Functional Analysis of Transmembrane Domain 3 in NKCC1

We sought to determine the functional roles of residues in transmembrane domain (TM) 3 of human NKCC1 using tryptophan and cysteine scanning mutagenesis. We generated a structural alignment of the transmembrane domains of NKCC1 to the related APC transporters ApcT and AdiC, and obtained 3-D alignments using Modeller. Based on these alignments, residues 368 to 380 were predicted to form part of the inner 2/3 of the translocation pore. We substituted these residues with tryptophan and cysteine and determined the impact of the changes on protein synthesis and cell surface delivery by Western blotting and immunofluorescence microscopy. Most of our mutants expressed and localized similar to wild type NKCC1 and these were analyzed in depth for transporter function by means of Rb86 influx assays. A working hypothesis is that tryptophan mutants that are much reduced in function are too hydrophobic for the solvent interface and those residues that retain function are either protein interior or lipid facing. The pattern of the tryptophan scan followed an alpha helical periodicity. Based on the tryptophan scan we deduced that the non-functional mutants I368W, G369W, F372W, A375W, N376W, A379W are pore lining residues. Cysteine scanning complemented the results of the tryptophan scan. Since cysteine is a mild mutation, most mutants were functional. However I368C, G369C, A379C showed dramatic reduction or loss of function which suggested that either the cysteine was too large or disrupted substrate binding. Also F372C and N376C residues showed reduced chloride and rubidium affinity. The combined results of the two scanning approaches are consistent with our predictions of TM3 being an alpha helical domain with I368, G369, F372, A375, N376, A379 being pore lining residues.