K+-Driven Cl-/HCO3- Exchange Mediated by Slc4a8 and Slc4a10

Abstract

Slc4a genes encode various types of transporters, including Na⁺-HCO₃^- cotransporters, Cl^-/HCO₃^- exchangers, or Na⁺-driven Cl^-/HCO₃^- exchangers. Previous research has revealed that Slc4a9 (Ae4) functions as a Cl^-/HCO₃^- exchanger, which can be driven by either Na⁺ or K⁺, prompting investigation into whether other Slc4a members facilitate cation-dependent anion transport. In the present study, we show that either Na⁺ or K⁺ drive Cl^-/HCO₃^- exchanger activity in cells overexpressing Slc4a8 or Slc4a10. Further characterization of cation-driven Cl^-/HCO₃^- exchange demonstrated that Slc4a8 and Slc4a10 also mediate Cl^- and HCO₃^--dependent K⁺ transport. Full-atom molecular dynamics simulation on the recently solved structure of Slc4a8 supports the coordination of K⁺ at the Na⁺ binding site in S1. Sequence analysis shows that the critical residues coordinating monovalent cations are conserved among mouse Slc4a8 and Slc4a10 proteins. Together, our results suggest that Slc4a8 and Slc4a10 might transport K⁺ in the same direction as HCO₃^- ions in a similar fashion to that described for Na⁺ transport in the rat Slc4a8 structure.

Keywords: K+-driven anion exchanger; chloride/bicarbonate exchangers; ion transport; sodium/bicarbonate cotransporters.

Figure 1

Slc4a8 and Slc4a10 localization in CHO-K1 cells. Immunofluorescence studies in CHO-K1 cells transfected with plasmids encoding Myc-DDK tagged versions of Slc4a8 (upper panels) and Slc4a10 (middle panels). Transfected cells with the empty plasmid were used as negative control (lower panels). Plasma membrane was labeled with WGA conjugated to Alexa Fluor 633^TM (shown in red, left panels), followed by protein detection using an anti-DYKDDDDK antibody (shown in green, middle panels). Pictures in the right panels were generated by merging WGA, DYKDDDDK and DAPI channels. Scale bar = 10 μm.

Figure 2

Slc4a8 and Slc4a10 mediate Cl⁻/HCO₃⁻ exchanger activity. Cl⁻ fluxes in Slc4a8- and Slc4a10-expressing cells loaded with SPQ. Electroporated CHO-K1 cells were perfused with HCO₃⁻-containing/high Cl⁻ solution (Solution A, Table 1) and then switched to a HCO₃⁻-containing/low Cl⁻ solution (Solution B, Table 1). Fluxes obtained from cells electroporated with Slc4a8- ((A), n = 9) or Slc4a10 ((B), n = 9) encoding plasmids are shown in black circles. Fluxes obtained from non-electroporated cells are shown as red dashed lines and are the same in A and B (n = 11). Experiments under HCO₃⁻-free conditions were also performed on Slc4a8 ((A), open circles; n = 5) and Slc4a10 ((B), open circles; n = 5) electroporated cells, which were initially perfused with HCO₃⁻-free/high Cl⁻ solution (Solution E) and then switched to HCO₃⁻-free/low Cl⁻ solution (Solution F). Fluxes in each condition are shown as the mean ± standard error of mean (SEM).

Figure 3

Slc4a8 and Slc4a10 display Na⁺- and K⁺- dependent Cl⁻/HCO₃⁻ exchanger activity. (A,C) Time course showing that Na⁺ and K⁺ elicited Cl⁻/HCO₃⁻ exchange in Slc4a8-(A) and Slc4a10-expressing cells (C) loaded with BCECF. Cells were perfused with NMDG-containing high Cl⁻ solution (Solution (C), Table 1) followed by NMDG-containing low Cl⁻ solution (Solution (D), Table 1) and finally switched to Na⁺ (Solution B, black circles; n = 10 for Slc4a8 and Slc4a10) or K⁺-containing solution (Solution G, open circles; n = 9 for Slc4a8 and n = 10 for Slc4a10). Fluxes obtained from non-electroporated cells are shown as red dashed lines and are the same in A and C (n = 16). (B,D) Slc4a8 (B) and Slc4a10 (D) activities were measured as the alkalinization rate in response to a reduction in external Cl⁻ concentration using Na⁺- and K⁺-containing solutions (change from solution A to solutions 5, 25, 50, 100, 125 and 150 mM [see Table 2 for ion composition of the Na⁺- and K⁺-containing solutions]). The normalized activities at different Na⁺ (black circles) and K⁺ concentrations (open circles) were plotted against the cation concentration and then fitted to a Hill function (shown as black lines; see Methods for the equation used). Values correspond to the average ± SEM.

Figure 4

Slc4a8− and Slc4a10−dependent K⁺- fluxes. (A) Experimental design used to assess K⁺ transport by Slc4a8 and Slc4a10. (B,C) Slc4a8- ((B), black circles, n = 7) and Slc4a10-expressing cells (C, black circles, n = 9) loaded with PBFI-AM were depleted of Cl⁻ by incubating the cells with low Cl⁻ solution and high K⁺ (solution G, Table 1) and then Cl⁻ uptake (and K⁺ efflux) was induced by switching to an external solution containing high Cl⁻ and low K⁺ (solution C, Table 1). Red dashed lines in (B,C) show the activity displayed by non-transfected cells (n = 16). The experiments shown in (B,C) were performed under Na⁺-free conditions to prevent Na⁺ contamination of the PBFI signal. Results are presented as the mean ± SEM.

Figure 5

K⁺ occupancy at the S1 site in the cryo-EM rSlc4a8 structure. Distances between K⁺ and the residues from S1 Na⁺-coordination site were computed within a stable range along three 100 ns MD simulations (referred to as r1, r2, and r3). Residues for distance calculation include D800 and its carbonyl side chain atoms OD1 and OD2 (A,B), and the respective hydroxyl OG1 side chain atoms from T804 (C), and T847 (D).

Figure 6

Residues at the S1 site involved in K⁺ coordination. Ribbon representation of (A) MD equilibrated structure of rat Slc4a8 where K⁺ replaces Na⁺ at S1 site from 100 ns MD running and (B) K⁺ interactions at S1 site with the residues originally described coordinating Na⁺ in [17]. The residue side chains are shown as sticks, CO₃²⁻ is shown as ball and sticks and, K⁺ is shown as a blue sphere. The transmembrane helices, TM8 and TM10, contributing to ion coordination are labeled. (C). The average distances between K⁺ and residues D800, T804, T847 and the CO₃²⁻ ion computed from MD simulations are indicated (referred as r1, r2, and r3). The K⁺-residue distances are measured to side chain atoms, in particular, the oxygen atoms (OD1 and OD2) from the carbonyl group of D800 and the oxygen (OG1) from hydroxyls of T804 and T847. The distance between K⁺ and CO₃²⁻ was computed between their center of mass. (D) Na⁺ contacting distance with residues D800, T804, T847, and the CO₃²⁻ ion was taken from Wang et al. [17].

Figure 7

Interaction between K⁺ and CO₃²⁻ at the S1 site. Distance between (A) K⁺ and center of mass of CO₃²⁻, (B) K⁺ to S1 site, and (C) CO₃²⁻ to S1 site along three 100 ns MD simulations (referred to as r1, r2, and r3). The S1-ion distance is defined as the distance between each ion and the Cα atom of residue A846, similar to the definition used by Wang et al. [17]. The residue A486 belongs to the S1 site.