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J Biol Chem. 2014 Mar 14;289(11):7569-79. doi: 10.1074/jbc.M113.542258. Epub 2014 Jan 22.

Molecular motions involved in Na-K-Cl cotransporter-mediated ion transport and transporter activation revealed by internal cross-linking between transmembrane domains 10 and 11/12.

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From the Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, Connecticut 06520.


We examined the relationship between transmembrane domain (TM) 10 and TM11/12 in NKCC1, testing homology models based on the structure of AdiC in the same transporter superfamily. We hypothesized that introduced cysteine pairs would be close enough for disulfide formation and would alter transport function: indeed, evidence for cross-link formation with low micromolar concentrations of copper phenanthroline or iodine was found in 3 of 8 initially tested pairs and in 1 of 26 additionally tested pairs. Inhibition of transport was observed with copper phenanthroline and iodine treatment of P676C/A734C and I677C/A734C, consistent with the proximity of these residues and with movement of TM10 during the occlusion step of ion transport. We also found Cu(2+) inhibition of the single-cysteine mutant A675C, suggesting that this residue and Met(382) of TM3 are involved in a Cu(2+)-binding site. Surprisingly, cross-linking of P676C/I730C was found to prevent rapid deactivation of the transporter while not affecting the dephosphorylation rate, thus uncoupling the phosphorylation and activation steps. Consistent with this, (a) cross-linking of P676C/I730C was dependent on activation state, and (b) mutants lacking the phosphoregulatory domain could still be activated by cross-linking. These results suggest a model of NKCC activation that involves movement of TM12 relative to TM10, which is likely tied to movement of the large C terminus, a process somehow triggered by phosphorylation of the regulatory domain in the N terminus.


Chloride Transport; Copper; Iodine; Loop Diuretics; Membrane Proteins; Membrane Transport; Na-K-Cl Cotransporter; Phenanthroline; Protein Cross-linking; Protein Structure

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