Diflunisal inhibits prestin by chloride-dependent mechanism

Abstract

The motor protein prestin is a member of the SLC26 family of anion antiporters and is essential to the electromotility of cochlear outer hair cells and for hearing. The only direct inhibitor of electromotility and the associated charge transfer is salicylate, possibly through direct interaction with an anion-binding site on prestin. In a screen to identify other inhibitors of prestin activity, we explored the effect of the non-steroid anti-inflammatory drug diflunisal, which is a derivative of salicylate. We recorded prestin activity by whole-cell patch clamping HEK cells transiently expressing prestin and mouse outer hair cells. We monitored the impact of diflunisal on the prestin-dependent non-linear capacitance and electromotility. We found that diflunisal triggers two prestin-associated effects: a chloride independent increase in the surface area and the specific capacitance of the membrane, and a chloride dependent inhibition of the charge transfer and the electromotility in outer hair cells. We conclude that diflunisal affects the cell membrane organization and inhibits prestin-associated charge transfer and electromotility at physiological chloride concentrations. The inhibitory effects on hair cell function are noteworthy given the proposed use of diflunisal to treat neurodegenerative diseases.

Fig 1. Effect of diflunisal on the NLC in HEKs expressing prestin.

(A) Representative non-linear capacitance curves recorded on the same HEK cell expressing prestin, with (○) or without (●) DFL. (B). Representative effect of DFL on the capacitance vs. voltage curve in the absence of prestin in HEKs. The membrane capacitance C_M of non-transfected HEKs was monitored at voltages from– 160 mV to +160 mV in the absence and presence of DFL. (C) Effect of DFL on the membrane capacitance in the presence (C_lin) and absence (C_M) of prestin. Both Clin and C_M were obtained from fits to the capacitance vs. voltage curves, using Eq 1 or a linear curve respectively. N = 4 for each condition. The error bars show the standard deviation. (D) Fold increase for each parameter obtained from fitting the NLCs to Eq 1; n≥3 cells for each condition. The error bar shows the standard deviation. The t-test is conducted between the values obtained upon perfusion of ECB with DFL versus ECB alone: * p<0.01; ** p<0.001.

Fig 2. Effect of DFL on OHCs.

The curves in A, C, E and in B, D F respectively are recorded on the same cells and in the same conditions, during hyperpolarizing voltage ramps. (A) & (B) Representative NLCs recorded on 2 different OHCs respectively with 140 mM and 5 mM chloride intracellularly. Increasing concentrations of DFL are perfused in the chamber, from 10⁻⁴ mM to 1 mM. The fits of the two-state C_SA model equation to the NLCs are shown as solid lines. In the absence of drug, the average parameters are: C_lin = 5.70±0.67 pF; C_max = 9.54±1.01 pF; Q_max = 0.47±0.07 pC; V_1/2 = -42.4±12.9 mV; z = 0.84±0.4. (C) & (D) Cell length recorded concomitantly in μm. The solid lines are the resulting fits of the data to the two-state CSA model equation (Eq 2). In the absence of drug, the average characteristics of eM are L₀ = 18±2.4 μm; L_max = 0.60±0.15 μm; V_1/2 = -38.90±15.31 mV; α = -31.55±3.99 V^-1. (E) & (F) Charge transfer against voltage, obtained from Q = Σ(ΔV.C). (G) The voltage sensitivity (α = ze/kT) for charge transfer and eM is determined at peak voltage and plotted against the DFL concentration. Each data point represents the average value from recordings on 3 cells or more, and the error bars are the standard deviation.

Fig 3. NLC and eM in the presence of DFL and in different chloride conditions.

(A) V_1/2 value obtained with OHCs from two-state Boltzmann fits of the NLC (○ at low & ● at high intracellular chloride) and the eM (◻ at low & $\mathbf{■}$ at high intracellular chloride), plotted against DFL concentration. (B) The difference V1/2(NLC)—V_1/2(eM) shows decoupling between charge transfer and electromotility as [DFL] increases. The t-test is conducted between the values obtained without DFL versus with DFL * p<0.01; ** p<0.001. (C) Inhibition of the NLC and (D) of the eM by DFL. The dotted line is the resulting fit of the data by a Hill equation (140mM Cl: n_H = -0.66, IC₅₀ = 331 μM; 5mM Cl^-: n_H = -1.12, IC₅₀ = 98 μM), The resulting eM is only inhibited at concentrations above 0.5 mM in low chloride conditions. The value for eM at 1mM DFL at low chloride concentration was obtained from direct measurement of cell length at hyperpolarized and depolarized voltages (star). For all plots, the data points represent the average and the error bar is the standard deviation for n≥3 cells per condition.

Fig 4. DFL affects the capacitance and the length of the OHC.

(A) The effect of DFL on absolute C_lin and C_max values and (B) relative C_lin (compiled on a cell to cell basis) are compared to (C) the change in total cell length, plotted as the ratio of OHC length with DFL to without. In order to have reliable data at high DFL concentrations, we used the fully extended length to calculate this ratio. (D) Specific capacitance C_lin/A (pF/μm²) is calculated for each OHC and showed in 3 distribution histograms. The diameter and length of each cell was measured and the resulting area was determined. Low DFL: [DFL]≤10⁻² mM & High DFL: [DFL]>10⁻² mM. The averages obtained from the probability distribution fitting of the raw data (grey line) are 9.45±1.9, 9.68±1.7 and 10.97±2.1 fF/μm². (E) The surface area specific capacitance C_SA as well as (F) the difference between C_SA and C_lin (ΔC_SA) are obtained from fitting the NLCs. ΔC_SA is normalized by dividing by the number of active motors N in the absence of drug on a cell to cell basis. Each value is averaged from 3 independent recordings or more, and the error bars represent the standard deviation. (t-test: * p<0.01; ** p<0.001).