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Proc Natl Acad Sci U S A
2006 Aug 15;10333:12613-8. doi: 10.1073/pnas.0602720103.
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Ouabain affinity determining residues lie close to the Na/K pump ion pathway.
Artigas P, Gadsby DC.
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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.
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