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, 26 (15), 3992-8

Control of Mammalian Cochlear Amplification by Chloride Anions

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Control of Mammalian Cochlear Amplification by Chloride Anions

Joseph Santos-Sacchi et al. J Neurosci.

Abstract

Chloride ions have been hypothesized to interact with the membrane outer hair cell (OHC) motor protein, prestin on its intracellular domain to confer voltage sensitivity (Oliver et al., 2001). Thus, we hypothesized previously that transmembrane chloride movements via the lateral membrane conductance of the cell, GmetL, could serve to underlie cochlear amplification in the mammal. Here, we report on experimental manipulations of chloride-dependent OHC motor activity in vitro and in vivo. In vitro, we focused on the signature electrical characteristic of the motor, the nonlinear capacitance of the cell. Using the well known ototoxicant, salicylate, which competes with the putative anion binding or interaction site of prestin to assess level-dependent interactions of chloride with prestin, we determined that the resting level of chloride in OHCs is near or below 10 mm, whereas perilymphatic levels are known to be approximately 140 mm. With this observation, we sought to determine the effects of perilymphatic chloride level manipulations of basilar membrane amplification in the living guinea pig. By either direct basolateral perfusion of the OHC with altered chloride content perilymphatic solutions or by the use of tributyltin, a chloride ionophore, we found alterations in OHC electromechanical activity and cochlear amplification, which are fully reversible. Because these anionic manipulations do not impact on the cation selective stereociliary process or the endolymphatic potential, our data lend additional support to the argument that prestin activity dominates the process of mammalian cochlear amplification.

Figures

Figure 1.
Figure 1.
TBT rescues the reduction of salicylate block in NLC under whole-cell voltage clamp. Only in the presence of a chloride gradient can TBT counter this reduction. A, B, Effects of graded concentrations of salicylate in the presence of a chloride chemical gradient (A) and in the absence of a gradient (B). Note also the shifts in operating voltages (Vpkcm) that accompany changes in intracellular chloride, an increase shifting NLC to hyperpolarizing voltages. Such shifts directly alter the gain of OHC electromotility at the normal resting potential of the cell (Kakehata and Santos-Sacchi, 1996).
Figure 2.
Figure 2.
Average dose–response curves (mean ± SE; n = 4–5) for extracellularly applied salicylate block of NLC. Each curve was collected at a different intracellular concentration of chloride. Chloride was unequivocally controlled by matching intracellular and extracellular levels, as we have done previously (Song et al., 2005). Note the leftward shift as intracellular chloride is reduced, indicating a more effective action of salicylate on the motor. Logistic fits provided the following slope and IC50Sal values (1, 5, 10, 20, and 140 mm chloride, respectively): 0.79, 29.5 μm; 0.83, 77.9 μm; 0.81, 124.0 μm; 0.86, 165 μm; 0.86, 964 μm. There is no significant difference between slopes at any concentration.
Figure 3.
Figure 3.
Estimates of OHC intracellular chloride levels using the gramicidin patch method. A, IC50Sal was determined in the presence of 140 mm extracellular chloride to mimic the in vivo condition. It should be stressed that IC50Sal measures only evaluate the interactions between intracellular salicylate and chloride on the motor and are not directly influenced by extracellular chloride levels; thus, the IC50Sal values determined above in Figure 2A serve as valid calibrations to estimate intracellular chloride levels regardless of extracellular chloride levels. The plot shows that as the length of time after an animal's death (AD) increased, intracellular chloride increased. For the cases in which quick measurements were made, IC50Sal was close to that of the value obtained with 10 mm intracellular chloride, indicating that OHC chloride levels are near or possibly <10 mm for cells under in vivo-like conditions. B, OHC diameter is a sensitive indicator of cell swelling and shows an increase as time AD increased, corresponding with the simultaneous estimates of intracellular chloride above. OHC images show one cell recorded at 30 min and another at 210 min, the latter showing pronounced swelling. The chloride loading after death probably indicates that our measures provide overestimates of intracellular chloride levels. Each symbol in the plots denotes an analysis from an individual cell from different animals (n = 8).
Figure 4.
Figure 4.
A, Schematic of in vivo preparation. See Materials and Methods for details. B, BM responses to acoustic stimulation during perfusion with artificial PL. The temporal order of listed perfusions is from top to bottom. This was a sensitive cochlea where CAP thresholds were little affected by surgery (<10 dB). In this case, a switch from normal PL to TBT-containing solution causes a decrease in BM motion. Salicylate produces a more pronounced reduction, and recovery follows reperfusion with TBT PL. C, This example shows a preparation that was less sensitive; thresholds deteriorated at >20 dB. TBT perfusion, in this case, augmented BM vibration and antagonized the detuning effects of salicylate. D, Perfusion of low chloride PL caused a profound loss of cochlear amplification, which was completely reversible. Again, in this animal, TBT increased BM vibration in the presence of normal chloride levels but was ineffective in the presence of low chloride levels, in which a chloride gradient across the OHC membrane would be small or absent. The small difference in the effects of low Cl treatment between the first perfusion and the fourth perfusion may stem from previous TBT treatment or the time between data collections. For visual clarity, sound pressure is plotted on a linear scale; frequency is plotted logarithmically.

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