Loop diuretic and ion-binding residues revealed by scanning mutagenesis of transmembrane helix 3 (TM3) of Na-K-Cl cotransporter (NKCC1)

Abstract

The Na-K-Cl cotransporter (NKCC) plays central roles in cellular chloride homeostasis and in epithelial salt transport, but to date little is known about the mechanism by which the transporter moves ions across the membrane. We examined the functional role of transmembrane helix 3 (TM3) in NKCC1 using cysteine- and tryptophan-scanning mutagenesis and analyzed our results in the context of a structural homology model based on an alignment of NKCC1 with other amino acid polyamine organocation superfamily members, AdiC and ApcT. Mutations of residues along one face of TM3 (Tyr-383, Met-382, Ala-379, Asn-376, Ala-375, Phe-372, Gly-369, and Ile-368) had large effects on translocation rate, apparent ion affinities, and loop diuretic affinity, consistent with a proposed role of TM3 in the translocation pathway. The prediction that Met-382 is part of an extracellular gate that closes to form an occluded state is strongly supported by conformational sensitivity of this residue to 2-(trimethylammonium)ethyl methanethiosulfonate, and the bumetanide insensitivity of M382W is consistent with tryptophan blocking entry of bumetanide into the cavity. Substitution effects on residues at the intracellular end of TM3 suggest that this region is also involved in ion coordination and may be part of the translocation pathway in an inward-open conformation. Mutations of predicted pore residues had large effects on binding of bumetanide and furosemide, consistent with the hypothesis that loop diuretic drugs bind within the translocation cavity. The results presented here strongly support predictions of homology models of NKCC1 and demonstrate important roles for TM3 residues in ion translocation and loop diuretic inhibition.

FIGURE 1.

Models of NKCC1 based on homology to APC transporters AdiC and APCT. A, extracellular face of the transporter showing the opening of the translocation pocket. Phe-372 is visible and marked within the pocket, and ICL1 (yellow) is visible at the intracellular end. TMs 1, 2, 3, 5, 6, 8, and 10 are labeled and are space-filling (except Phe-372 is in ball and stick), other helices are in schematic representation. B, extracellular face of occluded form of NKCC1. C, intracellular view, same model as in A. D, as in C, ICL1 residues removed. E, TM3 pore-lining residues highlighted in white; ball and stick TM3, and ICL1 as cartoon ribbon; TMs 1, 2, 6, 8, and 10 as spacefilling. F, lipid face of NKCC1. TM3 residues that misfold as tryptophan are in lime green, and residues that are fully functional as tryptophan are in white. G, cutaway view into TM3, pore-lining residues colored by element and labeled. Parts of TMs 1, 2, 5, 6, and 7 are removed.

FIGURE 2.

Western blots of cysteine and tryptophan substitution mutants. Triton X-100-soluble extracts of transfected cells were loaded (5 μg of protein), and immunoblots were probed with T4 antibody. The upper 190-kDa band is the mature fully glycosylated protein, and the lower band is a 140-kDa band immature protein.

FIGURE 3.

Maximum ⁸⁶Rb⁺ influx in cysteine and tryptophan scanning mutants. A, nontransfected HEK cells, wild-type hNKCC1, and cysteine-substituted mutants. B, tryptophan-substituted mutants. ⁸⁶Rb⁺ influx in stably transfected HEK293 cell lines was determined following low hypotonic, low chloride preincubation and addition of calyculin A to achieve maximal activation of the cotransporter. Dotted lines indicate the flux in nontransfected cells and in wild-type hNKCC1. Dark gray bars highlight residues different from the wild type by >50%, and open bars designate tryptophan substitutions that were poorly processed biosynthetically (Fig. 2). The shaded bar on the ordinate indicates α-helical periodicity of 100°/residue and is approximately aligned so that predicted TM3 pore residues are next to black. Data shown are mean ± S.E. (error bars; n = 3), and significant difference from wild-type hNKCC1 is indicated (*) when p < 0.05.

FIGURE 4.

K_m values for Na⁺, K⁺, and Cl⁻. K_m for Cl⁻, Rb⁺, Na⁺ in cysteine- and tryptophan-substitution mutants are expressed relative to the wild-type hNKCC1 values: K_m(Na) = 18.1 ± 1.6, K_m(Rb) = 1.6 ± 0.16, K_m(Cl) = 47. 9 ± 1.6. A, cysteine substitution mutants. B, tryptophan substitution mutants. Mutants indicated in light gray are nonfunctional and were not analyzed. The shaded bar indicates α-helical periodicity as in Fig. 3. Data shown are a mean ± S.E. (error bars; n = 3–5 experiments) with significant difference from wild-type hNKCC1 indicated (*) when p < 0.05.

FIGURE 5.

Maximal flux and K_m for ions of other pore lining mutants. A, maximal ⁸⁶Rb⁺ influx as in Fig. 3. B, K_m for ions as in Fig. 4. Data shown are mean ± S.E. (error bars; n = 3) with significant difference from wild-type hNKCC1 indicated (*) when p < 0.05.

FIGURE 6.

K_i for bumetanide and furosemide. Inhibition constants for bumetanide and furosemide were determined as described under “Experimental Procedures” and plotted relative to wild-type hNKCC1 values: K_i(furo) = 4.2 ± 0.21; K_i(bumet) = 0.42 ± 0.02 with bumetanide only in the preincubation medium, and K_i(bumet) = 0.38 ± 0.015 with bumetanide in both preincubation and flux medium. Nonfunctional mutants (light gray) were not analyzed. A, cysteine-substitution mutants. B, tryptophan-substitution mutants and two Met-382 mutants. The shaded bar indicates α-helical periodicity as in Fig. 3. Data are mean ± S.E. (error bars; n = 3) with significant difference from wild-type hNKCC1 indicated (*) when p < 0.05.

FIGURE 7.

Inhibition by MTS reagents. A, fractional inhibition of cysteine scanning mutants by 3 mm MTSET or 5 mm MTSES as described under “Experimental Procedures.” Nonfunctional mutants (light gray) were not analyzed. Data are corrected for base-line inhibition in wild-type hNKCC1 (−5.8 ± 2.1% with MTSET, −9.5 ± 5.2% with MTSES) and are presented as mean ± S.E. (error bars; n = 3) with significant difference from wild-type hNKCC1 indicated (*) when p < 0.05. The shaded bar indicates α-helical periodicity as in Fig. 3. B, concentration dependence of MTSET inhibition of M382C and V385C in a 10-min exposure in regular medium (●) or in the bumetanide-occluded condition (○). This experiment is one of three, error bars show the duplicate variation within the experiment. Data are fit with V = V_max*e − ^(k*^M*^t10), as described under “Experimental Procedures.” C, rate constant (k) for modification of M382C and V385C by MTSET in regular medium (open bars) or with bumetanide (shaded bars). The results are means ± S.E. from three experiments including the one in B.