Molecular Motions Involved in Na-K-Cl Cotransporter-mediated Ion Transport and Transporter Activation Revealed by Internal Crosslinking between Transmembrane Domains 10 and 11/12 [Molecular Biophysics]
January 22nd, 2014 by Monette, M. Y., Somasekharan, S., Forbush, B.
We examined the relationship between TM10 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 crosslink 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++ inhibition of the single-cysteine mutant A675C, suggesting that this residue and M382 of TM3 are involved in a Cu++ binding site. Surprisingly, crosslinking 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) crosslinking of P676C-I730C was dependent on activation state, and (b) mutants lacking the phosphoregulatory domain could still be activated by crosslinking. 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.