An anion antiporter model of prestin, the outer hair cell motor protein

Abstract

Cochlear amplification in mammalian hearing relies on an active mechanical feedback process generated by outer hair cells, driven by a protein, prestin (SLC26A5), in the lateral membrane. We have used kinetic models to understand the mechanism by which prestin might function. We show that the two previous hypotheses of prestin, which assume prestin cannot operate as a transporter, are insufficient to explain previously published data. We propose an alternative model of prestin as an electrogenic anion exchanger, exchanging one Cl(-) ion for one divalent or two monovalent anions. This model can reproduce the key aspects of previous experimental observations. The experimentally observed charge movements are produced by the translocation of one Cl(-) ion combined with intrinsic positively charged residues, while the transport of the counteranion is electroneutral. We tested the model with measurements of the Cl(-) dependence of charge movement, using SO(4)(2-) to replace Cl(-). The data was compatible with the predictions of the model, suggesting that prestin does indeed function as a transporter.

FIGURE 1

Nontransporting models of prestin. (A) An incomplete transport model of prestin. Prestin changes from a contracted to an expanded state when a Cl⁻ ion moves from the first binding site at the mouth of the pore to a second site at the top of the pore. All charge movement is provided by the translocation of the Cl⁻ ion. (B) The reaction scheme used to describe this model. (C) The NLC produced by the model depends on [Cl_i]. In the example shown K_D(Cl_i) = 10 mM, k₂(0) = k₋₂(0) = 10⁴ s⁻¹. There is a negative shift in V_o but no change in C_pk as [Cl_i] is reduced. Note that the traces for 150 mM and 1 M are superimposed. (D) The shift in V_o (ΔV_o) for the example shown in panel C. ΔV_o is always negative when [Cl_i] is reduced and approaches zero as [Cl_i] increases. The dotted line represents Eq. 10 with ΔV_o calculated relative to the V_o for 1 M [Cl_i]₁. (E) An alternative model of prestin, where prestin changes from an expanded to a contracted state when an intrinsic positively charged sensor is moved across the membrane. All charge movement is provided by the translocation of the intrinsic charged sensor. Cl_i binds to a distinct site, altering the rate of charge movement (k₋₂(V)/k₂(V) ≠ k₋₁(V)/k₁(V)). In this and subsequent figures, inside facing surfaces of the schematic molecule is down.

FIGURE 2

A chloride transporting model. (A) The reaction scheme used to describe a chloride transporting model. Cl⁻ binds to a binding site facing the intracellular medium. It is then transported across the membrane where it is released to the extracellular medium, before prestin returns to the unbound inward facing state. All charge movement is provided by the translocation of the Cl⁻ ion. (B) Since hyperpolarization increases the forward rate of the transition E1.Cl↔E2.Cl, we associate this transition with a conformational change of prestin into an expanded state (8). Furthermore, since the occupancy of the outward facing state E4 is increased by removal of Cl_i, and OHCs elongate in low Cl_e (33), the state E4 is provisionally assigned an expanded conformation. All other states are assigned conformations to fit with these constraints. (C) This transporter model reproduces aspects of previous experimental observations. C_pk decreases as [Cl_i] is reduced and there is little effect of removing Cl_e in the presence of high [Cl_i]. However, unlike experimental observations, a negative shift in V_o is produced as [Cl_i] is reduced.

FIGURE 3

A chloride/sulfate antiporter model. (A) The reaction scheme for a exchanger model. Prestin exchanges one Cl⁻ ion for one ion via an alternating-access mechanism, in which prestin can only change between inward and outward facing states with an anion bound. Charge movement is provided by the translocation of a Cl⁻ ion and some intrinsic positively charged residues. Thus net positive charge is moved across the membrane as the Cl⁻ ion is moved toward the extracellular surface. The reorientation of the intrinsic positively charged residues occurs with a ion bound, which neutralizes the positive charge, so the translocation of is voltage-independent. (B,C) Two alternative representations of the reaction scheme. Both assignments ensure that the critical voltage-dependent transition, E1.Cl↔E2.Cl, is associated with a conformational change of prestin into a compact state and symmetry is maintained (8). (D) The exchanger model reproduces most aspects of previous experimental observations. There are large positive shifts in the V_o of the NLC and C_pk decreases as [Cl_i] is reduced. There is little effect of removing Cl_e, in the presence of high [Cl_i]. For [Cl⁻] = 10, 1 or 0 mM, [] was 70, 74.5, or 75 mM, respectively. (E) Normalized strain versus membrane potential. Within the range of potentials usually measured (−100 mV to 100 mV), both representations B and C predict a sigmoidal dependence of length on potential. Depolarization causes shortening associated with an accumulation of compacted states, and hyperpolarization causes lengthening associated with an accumulation of expanded states.

FIGURE 4

The dependence of the NLC on [Cl_i] when Cl_i is replaced with . (A) NLCs were recorded in the whole-cell configuration with a [Cl_i] of 150 mM (n = 7), 10 mM (n = 5), or 1 mM (n = 4). Traces are shown at 54-second intervals, from the first record. The dotted lines show the fits of the derivative of the Boltzmann function (Eq. 1) to the NLC traces. The model predictions are shown in gray. The top panel shows an example of a cell patched with 150 mM [Cl_i]. No change in NLC over time was observed. The prediction of the model is scaled to the C_pk measured at break-in (t = 10 s). The middle panel shows an example of a cell patched with 10 mM [Cl_i]. After break-in there was a positive shift in the V_o of the NLC and C_pk decreased. The model predictions were scaled to the maximum C_pk, which was estimated for the instant of break-in (t = 0). The bottom panel shows the mean time courses of the shift in V_o(ΔV_o) and the relative change in C_pk (RelC_pk) after break-in, for cells patched with 150 mM (○), 10 mM (•), and 1 mM (*) Cl⁻. There was no shift in V_o or any change in C_pk for cells patched with 150 mM [Cl_i]. There was a positive shift in V_o and a decrease in C_pk when cells were patched with 10 mM or 1 mM. Note RelC_pk for 10 mM and 1 mM are superimposed. (B) A comparison of experimental measurements of the dependence of NLC on [Cl_i] with the predictions of the exchanger. The predictions of the model are shown in gray. Fitting a logistic Hill function to the shift in V_o gives a K_1/2 and Hill coefficient for Cl_i of 2.7 ± 0.4 mM and 0.97, respectively. Fitting RelC_pk gives a K_1/2 and Hill coefficient for Cl_i of 30 ± 3 mM and 1.6, respectively. The values for ΔV_o and RelC_pk from those measurements in excised patches (▴) taken from (19,20) and those obtained here from whole-cell recordings (•) are shown for comparison with the predictions of the model. All data is shown as mean ± SD.

FIGURE 5

Residual NLCs remain in the absence of chloride. NLCs were recorded in the whole-cell configuration with either Cl_i completely replaced with (n = 3), or with both Cl_i and Cl_e completely replaced with (n = 13). Where both Cl_i and Cl_e were replaced, cells were dissected and bathed in Cl⁻-free solution to ensure complete removal of Cl⁻ from both internal and external solutions. Traces are shown at 54 s intervals, from the first record. The dotted lines show the fits of the derivative of the Boltzmann function (Eq. 1) to the NLC traces. Model predictions are shown in gray. (A) Example of a cell patched with Cl⁻-free solution, in a high-Cl⁻ bath solution. The model predictions were scaled to the maximum C_pk, which was estimated for the instant of break-in (t = 0). A considerable NLC remained when Cl_i was completely removed. The V_o of the NLC shifted to a more positive potential and C_pk decreased. The shift in the NLC was much smaller than that predicted by the model. (B) Example of a cell patched in Cl⁻-free solution in a Cl⁻-free bath. An NLC was present in all cells bathed in Cl⁻-free solution at break-in. After break-in the V_o of the NLC shifted to a more positive potential, such that it was beyond the range of V measured. Attempts to fit the visible part of the trace were uncertain.

FIGURE 6

Predicted I(V)s for the chloride/sulfate exchanger model. No steady-state current is produced when [Cl_i] = [Cl_e] = 150 mM. Removal of Cl_i leads to a positive shift in the reversal potential and an increase in inward current at 0 mV. Removal of Cl_e leads to a negative shift in the reversal potential and an increase in outward current at 0 mV. 75 mM was used to replace Cl⁻ completely.