A single residue in transmembrane domain 11 defines the different affinity for thiazides between the mammalian and flounder NaCl transporters

Abstract

Little is known about the residues that control the binding and affinity of thiazide-type diuretics for their protein target, the renal Na(+)-Cl(-) cotransporter (NCC). Previous studies from our group have shown that affinity for thiazides is higher in rat (rNCC) than in flounder (flNCC) and that the transmembrane region (TM) 8-12 contains the residues that produce this difference. Here, an alignment analysis of TM 8-12 revealed that there are only six nonconservative variations between flNCC and mammalian NCC. Two are located in TM9, three in TM11, and one in TM12. We used site-directed mutagenesis to generate rNCC containing flNCC residues, and thiazide affinity was assessed using Xenopus laevis oocytes. Wild-type or mutant NCC activity was measured using (22)Na(+) uptake in the presence of increasing concentrations of metolazone. Mutations in TM11 conferred rNCC an flNCC-like affinity, which was caused mostly by the substitution of a single residue, S575C. Supporting this observation, the substitution C576S conferred to flNCC an rNCC-like affinity. Interestingly, the S575C mutation also rendered rNCC more active. Substitution of S575 in rNCC for other residues, such as alanine, aspartate, and lysine, did not alter metolazone affinity, suggesting that reduced affinity in flNCC is due specifically to the presence of a cysteine. We conclude that the difference in metolazone affinity between rat and flounder NCC is caused mainly by a single residue and that this position in the protein is important for determining its functional properties.

Fig. 1.

General topology and alignment analysis of transmembrane regions (TM) 8–12 in mammalian and flounder Na⁺-Cl⁻ cotransporters (NCC). The proposed topology for NCC includes a central hydrophobic domain made up of 12 putative TM segments divided into 2 fragments by a large extracellular glycosylated loop between segments 7 and 8. The central hydrophobic domain is flanked by intracellular short and long amino and carboxyl-terminal domains. Alignment analysis of TM segments 8–12 is shown. Amino acid residues in boxes are those less conserved between flounder and all mammalian sequences and were thus the residues chosen for study.

Fig. 2.

Mutant constructs for TM9, TM11, and TM12 are functional. Percentage of rat NCC activity in wild-type and mutant NCC. The thiazide-sensitive ²²Na⁺ uptake in wild-type NCC was set as 100%, and the values observed in mutant clones were normalized accordingly. At least 5 different 10-oocyte experiments were done. Normalization of data was done for each experiment before the groups were compared. The data shown represent the mean ± SE of 5 different experiments. *P < 0.05 vs. wild-type NCC.

Fig. 3.

The effect of TM mutations on rat NCC (rNCC) affinity for metolazone. Metolazone dose response analysis was assessed in 5 different experiments, in all of which the 5 different clones were tested simultaneously, using the same uptake solutions and metolazone dilutions. A: a compilation of the dose-response curves from the 5 experiments is shown. Wild-type rNCC (blue), wild-type flounder NCC (flNCC; red), TM9 mutant (orange), TM12 mutant (pink), and TM11 mutant (green). Data were fit to the Hill equation, and in all cases Hill coefficients close to unity were obtained. All group data are shown as means ± SE of %activity. The coefficient of determination (r²) was between 0.95 and 0.99 for the fits shown. B: metolazone IC₅₀ for rNCC and mutants calculated from the nonlinear regression fit of uptake data to the Hill equation. Data are shown as mean IC₅₀ ± SE of 5 different experiments. *P < 0.05 vs. rNCC.

Fig. 4.

The effect of individual mutations in the rNCC TM11 segment on the affinity for metolazone. Methods are similar to those explained in Fig. 3, and 5 different experiments were performed. The bars represent mean IC₅₀ ± SE for each tested clone, as stated. For each experiment, all Hill slopes were ∼1 and coefficients of determination (r²) were between 0.92 and 0.99. *P < 0.05 vs. rNCC.

Fig. 5.

The effect of TM mutations in flNCC on the affinity for metolazone. Metolazone dose-response analysis was assessed as explained in Fig. 3. Similar observations were done in 3 different experiments. A: a representative experiment is shown. Dose-response curves are shown for wild-type flNCC (■) and C576S-flNCC (▲). The coefficient of determination (r²) for wild-type flNCC and C576S-flNCC was 0.96 and 0.98, respectively. B: metolazone IC₅₀ for flNCC and C576S mutant was calculated from the nonlinear regression fit of uptake data to the Hill equation. Data are shown as mean IC₅₀ ± SE of 3 different experiments. *P < 0.05 vs. flNCC.

Fig. 6.

TM11 mutant rNCC and the single mutant S575C-rNCC exhibited higher activity than wild-type NCC or I574C-rNCC. Oocytes were injected with the same amount of cRNA of each clone, as explained in the text. A: 3 days later the ²²Na⁺ uptake was assessed in the absence (open bars) or presence (filled bars) of metolazone. Data presented are means ± SE of the uptake observed for each oocyte. Similar observations were done in at least 5 experiments. *P < 0.05 vs. control wild-type rNCC. B: proteins were extracted for a Western blot analysis using anti-FLAG or anti-actin antibody. A representative Western blot is shown in B. C: densitometric analysis of bands observed in the Western blot. OD, optical density.

Fig. 7.

Functional expression and metolazone affinity of mutant rNCCs S575C, S575A, S575D, and S575K. Oocytes were injected with cRNA from each mutant clone. A: 3 days later, the ²²Na⁺ uptake was assessed in the absence (open bars) or presence (closed bars) of 100 μM metolazone. Data are means ± SE. B: metolazone IC₅₀ values for rNCC and mutants were calculated from the nonlinear regression fit of uptake data to the Hill equation. Data are shown as mean IC₅₀ ± SE of 3 different experiments. *P < 0.05 vs. rNCC.

Fig. 8.

Electrophoretic behavior of rNCC and S575C-rNCC in the absence or presence of a reducing agent. Protein extracts from oocytes injected with water, rNCC cRNA, or rNCC-S575C cRNA were diluted for standard SDS-PAGE in Laemmli buffer without reducing agent (A) or containing either 0.5 (B), 2.5 (C), or 5% β-mercaptoethanol (D). In the absence of reducing agent (A), bands of expected size for monomeric and dimeric forms of the cotransporter were observed, and in both cases a diffused signal of slightly higher molecular weight was detected, which probably corresponds to glycosylated forms of the protein. This was seen for the wild-type and mutant form of rNCC. In the presence of reducing agent, the bands corresponding to the dimeric form of the cotransporter disappeared even at the lowest concentration of reducing agent used. At this concentration, we were able to observe a slight signal corresponding to the dimers, but no difference in intensity was observed between the wild type and the mutant.