Figure 6: SCN⁻ currents in prestin suggest an ancillary pathway independent of the transporter pathway.

(A) Mutation of residues critical for Cl⁻ binding, the first step in transport, has no effects on SCN⁻ currents. Average currents of CHO cells expressing prestin (n = 5), F137A (n = 4) and S398A (n = 7) show no differences (+/−SEM). Currents, after correction for background currents, were divided by linear capacitance to correct for differences in cell size and plotted against voltage (F137A did not have easily measurable NLC, so the more rigorous normalization to NLC was not possible). Cells show large inward currents in the presence of intracellular SCN⁻ (100 mM) at depolarizing voltages. Intracellular SCN⁻ was used since the residue corresponding to S398 lies closer to the inner cleft of the transporter pathway and binds to intracellular uracil in UraA; the residue corresponding to F137 acts as a barrier to the diffusion of uracil to the exterior surface. (B) Neutralization of charged residues lying outside the transporter pathway reduce SCN⁻ currents. Average currents of CHO cells expressing K227Q (n = 7), K359Q (n = 7), R463Q (n = 9), and D485N (n = 18) show significantly reduced currents in the presence of extracellular SCN⁻ (p < 0.001). To account for variations in expression, current averages for each mutant construct, after correction of average background currents, were normalized by the NLC charge Q_max, and plotted against voltage. (C) NLC traces from residues (K227Q, K359Q R463Q and D485N) where mutation affected SCN⁻ currents but had measurable NLC, and of mutations in the transporter pathway (F137A and S398A). (D–F) The location of residues that line a potential accessory pathway are shown on the structure of prestin modeled on the crystal structure of the bacterial uracil transporter UraA rendered with Chimera^11,12. The core domains are colored blue and the gate domains colored in pink. (D–F) are the molecule viewed from above, the side and below respectively. In the potential accessory pathway lined by alpha helices 5, 8 and 14, the contained amino acids K227, K359 and D485, and R463, that lies in the loop connecting helices 12 and 13, are colored purple. Residues modeled to bind intracellular chloride (F137 and S398) are colored orange.