The extracellular loop of pendrin and prestin modulates their voltage-sensing property

Abstract

Pendrin and prestin belong to the solute carrier 26 (SLC26) family of anion transporters. Prestin is unique among the SLC26 family members in that it displays voltage-driven motor activity (electromotility) and concurrent gating currents that manifest as nonlinear cell membrane electrical capacitance (nonlinear capacitance (NLC)). Although the anion transport mechanism of the SLC26 proteins has begun to be elucidated, the molecular mechanism of electromotility, which is thought to have evolved from an ancestral ion transport mechanism, still remains largely elusive. Here, we demonstrate that pendrin also exhibits large NLC and that charged residues present in one of the extracellular loops of pendrin and prestin play significant roles in setting the voltage-operating points of NLC. Our results suggest that the molecular mechanism responsible for sensing voltage is not unique to prestin among the members of the SLC26 family and that this voltage-sensing mechanism works independently of the anion transport mechanism.

Keywords: SLC26; anion transport; cell motility; electromotility; electrophysiology; membrane protein; molecular motor.

Figure 1.

Structural models of pendrin and prestin. A–C, extracellular views of pendrin and prestin models generated based on the solved structure of SLC26Dg. The N- and C-terminal cytosolic regions are not shown. The core and the gate domains are shown in cyan and orange, respectively. The two prominent extracellular loops (L₁ and L₂), which are not present in the bacterial SLC26 structure, are highlighted in magenta. D and E, partial amino acid sequences of SLC26A4 (pendrin) and SLC26A5 (prestin) that contain the L₁ (D) and L₂ (E) regions. Characters shown in cyan and magenta indicate acidic and basic residues, respectively. L₁, L₂, 14aa, and sL₂ indicate the segments that were swapped between pendrin and prestin in this study.

Figure 2.

NLC measurements for prestin and prestin-based constructs. A–D, schematic diagrams of WT prestin (WT-A5) (A) and prestin-based constructs (A5-L₁ (B), A5-L₂ (C), and A5-L₁/L₂ (D)) along with their representative NLCs. The parts of prestin and pendrin are indicated by magenta and dark blue, respectively, in the schematic diagrams. The magnitudes of NLCs (C_m − C_lin) are corrected for cell size (C_lin) because larger cells tend to express greater amounts of prestin in their cell membranes (NLC_sp ≡ (C_m − C_lin)/C_lin). Different colors indicate individual recordings. A two-state Boltzmann model (see “Experimental procedures”) was used to interpret the NLC data (solid lines). E, the V_pk values (mean ± S.D.) are as follows: WT-A5, −91 ± 24 mV (n = 9); A5-L₁, −132 ± 31 mV (n = 13); A5-L₂, −169 ± 26 mV (n = 9); A5-L₁/L₂, −170 ± 24 mV (n = 9). The asterisks indicate the degree of statistical significance (**, p < 0.01; ****, p < 0.0001). Error bars represent S.D. ns, not significant. F, the α values (mean ± S.D.) are as follows: WT-A5, 0.031 ± 0.006 mV⁻¹ (n = 9); A5-L₁, 0.026 ± 0.007 mV⁻¹ (n = 13); A5-L₂, 0.025 ± 0.005 mV⁻¹ (n = 9); A5-L₁/L₂, 0.026 ± 0.005 mV⁻¹ (n = 9). Error bars represent S.D.

Figure 3.

NLC measurements for pendrin and pendrin-based constructs. Representative NLC recordings for WT-A4 (A), WT-A4 + MβCD (B), A4-L₁ (C), A4-L₁(14aa) (D), A4-L₂ and A4-L₁/L₂ (E), A4-sL₂ (F), and A4-L₁/sL₂ and A4-L₁(14aa)/sL₂ (G) are shown. Schematic diagrams of these constructs are also shown above the NLC data. The parts of prestin and pendrin are indicated by magenta and dark blue, respectively. The magnitudes of NLCs are corrected for cell size as in Fig. 2. Different colors indicate individual recordings. A two-state Boltzmann model was used to interpret the NLC data (solid lines) for WT-A4 + MβCD (B) and A4-sL₂ (F). The V_pk and α values are summarized in Fig. 5, J and L.

Figure 4.

A summary of anion transport assay and qualitative assessment of subcellular localization for the pendrin-based constructs that did not show transport activity. A, HCO₃⁻/Cl⁻ antiport activities of noninduced negative control (n = 7), WT-A4 (n = 6), WT-A4 + MβCD (n = 3), A4-L₁ (n = 4), A4-L₁(14aa) (n = 4), A4-L₂ (n = 4), A4-L₁/L₂ (n = 4), A4-sL₂ (n = 3), A4-L₁/sL₂ (n = 3), and A4-L₁(14aa)/sL₂ (n = 4). Error bars represent S.D. The transport activities of A4-L₂, A4-L₁/L₂, A4-L₁/sL₂, and A4-L₁(14aa)/sL₂ were indistinguishable from that of the noninduced negative control (determined by Student's t test and indicated by “ns”). The results of the other constructs that showed significant transport activities (compared with the noninduced negative control) were further analyzed by one-way ANOVA and the Tukey–Kramer multiple comparison test to determine the adjusted p values. Only selected p values that are mentioned in the main text (under “Results”) are shown in the figure. B, the subcellular localizations of the pendrin constructs were microscopically examined. A4-L₂, A4-L₁/L₂, A4-L₁/sL₂, and A4-L₁(14aa)/sL₂, which did not show significant transport activity (A), exhibited predominantly cytosolic localizations, indicating severely impaired membrane targeting. A result for WT-A4 is also included as a positive control. Scale bars, 10 μm.

Figure 5.

The effects of the charged residues in L₂ on V_pk. A and B, schematic representations of the cell membranes (yellow) and surrounding electrolyte solutions. σ_o and σ_i are charge densities at the outer and inner surfaces of the cell membrane, respectively. Φ_o and Φ_i are electric potentials at the outer and inner surfaces of the cell membrane, respectively. C–I, representative NLC recordings for A4-E259Q (C), A4-E259R (D), A4-E259K (E), A4-D266N (F), A4-E259Q/D266N (G), A4-E259R/D266N (H), and A4-E259K/D266N (I). The magnitudes of NLCs are corrected for cell size as in Figs. 2 and 3. Different colors indicate individual recordings. A two-state Boltzmann model was used to interpret the NLC data (solid lines). J, the V_pk values (mean ± S.D.) are as follows: WT-A4 + MβCD, −166 ± 26 mV (n = 12); A4-sL₂, −173 ± 22 mV (n = 9); A4-E259Q, −184 ± 24 mV (n = 10); A4-E259R, −154 ± 19 mV (n = 9); A4-E259K, −134 ± 20 mV (n = 10); A4-D266N, −189 ± 20 mV (n = 11); A4-E259Q/D266N, −157 ± 16 mV (n = 11); A4-E259R/D266N, −132 ± 21 mV (n = 13); A4-E259KR/D266N, −149 ± 20 mV (n = 12). Error bars represent S.D. K, correlation between the net charge in L₂ versus V_pk. The means and standard deviations of the V_pk data shown in J are plotted against the calculated net charge in L₂ (at pH 7.3). Error bars represent S.D. L, the α values (mean ± S.D.) are as follows: WT-A4 + MβCD, 0.012 ± 0.002 mV⁻¹ (n = 12); A4-sL₂, 0.010 ± 0.001 mV⁻¹ (n = 9); A4-E259Q, 0.010 ± 0.002 mV⁻¹ (n = 10); A4-E259R, 0.011 ± 0.002 mV⁻¹ (n = 9); A4-E259K, 0.010 ± 0.002 mV⁻¹ (n = 10); A4-D266N, 0.011 ± 0.002 mV⁻¹ (n = 11); A4-E259Q/D266N, 0.010 ± 0.001 mV⁻¹ (n = 11); A4-E259R/D266N, 0.011 ± 0.001 mV⁻¹ (n = 13); A4-E259K/D266N, 0.012 ± 0.002 mV⁻¹ (n = 12). Error bars represent S.D.

Figure 6.

Eight NLC1/NLC2–focused pendrin/prestin chimeras used in this study. A, both schematic diagram and tertiary structural models (lateral and extracellular views) are shown for prestin-based (A4A5Ch1, A4A5Ch2, A4A5Ch3, and A4A5Ch4) and pendrin-based (A4A5Ch5, A4A5Ch6, A4A5Ch7, and A4A5Ch8) chimeras. The parts of prestin and pendrin are indicated by magenta and dark blue, respectively. The amino acids and their residue numbers at the boundaries of A4 and A5 are also provided. The C termini of all the constructs were tagged with ECFP (for prestin-based constructs, A4A5Ch1–A4A5Ch4) or mTurquoise2 (for pendrin-based constructs, A4A5Ch5–A4A5Ch8). B, qualitative assessments of the subcellular localizations of the eight chimeras (A4A5Ch1–8) heterologously expressed in HEK293T cells. Results for WT-A5 and WT-A4 are also included as positive controls. The scale bars indicate 10 μm. All eight chimeras show predominantly cytosolic localization, indicating severely impaired membrane targeting.