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.

Introduction

The solute carrier 26 (SLC26)⁴ proteins are ubiquitously expressed in a wide variety of species from bacteria to humans and constitute the SLC26/SulP family of ion transporters and channels (1). Although most of the members of this large gene family function as anion exchangers or channels, only prestin (SLC26A5) exhibits voltage-dependent motor activity referred to as electromotility. Electromotility of prestin and the anion transport mediated by pendrin (SLC26A4) are essential for normal development and operation of mammalian inner ears (2–4). Prestin-mediated electromotility confers a rapid voltage-induced force-generating cell length change on cochlear outer hair cells (5), which is indispensable for the frequency selectivity and sensitivity of mammalian hearing (4). Recent structural and biochemical studies (6, 7) have provided significant insights into the ion transport mechanism of the SLC26 proteins. However, the molecular mechanism of the voltage-driven motor activity, which is thought to be unique to prestin among the SLC26 family members, still remains largely unknown. Electromotility accompanies a movement of prestin-associated charge. Because this charge movement is rapid, it manifests as nonlinear electrical capacitance of the cell membrane (nonlinear capacitance (NLC)) (8, 9). Thus, NLC is regarded as the electric signature of electromotility, and its measurement often substitutes for direct electromotility measurement.

Because of the high degree of similarity in amino acid sequences among the SLC26 family members, it is conceivable that the tertiary structures of SLC26 proteins are quite similar among the family members and that a common molecular mechanism underlies their diverse physiological functions. Both pendrin and prestin are expressed in the mammalian inner ear, and they share 36% homology at the amino acid level. Using SLC26 models that were generated based on the solved structure of a bacterial SLC26 protein (see Fig. 1) (7), we carried out a comprehensive mutational analysis combined with whole-cell patch-clamp recordings and anion transport assays to understand the molecular basis for the functional difference between pendrin, a well-characterized anion transporter, and prestin, a voltage-driven motor. We found that pendrin also retains voltage-sensing ability, which becomes evident with addition of cyclodextrin to depolarize the peak voltage-operating point. We also found that the electrostatic property of the characteristic extracellular loop of pendrin and prestin (termed “L₂” in this study; see Fig. 1) influences the voltage-operating points of these proteins. Furthermore, the anion transport activity of pendrin was not affected by the alterations in operating voltage range, suggesting that the voltage-sensing and anion transport mechanisms are independent of each other. Our results suggest that the extracellular loop of prestin has adopted the optimal electrostatic property, which affects the membrane surface electric potential, so that the voltage-operating point resides within the physiologically relevant voltage range, and that the electromotility may have evolved independently of its anion transport mechanism.

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.

Results

The extracellular loops of pendrin and prestin

Recent structural and biochemical studies have provided significant insights into the ion transport mechanism of the SLC26 proteins (6, 7, 10). Because the basic architecture of the transmembrane (TM) region that accommodates the putative ion translocation pathway is likely quite similar among the SLC26 family members, it is possible that the diverse physiological functions of the SLC26 family of proteins have arisen from modifications of a common ancestral ion transport mechanism. Structural information often facilitates efforts to identify regions in proteins responsible for the expressions of their diverse physiological functions. Because the structure of the TM region has only been solved for a bacterial SLC26 protein, SLC26Dg (Fig. 1A) (7), we modeled the TM structures of pendrin and prestin based on the solved structure of SLC26Dg (Fig. 1, B and C). Two prominent extracellular loops, which are referred to as loop-1 (L₁) and loop-2 (L₂) in this study, are present in pendrin and prestin but not in the bacterial SLC26 protein (Fig. 1, B and C, magenta). Because many disease-associated pendrin mutations have been identified in the L₁ (F161I, V163I, V163L, S166N, and T178P) and L₂ (V250A, S252P, I253V, L257P, and D266N) regions (11–19), these extracellular loops may play significant roles in defining the physiological functions of pendrin (HCO₃⁻/I⁻/Cl⁻ exchanger) and prestin (voltage-operated motor).

The extracellular loops modulate the voltage-operating point of prestin

To examine the contributions of L₁ and L₂ to the function of prestin, we generated several prestin-based constructs whose L₁ and L₂ are replaced with those of pendrin individually (A5-L₁ and A5-L₂) or in combination (A5-L₁/L₂) (Figs. 1 and 2) and measured NLC in HEK293T cell lines expressing these constructs (Fig. 2). Replacement of L₁ did not abrogate NLC but resulted in significant hyperpolarization of the voltage-operating points (V_pk) (Fig. 2, B and E). Likewise, replacement of L₂ resulted in significant V_pk hyperpolarization but to a much greater degree compared with L₁ replacement (Fig. 2, C and E). No further hyperpolarization was observed upon double (L₁/L₂) replacement (Fig. 2, D and E). The voltage sensitivity (α) was not significantly affected by any of these loop replacements (one-way analysis of variance (ANOVA), p = 0.14) (Fig. 2F). These results suggest that the extracellular loops of prestin modulate its operating voltage range without affecting its voltage sensitivity.

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.

Pendrin possesses voltage-sensing ability

The voltage-driven motor function of prestin is thought to be unique among the members of the SLC26 family; however, the veracity of this presumption has not been tested rigorously. Given the high degree of similarity in amino acid sequences among the SLC26 family members, it is possible that many, if not most, other SLC26 proteins also retain a voltage-sensing mechanism, but it is not readily detectable within the experimentally measurable voltage range (typically ±150 mV). We noticed that some HEK293T cells expressing the WT pendrin construct tend to exhibit a gradual increase in cell membrane capacitance below ∼0 mV, hinting at the presence of large NLCs (Fig. 3A). We speculated that pendrin also retains voltage-sensing ability, but its V_pk is extremely hyperpolarized and thus not easily detected. To test this possibility, we repeated capacitance measurement for the WT pendrin construct in the presence of methyl-β-cyclodextrin (MβCD), which is known to depolarize V_pk of prestin by removing cholesterol from the cell membrane (20). As shown in Fig. 3B, NLCs of WT pendrin became quite evident upon application of MβCD due to its anticipated depolarizing effect, which brought the otherwise barely detectable NLC of pendrin into the experimentally measurable voltage range. We also performed NLC measurement in Sf9 cells (Fig. S1). The low membrane cholesterol level in this cell line (21) allowed detection of large NLC of pendrin without the use of MβCD.

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.

The importance of the extracellular loops of pendrin for membrane targeting and modulation of the voltage-operating point

We examined the contributions of L₁ and L₂ to the voltage-sensing function of pendrin as we did for prestin. To this end, we generated several pendrin-based constructs whose L₁ and L₂ are replaced with those of prestin individually (A4-L₁ and A4-L₂) or in combination (A4-L₁/L₂) (Figs. 1 and 3). We anticipated finding large NLCs for A4-L₁ because the L₁ region contains the “14-aa” segment that was reported to confer large NLC and electromotility on pendrin (22). However, the effect of L₁ replacement was ambiguous (Fig. 3C) as NLCs of A4-L₁ (and WT pendrin (WT-A4)) were only partially measurable to confidently estimate the NLC parameters. Qualitatively, the L₁ replacement did not seem to significantly affect V_pk of pendrin (Fig. 3, A and C). We also generated a construct in which only the 14-aa segment used in the previous study (22) was replaced (termed “A4-L₁(14aa)” in this study; Fig. 1D). Unexpectedly, however, we could not detect NLC for this construct (Fig. 3D). The reason for the discrepancy is unclear. At the least, the absence of NLC in A4-L₁(14aa) should not be solely ascribed to impaired membrane targeting of the construct because the HCO₃⁻/Cl⁻ antiport activity was detected for A4-L₁(14aa) (Fig. 4A). Although the transport activity of A4-L₁(14aa) was smaller than that of WT-A4 (p = 0.0016) (Fig. 4A), it was comparable with that of A4-sL₂ (p > 0.999; Fig. 4A), which exhibited a large NLC (Fig. 3F; see below).

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.

Replacement of L₂ (A4-L₂) or both L₁/L₂ (A4-L₁/L₂) resulted in loss of NLC (Fig. 3E) and HCO₃⁻/Cl⁻ antiport activity (Fig. 4A). This is very likely due to the lack of membrane targeting as they are predominantly cytosolic and are qualitatively very different from WT-A4 (Fig. 4B). To circumvent this membrane targeting issue, we generated additional pendrin constructs with a shorter L₂ (“sL₂”; Fig. 1E) segment replaced (termed “A4-sL₂,” “A4-L₁/sL₂,” and “A4-L₁(14aa)/sL₂” Fig. 3). The replacement of the shorter L₂ segment (A4-sL₂; Fig. 3F) resulted in the depolarization of V_pk (as in MβCD-treated WT-A4) (Fig. 3F), indicating that this segment similarly modulates the voltage-operating point in pendrin as in prestin. However, we did not detect any NLC in A4-L₁/sL₂ and A4-L₁(14aa)/sL₂ (Fig. 3G). Because the HCO₃⁻/Cl⁻ antiport activity was not detected for A4-L₁/sL₂ and A4-L₁(14aa)/sL₂ either (Fig. 4A), we examined the expression of A4-L₁/sL₂ and A4-L₁(14aa)/sL₂ and found that they failed to target the plasma membrane (Fig. 4B). These negative observations on A4-L₁/L₂, A4-L₁/sL₂, and A4-L₁(14aa)/sL₂ imply the importance of pendrin's extracellular loops for maintaining its structural integrity and/or membrane targeting.

The role of the charged residues in loop-2 in establishing V_pk

The L₂ swapping between prestin and pendrin resulted in hyperpolarization for prestin and depolarization for pendrin (Figs. 2 and 3). This inverse effect of the L₂ swapping can be explained by the electrostatic property of the L₂ region that is presumably expected to be exposed to the extracellular surface of the membrane. For example, the presence of acidic residues would decrease the surface charge density (σ_o), which should result in concomitant drops (hyperpolarization) of the membrane surface potential (Φ_o) (Fig. 5A). The opposite (depolarization of Φ_o) would be expected if basic residues were present in L₂ (Fig. 5B). If the inner surface potential (Φ_i) remained unchanged, hyperpolarization of Φ_o should result in hyperpolarization of V_pk and vice versa. This interpretation is compatible with the fact that the net calculated charges of L₂ are −1 and +4 for pendrin and prestin, respectively (Fig. 1E).

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.

Glu²⁵⁹ is the sole charged residue present in sL₂ of pendrin, whereas none is present in that of prestin (Fig. 1E). To test whether the significant V_pk depolarization found in A4-sL₂ (Fig. 3F) can be attributed to neutralization of the negative charge pertaining to Glu²⁵⁹, additional pendrin constructs harboring E259Q, E259R, or E259K mutation were generated, and their NLCs were measured. NLCs became clearly measurable upon neutralization of Glu²⁵⁹ (E259Q) (Fig. 5, C and J) and resemble those found in A4-sL₂ (Fig. 3F). Polarity reversal of Glu²⁵⁹ (E259R and E259K) also made NLC of pendrin clearly detectable within the measurable voltage range due to V_pk depolarization (Fig. 5, D, E, and J). We also examined the effect of the negative charge pertaining to Asp²⁶⁶, which is not located within the sL₂ region but located in L₂ (Fig. 1E). We found that neutralization of this negative charge (D266N) also depolarizes V_pk and makes large NLC of pendrin readily detectable (Fig. 5, F and J). Simultaneous introduction of charge-affecting mutations to Glu²⁵⁹ and Asp²⁶⁶ (A4-E259Q/D266N, A4-E259R/D266N, and A4-E259K/D266N) also depolarized V_pk but to greater extents (Fig. 5, G–J). These results suggest that the effects of these charge manipulations are additive. In fact, we found a statistically significant linear correlation (r = 0.85, p = 0.008) between the calculated net charge in L₂ versus V_pk (Fig. 5K). The α value and its variation were similar among the pendrin constructs (one-way ANOVA, p = 0.39) (Fig. 5L), which argues against the possibility that the V_pk values were significantly over- or underestimated. These observations are uniformly explained by depolarization of Φ_o as a result of reduction of the negative surface charge present in sL₂ or L₂ (Fig. 5, A and B).

Charge manipulations of Asp²⁷¹ (D271N, D271R, and D271K), which is located in the transmembrane helix that immediately follows L₂ (Fig. 1), significantly impaired the targeting of pendrin to the cell membrane (Fig. S2A). Consistently, these mutants showed little or no NLC and transport activity (Fig. S2, B and E), implying the importance of Asp²⁷¹ for maintaining the structural integrity of the transmembrane region.

The relationship between the voltage-sensing ability of pendrin and its anion transport function

The voltage-driven motor function of prestin has been presumed to stem from a common anion transport mechanism that is likely shared among the members of the SLC26 family (23, 24). To define the molecular mechanism underlying electromotility, it is important to examine whether the anion transport activity of prestin is affected by voltage. Unfortunately, prestin is not suitable for pursuing this possibility because we could not detect HCO₃⁻/Cl⁻ antiport activity for prestin (WT-A5) or any of our prestin-based constructs. We therefore examined the NLC–anion transport relationship in pendrin because we identified large NLC in pendrin (Fig. 3). The resting membrane potentials of HEK293T cells expressing WT pendrin (mean ± S.D.) were −15.0 ± 5.8 (n = 12) and −12.3 ± 4.4 mV (n = 16) in the presence and absence of MβCD, respectively, with no statistically significant difference among the groups (p = 0.17). If the voltage-sensing mechanism were intimately related to the anion transport mechanism, augmentation of the anion transport activity would be expected upon application of MβCD due to a significant shift of V_pk toward the resting membrane potential of the pendrin-expressing cells (Fig. 3, A and B). However, we did not find a statistically significant change in the anion transport activity upon application of MβCD (p > 0.999; Fig. 4A), suggesting independence of the transport and voltage-sensing mechanisms.

NLC1 and NLC2 segments of pendrin and prestin are not mutually interchangeable

Previous efforts to identify a segment(s) in the prestin molecule that is essential for the expression of electromotility have been inconsistent. One study (25) identified such segments, termed “NLC1” and “NLC2,” in the region forming the presumed anion translocation pathway (6), whereas other studies identified a short segment (termed “14aa” in the present study) located in L₁ (22, 26). These results are apparently irreconcilable because the short 14-aa segment is not a part of the large NLC1/NLC2 segments. Because we could not confirm the importance of the 14-aa short segment for the expression of NLC (Fig. 3D), we also generated eight chimeric constructs in which the NLC1 and NLC2 segments were swapped between pendrin and prestin (A4A5Ch1–A4A5Ch8) (Fig. 6A), which are similar to those generated by Schaechinger et al. (25) using electromotile and nonelectromotile prestin orthologues. Unfortunately, none of our pendrin/prestin chimeras showed NLC or HCO₃⁻/Cl⁻ antiport activity (Fig. S2, C–E), probably due to the lack of membrane targeting (Fig. 6B), suggesting a difference in the intramolecular interactions within the TM domain between pendrin and prestin.

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.

Discussion

The electromotile function of prestin (SLC26A5) is thought to be unique among the members of the SLC26 family. Although not all prestin orthologues exhibit motor activity, they all show NLC (27–30). NLC indicates the presence of a rapid voltage-sensing mechanism, and it is directly coupled to concurrent motor activity in electromotile prestin orthologues (31–34). In the present study, we show that pendrin (SLC26A4) also exhibits NLC but with extremely hyperpolarized V_pk. Given the high degree of similarity in amino acid sequences among the SLC26 proteins, the presence of NLC in other members in the family would not be surprising, but this possibility has not been thoroughly examined heretofore. A previous study also reported NLC parameters for pendrin-expressing cells (22). However, the magnitude of the NLCs were much smaller than those observed in the present study despite their apparent V_pk (∼−40 mV) located within an easily measurable voltage range (22). In addition, the small NLCs found in the previous study were qualitatively very different from those reported in this study as the V_pk values were depolarized, but not hyperpolarized, with respect to V_pk of prestin (22). We claim that the NLCs detected in the present study for the pendrin-expressing cells are indeed mediated by pendrin because we found that the V_pk of the NLCs responds to cholesterol depletion by MβCD (Fig. 3) in the same way as previously demonstrated for prestin (20) and that swapping of L₂ that contains opposite electrostatic property between pendrin (net negative) and prestin (net positive) results in V_pk shifts toward mutually opposing directions for pendrin (depolarization; Fig. 3) and prestin (hyperpolarization; Fig. 2). Successful identification of large NLCs in pendrin-expressing Sf9 cells without the use of any genetic or chemical manipulations further authenticates our claim (Fig. S1).

There is controversy over the identity of the voltage-sensing charge (6, 35–38). An early study proposed an incomplete transport model to explain the voltage-sensing mechanism of prestin where intracellular Cl⁻ or HCO₃⁻ is used as an extrinsic voltage sensor (35). The basic notion of this model does not disagree with the fact that the HCO₃⁻/Cl⁻ antiport activity of prestin is very low (39) or virtually undetectable. However, later studies found that electromotility can be conferred on nonelectromotile SLC26 proteins without diminishing or abrogating the transport activities (25, 26). Our finding that pendrin exhibits both significant HCO₃⁻/Cl⁻ antiport activity and NLC (Figs. 3 and 4) underscores that the anion transport function and voltage-sensing ability are not mutually exclusive. The identification of large NLC in pendrin provided a fortuitous opportunity to examine the functional relationship between voltage-sensing and anion transport mechanisms, which could not be pursued in prestin due to its extremely small anion transport activity to be compared with NLC. We found that the HCO₃⁻/Cl⁻ antiport activity of pendrin is not affected by a significant depolarizing shift of NLC toward the resting membrane potentials of pendrin-expressing cells upon MβCD application (Figs. 3 and 4). This observation suggests that voltage-sensing and anion transport mechanisms may not be intimately related, which is in agreement with a previous observation suggesting separation of voltage-sensing and anion transport functions for prestin (38).

It is possible that most, if not all, members of the SLC26 family retain voltage-sensing ability. It is important to note that the absence of measurable NLC does not necessarily mean inability of an SLC26 protein to sense voltage. Because the experimentally measurable range of membrane potential is limited, NLC with extremely hyper-/depolarized V_pk is difficult to identify. In the present study, we demonstrate that this is, in fact, the case for pendrin (Fig. 3A). Fast voltage-sensing kinetics is another important factor for a successful NLC recording because detection of capacitive currents is the basis of NLC measurement. If voltage-sensing charge movement is very slow, it would not manifest as NLC. For example, NLC of zebrafish prestin shows significant stimulus voltage frequency dependence, and it becomes unmeasurable at high stimulus frequencies due to its relatively slow voltage-sensing kinetics (27). It is conceivable that the voltage-sensing kinetics of some SLC26 members may be too slow to exhibit its voltage-sensing ability as NLC. Collectively, absence or abrogation of NLC by genetic or pharmacological manipulation needs to be concluded carefully.

For physiological electromotile function of prestin orthologues, it is important to set their V_pk within the physiological V_m range. Our study demonstrates that the charged residues present in L₂ greatly contribute to set distinct V_pk for prestin and pendrin (Figs. 2, 3, and 5) and that the directions of the V_pk shift induced by charge-affecting mutations in L₂ can be simply explained by changes in the membrane surface charge density (σ_o) that concomitantly affect membrane surface electrical potential (Φ_o) (Fig. 5, A and B). Furthermore, all previously reported V_pk shifts of prestin that were induced by charge-affecting mutations introduced to L₂, i.e. T234K (depolarization) (40), K233Q/K235Q/R236Q (hyperpolarization) (35), S238D (hyperpolarization) (41), and K255Q (hyperpolarization) (38), can be explained by their anticipated effects on the membrane surface potential. On the contrary, L₁ does not seem to contribute to confer distinct V_pk on pendrin (Fig. 3C). Although significant V_pk hyperpolarization was observed in prestin upon L₁ swapping (Fig. 2B), no additive effect on V_pk was found upon L₁/L₂ double swapping (Fig. 2D). In any case, the hyperpolarizing V_pk shift of prestin upon L₁ swapping (Fig. 2B) cannot be explained by the electrostatic property of L₁ because the net charge of L₁ is almost the same between prestin and pendrin (Fig. 1D). Also, hyperpolarizing V_pk shifts of prestin induced by D154N and D155N mutations (35) cannot be explained by an L₁-associated surface charge effect. These irregular and unpredictable consequences of L₁ swapping may imply a dynamic role of the L₁ region in prestin.

Both single (L₁ or L₂) and double (L₁ and L₂) loop swapping of the extracellular loops does not deprive prestin of its function (Fig. 2). Pendrin also tolerates single (L₁ or L₁(14aa)) swapping, but neither single L₂- nor double L₁/L₂-swapped pendrin constructs (A4-L₂ and A4-L₁/L₂) showed NLC or HCO₃⁻/Cl⁻ antiport activity, likely due to impaired membrane targeting (Fig. 4B). We eluded the membrane targeting issue for A4-L₂ by shortening the swapped L₂ region (sL₂) (Fig. 3F). However, double loop-swapped pendrin constructs with sL₂ (A4-L₁/sL₂ and A4-L₁(14aa)/sL₂) still fail to target the membrane (Fig. 4B). These observations, together with our negative observation on the eight pendrin/prestin chimeras (Fig. 6), suggest a difference in the intramolecular interactions between pendrin and prestin or may imply a difference in the TM domain architecture between pendrin and prestin.

It is noteworthy that the voltage sensitivity of pendrin, which is reflected in its α value, is 2–3 times smaller than that of prestin (Figs. 2F and 5L) and that the α values of nonelectromotile prestin orthologues are also smaller than those of electromotile prestin orthologues (27, 28). Because α values would indicate the magnitude and extent of voltage-induced conformational change (see “Experimental procedures”), it is interesting to examine whether and how the magnitudes of α correlate with the expression of electromotility. Although electrophysiological characterization has already been conducted for various prestin orthologues (27–30), similar efforts have not been expanded to the other members in the SLC26 family. Thorough comparative studies across the SLC26 family would facilitate efforts in defining the molecular mechanisms underlying the uniqueness and generality of electrophysiological characteristics among the SLC26 family members.

Experimental procedures

Generation of stable cell lines that express various SLC26 protein constructs

The pendrin (UniProt accession number O43511)- and prestin (UniProt accession number Q9JKQ2)-based constructs were generated by multistep PCR (A4-L₁, A4-L₂, A4-L₁/L₂, A5-L₁, A5-L₂, and A5-L₁/L₂) (42), site-directed mutagenesis (A4-E259Q, A4-E259R, A4-E259K, A4-D266N, A4-E259Q/D266N, A4-E259R/D266N, and A4-E259K/D266N), and de novo DNA synthesis combined with template vector PCR (A4-sL₂, A4-L₁/sL₂, A4-L₁(14aa), A4-L₁(14aa)/sL₂, and A4A5Ch1–A4A5Ch8). The complete DNA sequences of all these constructs, along with the PCR primers and synthesized dsDNA information, are provided in the supporting information. These constructs with C-terminally attached ECFP (for prestin-based constructs) or mTurquoise2 (for pendrin-based constructs) were cloned into a pSBtet-Pur vector (Addgene, Cambridge, MA) (43). The functions of pendrin and prestin are not abrogated by a C-terminally fused fluorescent protein (32, 44). Stable cell lines that express these recombinant protein constructs were established in HEK293T cells as described previously (43). Expressions of the pendrin/prestin constructs were induced by application of doxycycline (1 μg/ml) to the cell culture medium 1–3 days prior to experiments.

Protein structure modeling

The structural models of the transmembrane regions of pendrin and prestin were generated by Phyre2 (45) based on the SLC26Dg structure (Protein Data Bank code 5DA0) (7) using the partial amino acid sequences of pendrin (Pro⁷⁶–Arg⁵¹²) and prestin (Pro⁷²–Arg⁵⁰²). The models were rendered by PyMOL (Fig. 1) or CueMol software (http://www.cuemol.org/)⁵ (Fig. 6).

Electrophysiology

Whole-cell recordings were performed at room temperature using an Axopatch 200A/B amplifier (Molecular Devices, Sunnyvale, CA). Recording pipettes were pulled from borosilicate glass to achieve initial bath resistances averaging 3–4 megaohms. Whole-cell NLC recordings were performed using a 0-mV holding potential and a sinusoidal voltage stimulus (2.5-Hz, 120–150-mV amplitude) superimposed with two higher frequency stimuli (390.6 and 781.2 Hz, 10-mV amplitude). Recording pipettes were filled with an intracellular solution containing 140 mm CsCl, 2 mm MgCl₂, 10 mm EGTA, and 10 mm HEPES (pH 7.3 adjusted at room temperature). Cells were bathed in an extracellular solution containing 120 mm NaCl, 20 mm tetraethylammonium chloride, 2 mm CoCl₂, 2 mm MgCl₂, and 10 mm HEPES (pH 7.3 adjusted at room temperature). Osmolarity was adjusted to 310 mosmol liter⁻¹ with glucose (34, 46). Intracellular pressure was kept at 0 mmHg, and current data were collected by jClamp (SciSoft Co., New Haven, CT) using a fast Fourier transform–based admittance analysis to determine NLC (47). The resting cell membrane potentials were determined in Hanks' balanced salt solution (14025, Thermo Fisher Scientific, Waltham, MA) using recording pipettes filled with an intracellular solution containing 140 mm KCl, 2 mm MgCl₂, 10 mm EGTA, and 10 mm HEPES (pH 7.3 adjusted at room temperature).

NLC data analysis

Voltage-dependent cell membrane electric capacitance data were analyzed using the following two-state Boltzmann equation,

$$C_m=\frac{\alpha Q_{\max }\hspace{0.17em}\exp \left[\alpha \left(V_m-V_{\text{pk}}\right)\right]}{{\left\{1+\exp \left[\alpha \left(V_m-V_{\text{pk}}\right)\right]\right\}}^2}+C_{\text{lin}}$$ (1)

where α is the slope factor of the voltage dependence of charge transfer, Q_max is the maximum charge transfer, V_m is the membrane potential, V_pk is the voltage at which the maximum charge movement is attained, and C_lin is the linear capacitance (34, 46, 48, 49). In this two-state Boltzmann model, α is defined as z_appe/k_BT where z_app is the apparent valence of charge movement, e is electron charge, k_B is the Boltzmann constant, and T is absolute temperature. The magnitude of z_app reflects the valence (z) and the traveling distance (δ) along the direction of the electric field of a voltage-sensing charge.

Anion transport assay

A ratiometric fluorescent pH indicator, SNARF-5F (S23923, Thermo Fisher Scientific), was loaded into cells in a high-chloride buffer containing 140 mm NaCl, 4.5 mm KCl, 1 mm MgCl₂, 2.5 mm CaCl₂, and 20 mm HEPES (pH 7.4 adjusted at room temperature, 320 mosmol liter⁻¹) for 30 min in the presence of 5% CO₂ at room temperature. The cells were washed with the high-chloride buffer once and resuspended in 100 μl of the high-chloride buffer. Portions of the cell suspensions (50 μl) were transferred to wells in a 96-well plate (∼1.5 × 10⁵ cells/well). The HCO₃⁻/Cl⁻ antiport assay was initiated by an automated injection of 200 μl of a low-chloride buffer containing 125 mm sodium gluconate, 5 mm potassium gluconate, 1 mm MgCl₂, 1 mm CaCl₂, 20 mm HEPES, and 25 mm NaHCO₃ (pH 7.4 at room temperature under 5% CO₂) in a Synergy2 plate reader (BioTek, Winooski, VT). The fluorescence intensity of SNARF-5F was measured in a time-dependent manner. The excitation and emission filters used were 504/12 (excitation) (Semrock, Rochester, NY), 572/28 (F₁) (Semrock), and 709/167 (F₂) (Semrock), respectively. The fluorescence ratio, F₂/F₁, was converted into the intracellular H⁺ concentration using a pH calibration curve. The HCO₃⁻/Cl⁻ antiport activity (s⁻¹) was determined from exponential curve fitting.

Confocal imaging

The stable cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, high glucose, GlutaMAX, pyruvate, Thermo Fisher Scientific) with 10% FBS (French origin, BioWest, Riverside, MO) at 37 °C (5% CO₂) in glass-bottom dishes (D11130H, Matsunami Glass, Kishiwada, Japan). When cell confluence reached 20–30%, expressions of the pendrin/prestin constructs were induced by 1 μg/ml doxycycline hyclate (LKT Laboratories, St. Paul, MN) included in the medium for 2–3 days. Fluorescence images of the cells were acquired using an LSM710 confocal microscope (Carl Zeiss, Oberkochen, Germany) equipped with a 63× Plan-Apochromat objective lens (numerical aperture, 1.40 oil) and a filter set (BP 395–440 excitation filter, FT460 dichroic mirror, and LP470 emission filter). The cells were illuminated with a 30-milliwatt 405-nm diode laser (laser power, 2%) at room temperature, and the images were taken with ZEN software (Carl Zeiss). The pinhole size and pixel dwell time were set to be 1 airy unit (490 nm) and 1.27 μs, respectively. Four sequentially scanned images were averaged and then processed with a despeckle algorithm and contrast/brightness adjustment using Fiji software (50).

Statistical analyses

Statistical analyses were performed using Prism (GraphPad Software). Student's t test was used for comparisons between two groups. One-way ANOVA combined with the Tukey–Kramer test was used for multiple comparisons. p < 0.05 was considered statistically significant.

Author contributions

M. F. K., K. W., and K. H. formal analysis; M. F. K., K. W., S. T., J. B., T. K., and K. H. investigation; T. K., S. U., J. Z., T. S., and K. H. resources; T. K., S. U., T. S., and K. H. supervision; T. K., S. U., T. S., and K. H. funding acquisition; T. S. and K. H. conceptualization; K. H. writing-original draft; K. H. writing-review and editing.