The chloride-channel blocker 9-anthracenecarboxylic acid reduces the nonlinear capacitance of prestin-associated charge movement

Abstract

The basis of the extraordinary sensitivity and frequency selectivity of the cochlea is a chloride-sensitive protein called prestin which can produce an electromechanical response and which resides in the basolateral plasma membrane of outer hair cells (OHCs). The compound 9-anthracenecarboxylic acid (9-AC), an inhibitor of chloride channels, has been found to reduce the electromechanical response of the cochlea and the OHC mechanical impedance. To elucidate these 9-AC effects, the functional electromechanical status of prestin was assayed by measuring the nonlinear capacitance of OHCs from the guinea-pig cochlea and of prestin-transfected human embryonic kidney 293 (HEK 293) cells. Extracellular application of 9-AC caused reversible, dose-dependent and chloride-sensitive reduction in OHC nonlinear charge transfer, Qmax . Prestin-transfected cells also showed reversible reduction in Qmax . For OHCs, intracellular 9-AC application as well as reduced intracellular pH had no detectable effect on the reduction in Qmax by extracellularly applied 9-AC. In the prestin-transfected cells, cytosolic application of 9-AC approximately halved the blocking efficacy of extracellularly applied 9-AC. OHC inside-out patches presented the whole-cell blocking characteristics. Disruption of the cytoskeleton by preventing actin polymerization with latrunculin A or by decoupling of spectrin from actin with diamide did not affect the 9-AC-evoked reduction in Qmax . We conclude that 9-AC acts on the electromechanical transducer principally by interaction with prestin rather than acting via the cytoskeleton, chloride channels or pH. The 9-AC block presents characteristics in common with salicylate, but is almost an order of magnitude faster. 9-AC provides a new tool for elucidating the molecular dynamics of prestin function.

Keywords: auditory system; channelopathies; cochlea; electromechanical transduction; sensory hair cells.

Figure 1

9‐AC reversibly reduces the NLC of OHCs. (A) Capacitance of a control OHC (filled circles), voltage‐clamped with intracellular solution containing 135 mm CsCl. Extracellular application of 500 μm 9‐AC (open circles) reduces the NLC. The block is reversible (closed triangles). Lines show the results of the fits using Eqn (1); fit parameters are given in Table 1. (B) Time course of the 9‐AC block of NLC measured at −20 mV. Lines are single exponential fits with time constants of 1.7 ± 0.1 s and 3.4 ± 0.2 s for the on‐ and off‐drug phases, respectively. E104112011, acronym identifying the cell; the same convention is used in the following figures.

Figure 2

9‐AC block requires negative charge and appears independent of the integrity of the cytoskeleton. (A–C) Capacitances of three voltage‐clamped OHCs. Labelling conventions are as in Fig. 1A. (A) Extracellular application of 500 μm 9‐AM, an electrically neutral analog of 9‐AC. There is no detectable effect on cell capacitance, meaning that the negative charge of 9‐AC is mandatory for 9‐AC block. (B) Application of 500 μm 9‐AC to an OHC treated extracellularly with diamide. NLC is reversibly reduced; fit parameters are given in Table 3. (C) Application of 500 μm 9‐AC to an OHC treated intracellularly and extracellularly with latrunculin A. NLC is reversibly reduced; fit parameters are given in Table 4. (D) The relative reduction in Q _max in response to extracellular application of 500 μm 9‐AC, DMSO or 500 μm 9‐AM. Labelling on the abscissa has the following meaning. Control, extracellular application of 500 μm 9‐AC to control OHCs (n = 11). DMSO, extracellular application of DMSO to control OHCs (n = 6). 9‐AM, extracellular application of 500 μm 9‐AM to control OHCs (n = 6). Diam, diamide‐treated OHCs (n = 5) with extracellular application of 500 μm 9‐AC. Latr, latrunculin‐treated OHCs (n = 7) with extracellular application of 500 μm 9‐AC. There is no significant difference for relative Q _max reductions for cells treated with diamide or latrunculin A compared with untreated cells, indicating that the cytoskeleton is not a primary target of 9‐AC. Relative reductions for DMSO and 9‐AM applications are not significantly different from zero and there was also no effect on the other capacitance‐defining parameters; that is, neither DMSO nor 9‐AM had a detectable effect on NLC.

Figure 3

9‐AC reversibly reduces the NLC of prestin‐transfected HEK 293 cells. (A) Prestin‐transfected HEK 293 cell possessing the typical dotted fluorescence in the plasma membrane. Cells were voltage‐clamped using 135 mm CsCl intracellular solution (upper left/DIC image). GFP labelling was recorded with confocal microscopy using an argon laser with excitation wavelength of 488 nm. The emitted light was detected above 505 nm. Numbers in the bottom right corners of the fluorescence images indicate the level of the confocal section in microns. (B) Capacitance of the prestin‐transfected HEK 293 cell in A. Extracellular application of 500 μm 9‐AC reversibly reduces the NLC. Lines indicate fits using Eqn (1) with parameters given in Table 5. (C) I‐V characteristics of a non‐transfected HEK 293 cell, indicating that 9‐AC does not influence voltage‐gated chloride conductances in this cell line. Scale bar, 10 μm.

Figure 4

The 9‐AC block is chloride‐sensitive and 9‐AC can access from both sides of the plasma membrane. (A) Dose dependency of the extracellularly applied 9‐AC on the maximum amount of motor charge moved, Q _max, measured with extracellular 9‐AC concentrations of 0.05, 0.5, 5, 50, 500 and 5000 μm. The effect on Q _max is presented as relative reduction. Circles labelled ‘CsCl’, the control condition with intracellular solution containing 135 mm CsCl. Triangles labelled ‘Cs₂ SO ₄’, intracellular chloride concentration reduced by substituting 115 mm chloride with sulfate in the patch pipette. Squares labelled ‘redCl’, both intracellular and extracellular chloride concentrations reduced by substituting 130 mm CsCl with gluconate in both the patch‐pipette and bath solutions. This latter condition significantly shifted the dose dependency to the left. Lines indicate logistic‐function fits (Eqn (3)) with estimates of the IC ₅₀ and the slope parameter, respectively, of 928 ± 66, 1012 ± 192, 79 ± 14 μm and 0.82 ± 0.04, 0.56 ± 0.06, 0.71 ± 0.08 for the three conditions. (B) The relative reduction in Q _max in response to extracellular application of 500 μm 9‐AC, using the indicated intracellular solutions, patch configuration, drugs and cell types. Labelling on the abscissae has the following meaning. ‘CsCl’ (n = 11 for OHCs, n = 11 for HEK 293 cells), ‘Cs₂ SO ₄’ (n = 5), and ‘redCl’ (n = 4), as in panel A. ‘9‐AC _i’, 500 μm 9‐AC in the patch solution (n = 5 for OHCs, n = 5 for HEK 293 cells). ‘InOut’, inside‐out excised‐patch configuration (n = 3); all other measurements were made in the whole‐cell configuration. Data for the bar labelled ‘OHC/CsCl’ are the same as those for the bar labelled ‘Control’ in Fig. 2D. Notice for OHCs, only intracellular and extracellular chloride substitution with gluconate (redCl; n = 4) increased the relative reduction in Q _max compared with CsCl control. That is, the blocking efficacy of extracellular 9‐AC was increased by lowering the OHC [Cl⁻] gradient by reducing the intra‐ and extracellular [Cl⁻]. Notice for HEK 293 cells, the addition of 9‐AC to the cytosol (9‐AC _i; n = 5) decreased the blocking efficacy of extracellularly applied 9‐AC.

Figure 5

Extracellular 9‐AC reduces intracellular pH. (A) Fluorescence monitoring of the time course of intracellular pH, using the fluorescence dye BCECF, in response to extracellular 500 μm 9‐AC. Numbers in the bottom right corners of the fluorescence images indicate time in seconds. The region of interest indicated by the red rectangle in the middle of the isolated OHC was chosen to calculate fluorescence intensity values, F, normalized to the averaged initial value F₀ recorded at the beginning of the experiment. (B) Time course of the relative fluorescence intensity for the cell in A. Including DMSO in the perfusion fluid resulted in a larger and faster fluorescence signal (right panel) than when 9‐AC alone was present (left panel). Red lines indicate single exponential fits with time constants of 6.7 ± 1.1 s and 1.5 ± 0.3 s, respectively, in the absence and presence of DMSO. Scale bar, 10 μm.