Takeuchi A et al. (2008), The ion pathway through the opened Na(+),K(+)-A...

XB-ART-38689

Nature 2008 Nov 20;4567220:413-6. doi: 10.1038/nature07350.

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The ion pathway through the opened Na(+),K(+)-ATPase pump.

Takeuchi A , Reyes N , Artigas P , Gadsby DC .

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P-type ATPases pump ions across membranes, generating steep electrochemical gradients that are essential for the function of all cells. Access to the ion-binding sites within the pumps alternates between the two sides of the membrane to avoid the dissipation of the gradients that would occur during simultaneous access. In Na(+),K(+)-ATPase pumps treated with the marine agent palytoxin, this strict alternation is disrupted and binding sites are sometimes simultaneously accessible from both sides of the membrane, transforming the pumps into ion channels (see, for example, refs 2, 3). Current recordings in these channels can monitor accessibility of introduced cysteine residues to water-soluble sulphydryl-specific reagents. We found previously that Na(+),K(+) pump-channels open to the extracellular surface through a deep and wide vestibule that emanates from a narrower pathway between transmembrane helices 4 and 6 (TM4 and TM6). Here we report that cysteine scans from TM1 to TM6 reveal a single unbroken cation pathway that traverses palytoxin-bound Na(+),K(+) pump-channels from one side of the membrane to the other. This pathway comprises residues from TM1, TM2, TM4 and TM6, passes through ion-binding site II, and is probably conserved in structurally and evolutionarily related P-type pumps, such as sarcoplasmic- and endoplasmic-reticulum Ca(2+)-ATPases and H(+),K(+)-ATPases.

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	Figure 2. Effects of MTSET+ on current through palytoxin-bound Na+,K+ pump-channels with cysteines in TM5 or TM5-TM6 loopa-d, Current at -50 mV in outside-out patches exposed to symmetrical Na+ concentrations. Application of 50 nM palytoxin (black arrowheads) generated inward (negative) current, Ipalytoxin (dashed line marks zero total membrane current). Temporary substitution (asterisk) of less permeant TMA+ (tetramethylammonium) for external Na+ monitored patch integrity. Application of 10 mM dithiothreitol (grey arrows, grey traces) caused a small, reversible, poorly understood current decrease. Then, 1 mM MTSET+ (blue arrows, blue traces) was applied until the current became steady. e, Summary of mean (± s.e.m., n=3-6 patches) % inhibition of Ipalytoxin by 1 mM MTSET+ at -50 mV for each single-cysteine mutant.
	Figure 3. Effects of MTSET+ on current through palytoxin-bound Na+,K+ pump-channels with cysteines in TM1, TM2, or TM1-TM2 loopa, b, d, e, Representative current recordings in outside-out patches under same conditions, and with applications of palytoxin, TMA+, dithiothreitol, and MTSET+, as in Fig. 2. c, f, Summary of % inhibition of Ipalytoxin by 1 mM MTSET+ at -50 mV for each single-cysteine mutant, given as mean ± s.e.m. (n=3-11 patches, except for Q120C [n=2] and N131C [n=1], both previously shown18 to be MTSET+-accessible). C113C indicates data from wild type, ouabain-sensitive, Xenopus Na+,K+ pumps tested (in the absence of ouabain) in patches from non-injected control oocytes.
	Figure 4. Structural model and characteristics of ion pathway through the palytoxin-bound Na+,K+-ATPaseResults (including reactive and non-responsive positions from ref. 5) mapped onto a homology model of the Na+,K+-ATPase TM domain (helices coloured as in Fig. 1) based on the SERCA E2·BeF3- structure12, viewed from the extracellular surface (a) or from the membrane plane (b). Dashed line in a indicates plane of cut in Supplementary Fig. 2a. Red sticks mark reactive positions (Ipalytoxin altered >10% by MTSET+), and yellow sticks mark non-responsive positions. Reaction rate constants for MTSET+ declined from ≥104 M-1s-1 for superficial positions to ≥10 M-1s-1 for deep positions (Supplementary Fig. 8). c, Accessibility of cysteines beyond the cation selectivity filter depends on charge of MTS reagent; summary of mean % inhibition (± s.e.m., n=3-8 patches) of Ipalytoxin at -50 mV by ∼2.5-min applications (all 1 mM) of MTSES- (red bars), MTSET+ (blue bars), or MTSACE (green bars).

References [+] :

Artigas, Ouabain affinity determining residues lie close to the Na/K pump ion pathway. 2006, Pubmed, Xenbase