Figure 7. Substrate binding site.

(A) Structure of the Cl^– binding site. The molecular surface of the binding pocket is shown. Selected residues are displayed as sticks. Bound Cl^– is shown as a green sphere. (B) I-V relationships of selected Cl^– binding site mutants (Q88A, n = 5; F128A, n = 8; S392A, n = 5). Data for WT Slc26a9^T is shown as dashed line for comparison. (C) Conductance-concentration relationships of anion transport across the Slc26a9^T mutant F128A (n = 3). For comparison, SCN^– conductance–concentration relationship for non-mutated Slc26a9^T is shown as a pink dotted line. (D) Permeability (P_x/P_Cl) and (E) conductance ratios (G_x/G_Cl) of the Slc26a9^T mutant F128A obtained from bi-ionic substitution experiments (Cl^–, Br^–, NO₃^–, SCN^–, n = 8; I^–, n = 3). The light-blue bars indicate corresponding values for WT Slc26a9^T. (F) Conductance-concentration relationships of anion transport across the Slc26a9^T mutant S392A (n = 5). For comparison, SCN^– conductance–concentration relationship for non-mutated Slc26a9^T is shown as a pink dotted line. (G) Permeability (P_x/P_Cl) and (H) conductance ratios (G_x/G_Cl) of the Slc26a9^T mutant S392A obtained from bi-ionic substitution experiments (Cl^–, Br^–, n = 5; NO₃^–, SCN^–, I^–, n = 4). The light-blue bars indicate corresponding values for WT Slc26a9^T. Data was recorded from excised patches either in symmetric 150 mM Cl^– (B), the indicated intracellular anion-concentrations with 7.5 mM extracellular Cl^– (C and F), or at equimolar bi-ionic conditions containing 150 mM extracellular Cl^– and 150 mM of the indicated intracellular anion (D, E, G, H). Data show mean values of the indicated number of biological replicates, errors are s.e.m.. C-H, Data were recorded at −100 mV.

Figure 7—figure supplement 1. I-V-relationships of anion binding site mutants.

(A–G) Alanine mutants investigated by inside-out patch-clamp electrophysiology of HEK392T cells expressing Slc26a9^T anion binding-site mutations. Binding-site mutations do not alter Cl^– selectivity, but some mutants show rectifying behavior. Current traces (left) demonstrate that the shortening of side-chains does not severely alter the current magnitude. I-V relationships (right) in symmetric 150 mM chloride concentrations (blue) shows different degree of rectification. Currents recorded in a 5-fold inwardly-directed NaCl gradient (green) reverse at the Nernst potential for Cl^– (indicated by arrow). (A) Q88A, n = 5; (B) F92A, n = 3; (C) T127A, n = 5; (D) F128A, n = 8; (E) L391A, n = 4; (F) S392A, n = 5; (G) N441A, n = 3. For panels A-G, data is represented as averages of the indicated number of biological replicates, and errors are s.e.m.. WT is shown as red dashed line for comparison. (H) The Slc26a9^T-Q88E mutation displays strong outward rectification in the whole-cell configuration, along with severely attenuated current magnitude. Left, Whole-cell currents at symmetrical 150 mM NaCl. Middle, I–V plot for whole-cell Slc26a9^T-Q88E currents at symmetrical 150 mM NaCl (blue, n = 4) and with a 5-fold outward-directed NaCl gradient (green, n = 4). The triangle indicates the Nernst potential for Cl^–. Right, Strong reduction of Cl^– currents by Q88E, relative to WT Slc26a9^T. Shown are Cl^– influx currents at 100 mV for Slc26a9^T (black bar, n = 5), full-length Slc26a9 (FL) (gray bar, n = 8), and Slc26a9^T-Q88E (blue bar, n = 4). (I) The Slc26a9^T-Q88E mutation does not compromise surface expression or stability of Slc26a9^T. Left, Fluorescence microscopy image of Slc26a9^T-Q88E-transfected HEK293T cells shows that the Q88E mutation displays similar membrane-attributable fluorescence as for Slc26a9^T. Right, FSEC profiles of detergent extracts of Slc26a9^T and the Slc26a9^T-Q88E mutant show that the mutation has not apparently compromised protein stability, and in fact the mutation may result in more stable protein, as evidenced by the larger monodisperse peak relative to Slc26a9^T.

Figure 7—figure supplement 2. Anion selectivity of binding site mutants.

Currents were recorded by inside-out patch-clamp electrophysiology at asymmetric conditions containing 150 mM extracellular Cl^– and 150 mM of the indicated anion. (A–G) Top, Bi-ionic I–V relationships for equimolar replacement of intracellular Cl^– with permeable anions Br^– (orange), I^– (violet), NO₃^– (black), and SCN^– (red), and with symmetrical Cl^– shown as reference (blue). Mutants Q88A, T127A, F128A, L391A, and S392A show considerable changes in the I–V relationships of intracellularly substituted permeable anions, relative to WT-Slc26a9^T, while the mutants F92A and N441A reveal less severe alterations. Bottom, Bi-ionic I–V relationships for equimolar replacement of intracellular Cl^– with impermeable anions HCO₃^– (pink), SO₄^2– (chartreuse), and F^– (gray). All mutants remain poorly permeable to bicarbonate, sulfate, and fluoride. (A), Q88A, Cl^–, Br^–, SCN^–: n = 5, I^–, NO₃^–, HCO₃^–, SO₄^2–n = 4, F^–: n = 3; (B) F92A, Cl^–, Br^–, I^–, NO₃^–, SCN^–, HCO₃^–, SO₄^2–, F^–: n = 3; (C) T127A, Cl, NO₃^–, SCN^–, F^–: n = 5, Br^–, I^–: n = 4; HCO₃^–, SO₄^2–n = 3); (D) F128A, Cl^–, Br^–, SCN^–: n = 8, I^–, HCO₃^–, SO₄^2–, F^–: n = 3; (E) L391A, Cl^–, Br^–, I^–, NO₃^–, SCN^–, HCO₃^–, SO₄^2–, F^–: n = 4; (F) S392A, Cl^–, Br^–: n = 5, I^–, NO₃^–, SCN^–, HCO₃^–, SO₄^2–, F^–: n = 4; (G) N441A, Cl^–, Br^–, I^–, NO₃^–, SCN^–, HCO₃^–, SO₄^2–, F^–: n = 3. For panels A-G, data is represented as averages of the indicated number of biological replicates, and errors are s.e.m.. (H) Data for Slc26a9^T displayed in Figure 1—figure supplement 3B–F is shown for comparison.

Figure 7—figure supplement 3. Kinetic properties of anion binding site mutants.

Data was recorded from excised patches. (A–E) Relative permeabilities (P_x/P_Cl, green) and macroscopic conductivities (G_x/G_Cl, orange) for (A) Q88A, (B) F92A, (C) T127A, (D) L391A and (E) N441A. Number of replicates for each mutant/ion combination corresponds directly to the values listed for the bi-ionic data in Figure 7—figure supplement 2. The black horizontal line for each bar indicates the corresponding value in WT Slc26a9^T. (F–J) Mutant conductance–concentration relationships. Data was recorded from excised patches. Extracellular pipette NaCl concentration was kept constant at 7.5 mM, and patches were intracellularly perfused sequentially with 7.5–300 mM Cl^– and SCN^–, which allowed measurement of apparent K_m values as well as assessment of relative V_max for both anions. Macroscopic conductance corresponds to measured efflux currents at –100 mV, and data is normalized to efflux at 300 mM intracellular Cl^–. Cl^– and SCN^– are displayed in blue and red, respectively. SCN^– conductance data for WT-Slc26a9^T is shown (pink) for comparison. K_m, V_max(SCN^–)/V_max(Cl^–) ratios, and number of replicates were (F), Q88A: K_m(Cl^–)=2.3 mM, K_m(SCN^–)=0.2 mM, V_max(SCN^–)/V_max(Cl^–)=0.63, n = 3; (G), F92A: K_m(Cl^–)=19.2 mM, K_m(SCN^–)=0.8 mM, V_max(SCN^–)/V_max(Cl^–)=0.45, n = 3; (H), T127A: K_m(Cl^–)=4.7 mM, K_m(SCN^–)=1.6 mM, V_max(SCN^–)/V_max(Cl^–)=0.74, n = 3; (I), L391A: K_m(Cl^–)=44.0, K_m(SCN^–)=29.4, V_max(SCN^–)/V_max(Cl^–)=1.1, n = 4, and (J), N441A: K_m(Cl^–)=31.3, K_m(SCN^–)=1.8, V_max(SCN^–)/V_max(Cl^–)=0.68,n = 5. Relative to WT-Slc26a9^T, L391A showed a considerable deviation in the parameters, Q88A and T127A displayed moderate shifts, and F92A and N441A had the least effective changes. For panels A-J, data is represented as averages of the indicated number of biological replicates, and errors are s.e.m.