Prestin's anion transport and voltage-sensing capabilities are independent

Abstract

The integral membrane protein prestin, a member of the SLC26 anion transporter family, is responsible for the voltage-driven electromotility of mammalian outer hair cells. It was argued that the evolution of prestin's motor function required a loss of the protein's transport capabilities. Instead, it was proposed that prestin manages only an abortive hemicycle that results in the trapped anion acting as a voltage sensor, to generate the motor's signature gating charge movement or nonlinear capacitance. We demonstrate, using classical radioactive anion ([(14)C]formate and [(14)C]oxalate) uptake studies, that in contrast to previous observations, prestin is able to transport anions. The prestin-dependent uptake of both these anions was twofold that of cells transfected with vector alone, and comparable to SLC26a6, prestin's closest phylogenetic relative. Furthermore, we identify a potential chloride-binding site in which the mutations of two residues (P328A and L326A) preserve nonlinear capacitance, yet negate anion transport. Finally, we distinguish 12 charged residues out of 22, residing within prestin's transmembrane regions, that contribute to unitary charge movement, i.e., voltage sensing. These data redefine our mechanistic concept of prestin.

Figure 1

Prestin-transfected CHO cells indicate anion transport. (A) CHO cells transfected with prestin show a time-dependent uptake of [¹⁴C]formate that was greater than that of CHO cells transfected with empty vector alone (control). Uptake of [¹⁴C]formate by prestin-transfected cells at 2, 4, and 8 min was 60 (±8.2), 135 (±10.4), and 160 (±9.5), respectively. In contrast, the uptake of control cells at corresponding time points was 44 (±8.2), 84 (±2.2) and 100 (n = 3). Data were normalized to vector-only controls at 8 min, which was given a value of 100. (B) There is a dose-dependent increase in [¹⁴C]formate uptake in cells transfected with increasing quantities of prestin-YFP plasmid. The mean uptake for cells transfected with 0.12 μg, 0.6 μg, and 1.2 μg of prestin plasmid DNA were 107 (±4), 131 (±8.5), and 166 (±31), respectively. Control wells were transfected with 1.2 μg of YFP plasmid DNA (n = 3). (Inset) Linear relationship between transport induced by prestin (after subtracting background) and amount of DNA used in transfection. (C and D) CHO cells transfected with prestin and Slc26a6 show increased uptake of [¹⁴C]formate (C) and [¹⁴C]oxalate (D) compared with control (YFP vector only). The plot shows mean uptake per 200,000 cells contained in a well (of a 24-well plate) ± SE. Data were normalized to vector-only controls (n = 3, in each case). Controls were assigned a value of 100. The uptake of [¹⁴C]formate by prestin and Slc26a6 was 183 (±0.7) and 171 (±17.3), respectively, i.e., significantly different from controls (p < 0.01, one-way ANOVA). The uptake of [¹⁴C]oxalate by prestin and Slc26a6 was 198 (±13.5) and 187 (±24.0), respectively, i.e., significantly different from vector-only controls (p < 0.05, one-way ANOVA). The absolute prestin-induced uptake of oxalate was 1.5-fold greater than formate uptake; this was obscured by normalization. Uptake is denoted in relative counts (see Methods).

Figure 2

Prestin-induced uptake of [¹⁴C]formate can be blocked. (A) [¹⁴C]formate uptake in cells transfected with prestin and vector only, and similarly transfected cells treated with 10 mM salicylate and 1 mM DIDS in incubation/preincubation medium. Prestin-induced uptake of [¹⁴C]formate was reduced by both agents. Mean values for the six groups were: prestin, 183 (±37); control, 100, prestin with salicylate, 87 (±16); control with salicylate, 61 (±13.0); prestin with DIDS, 53.9 (± 13.0); and control with DIDS, 44 (±8.5) (n = 3). (B) Effects of various test anions on prestin-induced uptake of [¹⁴C]formate. Prestin-transfected cells were incubated with Na gluconate and Na gluconate to which various indicated test anions were then added. Both chloride and malate decreased prestin uptake, whereas SO₄²⁻ had minimal effects on prestin-induced [¹⁴C]formate uptake. Mean values for the eight groups were: prestin, 237 (±11.8); control, 100; prestin + chloride, 130 (±8.6); control + chloride, 61 (±6.7); prestin + malate, 142 (±15); control + malate, 70 (±5.7); prestin + SO₄²⁻, 209 (±35); and control + SO₄²⁻, 75 (±6.5). Uptake of [¹⁴C]formate by prestin was significantly reduced by chloride and malate (p < 0.05, one-way ANOVA), but not by SO₄²⁻ (p > 0.05) (n = 3). Data were normalized to vector-only controls. Uptake is denoted in relative counts (see Methods).

Figure 3

Effects of truncations and mutations in prestin on prestin-induced uptake of [¹⁴C]formate. (A) Single mutations in potential chloride-binding motif decrease anion transport. All mutants except P327A show decreased anion transport. Mean uptake of [¹⁴C]formate of prestin, G324W, L325A, L326A, P327A, P328A, and vector-only control cells were 269 (±9), 113 (±3), 100 (±9), 117 (±20), 241 (±40), 93 (±3.5), and 100, respectively. The uptake of [¹⁴C]formate by prestin and P327A-transfected cells was significantly greater than in controls (p < 0.05, one-way ANOVA) (n = 4). (B) Effects of these mutations on NLC. Two mutants P328A and L326A show preserved NLC but decreased anion transport (indicated by asterisk in A). P327A has preserved NLC and anion transport, whereas remaining mutants eliminated (G324W) or significantly decreased (L325A) NLC, while also decreasing anion transport. (C) Confocal microscopy of YFP fusions of prestin and individual mutants (G324W, L325A, L326A, P327A, and P328A) shows membrane targeting. Arrows indicate filopodia containing prestin-YFP fluorescence. (D) Truncation of C-terminus at amino acid 709 (stop 709) that eliminates NLC shows preserved anion transport. Mean [¹⁴C]formate uptake values for prestin, stop 709, and vector-only control were 188 (±18), 197 (±28), and 100, respectively. The [¹⁴C]formate uptakes in prestin and stop 709-transfected cells were significantly greater than in controls (p < 0.05) (n = 3). Uptake is denoted in relative counts (see Methods).

Figure 4

Effects of single amino-acid substitutions of charged residues that are within or in close proximity to predicted membrane-spanning regions. (A) Effects on unitary charge (z) are shown and plotted against amino-acid position. Data include only those mutants that showed nonlinear capacitance. Residues are grouped according to how significantly different their z values were, compared with wild-type prestin (red). Those residues are in blue where p < 0.05 (R130Q, R211Q, K255Q, K359Q, D269N, and D457N), and in green where p < 0.005 (E207Q, K276Q, E280Q, K364Q, K449Q, and R463Q). (B) Average membrane resistance of prestin and prestin mutants in A. These data confirm that changes in z were independent of membrane resistance. (C) Normalized examples of NLC of prestin and four mutants that showed a reduction in z. Data points are fitted with Eq. 1. (D) Normalized average fits of individual mutants are aligned to wild-type prestin's V_h, for a better demonstration of decrease in voltage sensitivity evidenced by a broadening of the functions.

Figure 5

Residue locations. Placement of charge residue mutations in 12 (A) and 10 (B) transmembrane models of the protein. The residues and their traces in Fig. S1 are correspondingly color-coded. Also indicated are specific amino-acid numbers, which in turn are color-coded as in Fig. 4A, to indicate residues and their effects on z.

Figure 6

Relationship between voltage of peak capacitance (V_h) and unitary charge (z). There is no relationship between z values and V_h, although most of the mutants caused a negative shift in V_h.

Figure 7

Relationship between unitary charge z and specific capacitance. There is a positive trend between z and Q_sp (Q_max/linear capacitance).