Loop Diuretic and Ion-binding Residues Revealed by Scanning Mutagenesis of Transmembrane Helix 3 (TM3) of Na-K-Cl Cotransporter (NKCC1)

Abstract

Background: Na-K-Cl cotransporters (NKCCs) are essential in chloride homeostasis and salt transport. Results: Mutations in NKCC1 transmembrane domain 3 (TM3) alter transport activity, ion binding, and inhibitor affinities. Conclusion: This demonstrates a role for TM3 in the NKCC1 transport pathway. Significance: This is the beginning of a systematic analysis of the Na-K-Cl cotransporter function in the context of structural models.

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

“…The key feature of the TM domain is an inverted repeat of TMs 1-5 and 6 -10 that comprise a translocation domain surrounding a central ligand-binding cavity (3)(4)(5)(6), a motif that is the structural core in five superfamilies of transporters (7). Our recent scanning analysis of highly conserved TM3 provides very strong support for the proposed alignment of TM3 as a porelining helix in structural homology between NKCC and amino acid transporters (8); however, there is lower confidence in the proposed alignments of other regions of the proteins. We have begun to further test the homology models utilizing a cysteine cross-linking approach, beginning here with an examination of the predicted relationship of TMs 10 and TM11/12.…”

“…It can be argued that the data do not uniquely pinpoint the occlusionrelated conformational change in NKCC1 as the inhibited step because other conformational transitions may also involve TM10 movement. However, it may be noted that models of an inward open transporter based on the GadC crystal structure (23) do not display further movement of TM10 relative to TM12, and an alternative suggestion for inward opening of NKCC1 involving movement of intracellular loop 1 out of the pore would also not involve TM10 (8). Thus it is reasonable to propose that the movement of TM10 relative to TM12 that is inhibited by CuPhe cross-linking is the movement involved in formation of the occluded state from the outward open state.…”

“…8, using multiple sequence alignments of truncated NKCC1 with AdiC (4 -6, 11) and the program Modeler (18). The degree of AdiC-NKCC homology is very low in the TM11-12 region, and the predictions here utilized a TM11-TM12 sequence alignment supported by homodimeric cross-linking studies (see Fig.…”

“…would be especially intriguing because based on structural models as well as on conformation-sensitivity of MTSET block and reduced bumetanide affinity of M382W, Met 382 appears to be part of the extracellular gate determining access to the translocation pathway (8).…”

Diuretic Treatment in Heart Failure

“…Furosemide, bumetanide, and torsemide are prototypic loop diuretics; they bind to the translocation pocket at the extracellular surface of sodium-potassium-chloride symporters (NKCCs), blocking ion transport directly 3 (Fig. 1).…”