analysis of even a single Na/K pump. We used outside-out patch recording of Xenopus alpha1beta3 Na/K pumps, which were made ouabain-resistant by point mutation, after expressing them in Xenopus oocytes. Endogenous, ouabain-sensitive, Xenopus alpha1beta3 Na/K pumps were silenced by continuous exposure to ouabain. We found that side-chain charge of two residues at either end of the alpha subunit's first extracellular loop, known to make a major contribution to ouabain affinity, strongly influenced conductance of single palytoxin-bound pump-channels by an electrostatic mechanism. The effects were mimicked by modification of cysteines introduced at those two positions with variously charged methanethiosulfonate reagents. The consequences of these modifications demonstrate that both residues lie in a wide vestibule near the mouth of the pump's ion pathway. Bound ouabain protects the site with the strongest influence on conductance from methanethiosulfonate modification, while leaving the site with the weaker influence unprotected. The results suggest a method for mapping the footprint of bound cardiotonic steroid on the extracellular surface of the Na/K pump.

Fig. 1.

Different conductances of alternative ouabain-resistant PTX-bound pump-channels. (A and B) Currents at –50 mV in outside-out patches excised from oocytes expressing rat α1 (A) or Xenopus DR-α1 (B) Na/K pumps in symmetrical 125 mM Na solutions with 100 μM external ouabain but no pipette ATP, briefly exposed to PTX (50 pM in A and 100 pM in B) until the first channel opening was observed. Lower traces show gating transitions of PTX-bound pump-channels at indicated voltages after washout of unbound toxin; dotted lines mark 0, 1, 2, or 3 open channels. (C) Current amplitudes for channels in A (circles) and B (triangles) plotted against voltage; fits (straight lines) between –100 and –20 mV gave channel conductances γ = 7.5 pS for A and γ = 1.8 pS for B. (D) Sequence alignment of WT Xenopus α1, Xenopus DR-α1, rat α1, and sheep α1 Na,K-ATPases, and rabbit SERCA Ca-ATPase; all residues are numbered from Met 1. Loop 1-2 assignment is from cardiotonic steroid-binding studies (e.g., ref. 14), not later SERCA structures.

Fig. 2.

Charge substitutions at position 131 influence single pump-channel conductance. (A–C) Unitary current transitions of PTX-bound pump-channels at the indicated voltages in outside-out patches expressing C113Y Xenopus α1 Na,K-ATPase mutants with native Asn (A), or with Asp (B) or Arg (C), at position 131. Solutions are as in Fig. 1; brief PTX (100 pM) application is not illustrated in B and C. (D) Current–voltage plots for channels in A (N131; circles, γ = 3.6 pS), B (N131D; triangles, γ = 5.5 pS), and C (N131R; squares, γ = 0.95 pS).

Fig. 3.

Dependence on Na activity of conductance of single pump-channels with neutral N131 (circles), negative N131D (triangles), or positive N131R (squares) residue at position 131. Experiments are as in Figs. 1 and 2 but with varying external (and internal; Materials and Methods) Na concentration (upper abscissa). Na activity coefficients (from 0.90 to 0.47 for [Na] 7.5 to 1,000 mM) were estimated from the osmolality of Na-sulfamate solutions (25). Curves show Michaelis–Menten fits (see Results for parameters) to mean data.

Fig. 4.

Residue 131 is water accessible in PTX-bound pump-channels. (A–C) Macroscopic current recordings of N131C Na/K pumps at −50 mV in outside-out patches in 125 mM Na solutions with 100 μM ouabain and 5 mM pipette MgATP, exposed (as indicated) to 100 nM PTX to open thousands of pump-channels. Brief switches from Na to poorly permeant N-methyl-d-glucamine (NMG) or tetramethylammonium (TMA) served to verify pipette-membrane seal integrity. Repeated triangular disturbances of current records (also Figs. 5 and 6) mark application of 100-ms voltage steps to gather current-voltage data. (A) One millimolar MTSET⁺ diminished PTX-induced inward current, but 10 mM DTT slowly restored it; DTT also caused a rapid and reversible current decrease, which was not studied further. (B) One millimolar MTSACE did not alter PTX-induced current but prevented MTSET⁺ from decreasing it. (C) One millimolar MTSES⁻ increased PTX-induced current. (D) Mean steady-state PTX-induced current, normalized to control at −100 mV, plotted vs. voltage, before MTS reaction (control; circles, n = 11) or after MTSET⁺ (squares, n = 7), MTSACE (stars, n = 2), or MTSES⁻ (triangles, n = 2). Linear fits between −100 and −20 mV yielded slopes (in mV⁻¹) of 0.011 for control, 0.003 after MTSET⁺, 0.010 after MTSACE, and 0.019 after MTSES⁻.

Fig. 5.

Residue 131 is water accessible without PTX. (A) Outside-out patch with N131C Na/K pumps were held at –50 mV and exposed for 5 min to 2 mM MTSET⁺ in the presence of 1 μM ouabain. After [ouabain] was raised to 100 μM, PTX (100 nM) elicited a small inward current that was unaltered by reexposure to MTSET⁺ but was greatly increased by 20 mM DTT; a third exposure (≈20 s) to MTSET⁺ abolished that current. (B) Similar to A except that [ouabain] was much higher, 10 mM, during the 5-min exposure to 2 mM MTSET⁺. After lowering [ouabain] to 100 μM, PTX (100 nM) induced a large inward current that was rapidly decreased by a second exposure (≈20 s) to MTSET⁺.

Fig. 6.

MTS modification of Q120C Na/K pump-channels. (A) Mean steady-state PTX-induced current–voltage plots, normalized to control current at −90 mV; experiment and solutions as in Fig. 4. Relative to control (circles, n = 8), MTSET⁺ decreased current ≈30% (squares, n = 4) and MTSES⁻ increased current ≈25% (triangles, n = 4). Linear fits between −100 and −20 mV gave slopes 0.011 mV⁻¹ before MTS application, 0.008 mV⁻¹ after MTSET⁺, and 0.016 mV⁻¹ after MTSES⁻. (B) Bound ouabain (10 mM) does not protect Q120C Na/K pumps from modification by MTSET⁺; experiment and solutions as in Fig. 5B. After lowering [ouabain] to 100 μM, PTX induced current that was unaffected by reexposure to MTSET⁺, indicating that all Cys had been modified by MTSET⁺ in 10 mM ouabain.

Fig. 7.

Luminal view of E2P-related Ca-ATPase structure (PDB ID code 1WPG; ref. 7). The backbone carbons of E79 and A87 (equivalent to Q120 and N131 in Xenopus α1 Na,K-ATPase and to R118 and D129 in rat α1 Na,K-ATPase; see Fig. 1D) are 15.9 Å and 10.8 Å, respectively, distant from P789 (equivalent to T806 in Xenopus α1). Figure prepared with PyMOL (www.pymol.org).