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Intracellular Anions as the Voltage Sensor of Prestin, the Outer Hair Cell Motor Protein

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Science  22 Jun 2001:
Vol. 292, Issue 5525, pp. 2340-2343
DOI: 10.1126/science.1060939

Abstract

Outer hair cells (OHCs) of the mammalian cochlea actively change their cell length in response to changes in membrane potential. This electromotility, thought to be the basis of cochlear amplification, is mediated by a voltage-sensitive motor molecule recently identified as the membrane protein prestin. Here, we show that voltage sensitivity is conferred to prestin by the intracellular anions chloride and bicarbonate. Removal of these anions abolished fast voltage-dependent motility, as well as the characteristic nonlinear charge movement (“gating currents”) driving the underlying structural rearrangements of the protein. The results support a model in which anions act as extrinsic voltage sensors, which bind to the prestin molecule and thus trigger the conformational changes required for motility of OHCs.

Electromotility of OHCs (1, 2) occurs at acoustic frequencies and is assumed to produce the amplification of vibrations in the cochlea that enables the high sensitivity and frequency selectivity of the mammalian hearing organ (3, 4). This motility results from a protein in the OHC basolateral membrane that undergoes a structural rearrangement in response to changes in the transmembrane voltage (5–7). Coupling of motility and transmembrane voltage is mediated by a charged voltage sensor within the protein that moves through the electrical field and thus gives rise to a gating current similar to that observed in voltage-gated ion channels (8, 9). Recently, the gene coding for an integral membrane protein of OHCs termed prestin (Fig. 1A) has been identified (10). Upon heterologous expression, the protein reproduces all hallmarks of the motor protein including voltage-dependent charge movement and cell motility (10–13). Because of its fundamental role in OHC electromotility, we examined the mechanism underlying the voltage sensitivity of prestin.

Figure 1

Effect of neutralizing charged residues in the prestin sequence on the voltage dependence of nonlinear charge movement. (A) Membrane topology of prestin as suggested by hydrophobicity analysis and epitope-tagging (28) indicating location of the charged residues tested for involvement in the voltage sensor. Circles and squares denote negatively and positively charged amino acids, respectively; overlaid squares represent clusters of positively charged residues (C1 to C4); filling indicates significant effect on nonlinear charge movement. Asterisks mark position and location of HA tags introduced into the prestin molecule. (B) Relative Cnonlin measured in response to voltage ramps in CHO cells expressing either wild-type (WT) or mutant prestin or SLC26A6. Prestin traces are normalized to peak capacitance, and the SLC26A6 trace is scaled with respect to the WT prestin trace. Continuous lines represent fit of Eq. 1 to the prestin data [values for V 1/2 and slope (α) were −75.3 mV and 38.1 mV for WT prestin, −146.7 mV and 41.3 mV for D154N, and 14.2 mV and 38.5 mV for D342Q] (29). (C) V 1/2 values (means ± SD) determined in 5 to 10 experiments as in (B) for the prestin mutants indicated; C1 is K233Q, K235Q, and R236Q; C2 is R281Q, K283Q, and K285Q; C3 is K557Q, R558Q, and K559Q; and C4 is R571Q, R572Q, and K577Q. Continuous line and dashed area represent meanV 1/2 ± SD obtained for WT prestin.

Functionality of the voltage sensor of prestin was probed by measuring the nonlinear capacitance (Cnonlin) arising from its gating currents with the phase-tracking technique (14). Prestin-expressing CHO cells exhibited a bell-shaped Cnonlin in response to the transmembrane voltage ramped from –130 mV to 60 mV (Fig. 1B). This electrical signature was well fitted with the derivative of a first-order Boltzmann function [Eq. 1in (14)] yielding values forV 1/2 and α of –75.5 ± 9.1 mV and 35.5 ± 2.3 mV (n = 16), respectively.

In contrast, no Cnonlin was observed in cells expressing SLC26A6 (15) (n = 12), another member of the family of pendrin-related transporters that exhibits closest homology to prestin (∼40% identity). It is thus likely that the voltage sensor of prestin is made up of a charged residue present in the prestin sequence but absent in SLC26A6. We mutated each of the nonconserved negatively or positively charged residues in the putative membrane domain of the prestin molecule to a neutral amino acid, glutamine or asparagine, either individually or in groups [(16) and Fig. 1A]. In no case was the electrical signature of the voltage sensor abolished, although in some prestin mutants Cnonlin was shifted along the voltage axis by up to 100 mV (Fig. 1, B and C). The slope factor α characterizing the voltage dependence of Cnonlin was not significantly different between wild type and any of the mutants tested (17).

These mutagenesis results led to the idea that, instead of being an intrinsic property of the prestin molecule, the voltage sensor may be a charged particle extrinsic to the protein. We thus replaced cations and anions on either side of the membrane byN-methyl-d-glucamine (NMDG+) or tetra-ethyl-ammonium (TEA+) and pentane-sulfonate or sulfate, respectively. Cnonlin of a prestin-expressing CHO cell largely decreased on reduction of the Clconcentration in the whole-cell recording pipette from 150 mM to 2 mM (Fig. 2, A and B). This decrease was fully reversible, as Cnonlin was completely restored when Cl was increased back to 150 mM (Fig. 2B). These findings were confirmed in patches excised from rat OHCs (18). Removal of Cl from the cytoplasmic side led to a complete but reversible loss of Cnonlin in all inside-out patches tested (n = 15, Fig. 2C). In contrast, replacement of Cl on the extracellular side by sulfate or pentane-sulfonate had no detectable effect on Cnonlin(n = 9, Fig. 2D). Similarly, Cnonlin was not affected by the cation species, K+, Na+, NMDG+, or TEA+, present on either side of the membrane (19).

Figure 2

Removal of Cl abolishes voltage-dependent charge movement of prestin in CHO cells and OHCs. (A and B) Successive whole-cell measurements of Cnonlin in a prestin-expressing CHO cell with alternating high and low Cl concentration in the recording pipette; solutions were either (millimolar concentration) 150 KCl, 10 Hepes, and 1 EGTA, or 2 KCl, 148 Na-pentane-sulfonate, 10 Hepes, and 1 EGTA. After measuring Cnonlin with one intracellular solution, the pipette was gently withdrawn to allow resealing of the cell before repatching with the next solution [insets in (B)]. Note the reversible decrease in Cnonlin when intracellular Cl was reduced. (C) Cnonlinmeasured in an inside-out patch excised from a rat OHC with and without Cl present at the cytoplasmic side of the patch; 150 mM Cl was replaced by 50 mM SO4 2−. (D) Cnonlin measured in an outside-out patch excised from a rat OHC with and without Cl present at the extracellular side of the patch; Cl replacement as in (C).

Next, we studied the significance of cytoplasmic Cl for OHC electromotility by measuring cell-length changes in response to voltage steps (20). With 150 mM Cl in the recording pipette, OHCs displayed normal length changes with amplitudes up to 1 μm (n = 8). However, no electromotility was detected with 150 mM pentane-sulfonate replacing Cl in the intracellular solution (n = 14; Fig. 3). After removal of cytoplasmic Cl, the cells remained in the contracted state (21).

Figure 3

Removal of Cl eliminates voltage-dependent motility of OHCs. (A) Electromotility of a gerbil OHC measured in whole-cell voltage-clamp mode with high (150 mM Cl; upper panel) or low Cl (150 mM pentane-sulfonate; lower panel) in the patch pipette. Holding potential was −70 mV, membrane potential was stepped between −140 mV and 100 mV in 20-mV step increments. Pipette solutions were (millimolar concentration): 150 KCl, 10 Hepes, and 1 EGTA, or 150 Na-pentane-sulfonate, 0.5 KCl, 10 Hepes, and 1 EGTA. (B) Steady-state motility-voltage relation determined from the experiments in (A).

These results indicated that intracellular Cl was sufficient to confer both voltage-dependent charge movement and electromotility onto prestin, most likely by acting on a binding site within the molecule. We tested various halides and small organic anions for their ability to induce Cnonlin. All monovalent anions tested induced voltage-dependent charge movement when applied to inside-out patches at 150 mM (Fig. 4A) with an order of Q max of I≈ Br > NO3 > Cl > HCO3 > F, which is similar to that observed for anion-binding to pendrin and some chloride channels (22, 23). The characteristics of the charge movement differed somewhat among the various anions. Although the slope values were not significantly different from that obtained with Cl, theV 1/2 values covered a wide range from –138.5 ± 14.4 mV (n = 5) observed for I to 0.0 ± 19.6 mV (n = 4) determined for F. In contrast, the divalent SO4 2– did not induce any detectable Cnonlin (Fig. 2C).

Figure 4

Functional characteristics of the voltage sensor are determined by the anion species. (A) Relative peak of Cnonlin and Q maxmeasured in inside-out patches from rat OHCs with the anions indicated present at the cytoplasmic side of the patches. Values are means ± SD of three to seven experiments; Cnonlin andQ max values for Cl were used for normalization. (B) Concentration-Q max curves for Cland HCO3 determined in inside-out patches from rat OHCs (30). Lines represent fit of a logistic function to the data (means ± SD of four to eight experiments) with values for EC50 and Hill coefficient of 6.3 mM and 0.89 for Cl and 43.6 and 0.87 for HCO3 , respectively. (C) Cnonlin measured in an inside-out patch from a rat OHC with Cl and the various carboxylic acids present at the cytoplasmic surface of the patch. Form is formate, acet is acetate, prop is propionate, and but is butyrate. (D) Slope (α) of Cnonlin determined from the experiment in (C). Note the decrease in slope with increase in chain length of the carboxylic acid.

Although all halides were able to induce voltage-dependent charge movement, only Cl and bicarbonate (HCO3 ) are thought to be present in the cytoplasm at millimolar concentrations. Such concentrations are indeed necessary as demonstrated in experiments measuring the affinity of prestin for both anions. The concentrations required for half-maximal charge movement were 6.3 mM and 43.6 mM for Cl and HCO3 , respectively (Fig. 4B). Together these findings suggest that Cl and HCO3 work as the voltage sensor of prestin, which is translocated when the membrane potential is changed. This view is further supported by experiments characterizing the Cnonlin induced by carboxylic acids of increasing chain length. Formate, acetate, propionate, and butyrate were all able to induce nonlinear charge movement when applied to inside-out patches (Fig. 4, C and D). However, although the slope of Cnonlininduced by formate was almost identical to that obtained with Cl (α was 37.5 ± 2.1 mV and 34.9 ± 2.4 mV for formate and Cl, respectively; n = 3), it decreased significantly with increasing chain length of the carboxylic acid (α for acetate, propionate, and butyrate was 51.1 ± 2.2 mV, 60.6 ± 4.1 mV, and 71.7 ± 6.9 mV;n = 3; see also inset Fig. 4C). Accordingly, the transmembrane voltage required for moving butyrate through the electrical field is more than twice that necessary for translocating Cl or formate.

These results support a model in which the intracellular anions Cl and HCO3 act as the voltage sensor of prestin (24). We propose that after binding to a site with millimolar affinity, these anions are translocated across the membrane by the transmembrane voltage: toward the extracellular surface upon hyperpolarization, toward the cytoplasmic side in response to depolarization. Subsequently, this translocation triggers conformational changes of the protein that finally change its surface area in the plane of the plasma membrane. The area decreases when the anion is near the cytoplasmic face of the membrane (cell contraction), it increases when the ion has crossed the membrane to the outer surface (cell elongation). As concluded from the lack of effect on exchanging anions on the extracellular side (Fig. 2D), this outer position of the voltage sensor is inaccessible from the extracellular space.

According to this model, any molecule interacting with the anion-binding site and repelling Cl should reduce the structural rearrangements required for cell motility. We thus performed experiments with salicylate, a membrane-permeable inhibitor of electromotility usually applied to OHCs from the extracellular side (25, 26). Salicylate induced a Cnonlin when applied to the cytoplasmic side, indicating interaction with the anion-binding site (Fig. 5A). However, the voltage dependence of salicylate-induced Cnonlin was shallow, similar to that of the large carboxylic acids, and resulted in only small charge movement over the entire physiological voltage range (inset of Fig. 5A). Coapplication of 0.5 mM salicylate with Cl shifted the concentration-charge relation for Cl toward higher concentrations by more than 20-fold, indicating competition between these anions. The affinity of the anion-binding site for salicylate as calculated from this shift is about 300 times that for Cl[K Sal = 21 μM; (27)]. This provides a straightforward explanation for the efficacy of salicylate in blocking OHC electromotility.

Figure 5

Salicylate acts as a competitive antagonist at the anion-binding site. (A) Nonlinear capacitance induced by application of 10 mM salicylate (ionic strength maintained with 47 mM SO4 2−) or 150 mM Cl at an inside-out patch excised from a rat OHC. Holding potential was −50 mV, applications as indicated by horizontal bars. Inset: Cnonlin measured in an inside-out patch from a rat OHC in response to a voltage ramp from −150 mV to 150 mV with 150 mM salicylate present at the cytoplasmic side of the patch. Continuous line represents fit of Eq. 1 to the data yielding values forV 1/2 and α of −65.3 mV and 83.3 mV, respectively. (B) Concentration-Q maxrelations for Cl determined in inside-out patches from rat OHCs in the absence (control) and presence of 0.5 mM salicylate. Lines represent fit of a logistic function to the data (means ± SD of five to eight experiments) with values for EC50 and Hill coefficient of 6.3 mM and 0.89 (control) and 138.4 mM and 0.90 (presence of salicylate).

The present findings offer a molecular framework for understanding the voltage sensitivity and resulting structural changes of the OHC motor protein. Direct experimental access to anion binding and translocation makes prestin a model for investigating functionality of anion-transporting membrane proteins.

  • * To whom correspondence should be addressed. E-mail: bernd.fakler{at}uni-tuebingen.de

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