Cl- flux through a non-selective, stretch-sensitive conductance influences the outer hair cell motor of the guinea-pig

Abstract

Outer hair cells underlie high frequency cochlear amplification in mammals. Fast somatic motility can be driven by voltage-dependent conformational changes in the motor protein, prestin, which resides exclusively within lateral plasma membrane of the cell. Yet, how a voltage-driven motor could contribute to high frequency amplification, despite the low-pass membrane filter of the cell, remains an enigma. The recent identification of prestin's Cl- sensitivity revealed an alternative mechanism in which intracellular Cl- fluctuations near prestin could influence the motor. We report the existence of a stretch-sensitive conductance within the lateral membrane that passes anions and cations and is gated at acoustic rates. The resultant intracellular Cl- oscillations near prestin may drive motor protein transitions, as evidenced by pronounced shifts in prestin's state-probability function along the voltage axis. The sensitivity of prestin's state probability to intracellular Cl- levels betokens a more complicated role for Cl- than a simple extrinsic voltage sensor. Instead, we suggest an allosteric modulation of prestin by Cl- and other anions. Finally, we hypothesize that prestin sensitivity to anion flux through the mechanically activated lateral membrane can provide a driving force that circumvents the membrane's low-pass filter, thus permitting amplification at high acoustic frequencies.

Figure 11. Whole-cell current responses evoked by stiff probe deformations of the lateral membrane

A, digitally captured image of OHC under whole-cell voltage clamp with stiff probe placed across the middle of the cell. B, step mechanical deformation of the lateral membrane evokes a transient gating current and a DC ionic current at the fixed potential of 50 mV. C, relationship of motor gating charge (integrated transient current, Q) and ionic current magnitude (I, difference between steady-state levels before and after membrane stress) with membrane voltage. Motor charge peaks at V_pkcm, whereas I reverses at the potential (−10.5 mV) where total current reverses. D, sinusoidal mechanical stimulation at various potentials produces a corresponding current whose phase reverses at the cell's steady ionic current reversal potential. Extracellular solution was 140 Cl and intracellular was Tris-Hepes + 1 mm Tris-Cl⁻.

Figure 1. Effects of intracellular Cl⁻ on prestin-generated non-linear capacitance in outer hair cells (OHCs)

A, top, C–V functions of an OHC during washout of Cl⁻ with Tris-sulfate patch-pipette solution, in the presence of 140 mm Tris-Cl extracellular solution. Bottom, same as above except with Tris-Hepes pipette solution. B, relative peak non-linear capacitance (C_mpk (rel)) as a function of time after patch rupture with either Tris-sulfate (n = 4; mean ±s.e.m.) or Tris-Hepes (n = 5) Cl⁻-free intracellular solutions and Tris-Cl 140 mm (140 Cl) extracellular solution. Recordings began at the moment of patch rupture at a holding potential of 0 mV. Data were obtained from full C–V functions, and peak non-linear membrane capacitance C_m was obtained by subtracting linear capacitance determined by fits (see Methods) to the data. [Cl]_in, intracellular Cl⁻ concentration.

Figure 2. Boltzmann parameters of motor charge movement determined from capacitance functions during washout of intracellular Cl⁻

Horizontal bar at top represents extracellular Cl⁻ concentrations ([Cl]_out) during whole-cell recording. Pipette contained either Tris-sulfate or Tris-Hepes solutions (n = 5 for each condition; mean ±s.e.m.). The dominant effect of Cl⁻ washout is a shift in V_pkcm, with small changes in Q_max and z. Error bars are within symbol dimensions for most data points. After removal of extracellular Cl⁻, steady-state levels of parameters show changes that depend upon species of the intracellular substitute anion. Recordings began at the moment of patch rupture at a holding potential of 0 mV.

Figure 3. Dependence of peak C_m and V_pkcm on extracellular Cl⁻ concentration

A, continuous line is sigmoidal fit to data points (mean ±s.e.m.; n = 5). Intracellular solution was Tris-sulfate (Table 1) and initial extracellular solution was 140 Cl (Table 2). Extracellular Cl⁻ ([Cl]_out) reductions were made by replacement with Hepes. For comparison, we show relative Q_max (determined from capacitance measures) vs. intracellular Cl⁻ ([Cl]_in) concentration published by Oliver et al. (2001). B, same procedures as above. Line is linear regression (r²= 0.98) with a slope of 0.37 mV mm⁻¹.

Figure 4. OHC non-linear capacitance and mechanical response with sodium pentane sulfonate patch-pipette solutions

In the presence of 140 Cl extracellular solution, and after steady– state intracellular perfusion of Cl⁻-free pentane sulfonate solution, non-linear capacitance remains significant, and mechanical responses during voltage stimulation are prominent. Smooth line is fit to eqn (1) (V_pkcm, −96 mV; z, 0.31; Q_max, 2.1 pC and C_lin, 23.2 pF, respectively. Inset: image of OHC at negative (A) and positive (B) extremes of voltage ramp. Note displacement of cuticular plate.

Figure 5. Correspondence among Cl⁻ current, peak non-linear C_m and V_pkcm

Horizontal bars at top represent extracellular Cl⁻ concentrations ([Cl]_out) during whole-cell recording. Recordings began at the moment of patch rupture with Tris-Hepes Cl⁻-free pipette solution at a holding potential of 0 mV. Despite a large sustained inward Cl⁻ flux (outward current; I_Cl, top), non-linear C_mpk decreases (middle), and V_pkcm shifts to depolarized levels (bottom) during Cl⁻ washout through the patch pipette. Subsequent removal of extracellular Cl⁻ shifts C_mpk and V_pkcm values further, showing their dependence on Cl⁻ influx into the cell. The treatments are reversible and responses are graded with magnitude of Cl⁻ current. ([Cl]_in), intracellular [Cl⁻].

Figure 6. Localization of the Cl⁻ conductance to the lateral membrane

A 1 m Cl⁻ point-diffusion source (high impedance pipette), briefly positioned close to different OHC regions (image insets), revealed a maximum increase in I_Cl and C_m in the mid region of the cell. After each placement, the pipette was moved away from the cell and is indicated as a return of current and capacitance to near baseline.

Figure 7. OHCs possess a voltage-dependent, cation-anion non-selective conductance

All recordings were made with Tris-Hepes intracellular solution (Table 1). A, currents recorded in the same OHC subjected to voltage pre-conditioning step protocol (a,c: holding potential, V_hold=−20 mV, 150 ms conditioning steps from −100 to +60 mV (ΔV = 20 mV), 150 ms test step to +60 mV) and pre-depolarizing step protocol (b,d: V_hold=−20 mV, 150 ms conditioning step to +60 mV, 150 ms test steps from −100 to +60 mV (ΔV = 20 mV)) in Tris-Hepes reference extracellular solution (a, b) or 140 mm TrisCl-based (140 Cl) extracellular solution (c, d). Horizontal lines at left of traces indicate zero-current level. B, series-resistance corrected I–V curves from currents in A, trace averaged between vertical marks. C, OHC currents recorded in (top to bottom) Tris-Hepes, 80 K, 80 Na and 80 Cl extracellular solutions (Table 2) in the presence of 1 mm 4AP + 200 μm linopirdine, evoked by voltage steps from +60 to −120 mV (ΔV=−20 mV) from V_hold=−20 mV. Records are from the same OHC. D, Deiters' cell currents recorded in (top to bottom) Tris-Hepes, 80 Na and 80 Cl extracellular solutions, evoked by the same voltage protocol as in C. Scale is same as in C. E, relative currents for OHC and Deiters' cell from C and D above, normalized by their membrane capacitance (24.7 pF linear capacitance for OHC and 21.8 pF linear capacitance for Deiters' cell).

Figure 8. Cl⁻ channel blockers increase the lateral membrane conductance but block non-linear capacitance

A, currents were evoked by prolonged (5 s) pulses from +20 to −100 mV (step =−20 mV) from V_hold= 0 mV. Data were collected from the same OHC in 80 Na (left) or 80 Cl (right) extracellular solutions (Table 2), in the absence (top) or in the presence (bottom) of 200 μm niflumic acid (NiA). Horizontal lines at left of traces indicate zero-current level. Note the identical kinetics, voltage dependence and increase by NiA for Na⁺ and Cl⁻ currents. B, I–V plots obtained from trace averages (between vertical marks) in A above. C, C–V functions obtained from another OHC after treatments as in A above. Note potent block of niflumic acid of prestin-related non-linear capacitance, despite enhanced inward-outward Cl⁻ currents.

Figure 9. Partial block of the lateral membrane conductance by gadolinium

OHC I–V plots with extracellular solutions 80 Na (A) or 80 Cl (B) without (•) and with (^) the stretch-channel blocker gadolinium (500 μm) obtained in the same cell.

Figure 10. Membrane tension dependence of the Cl⁻ current

A, sigmoidal I–V curves were derived from trace-averaged currents recorded with Cl⁻-free Tris-Hepes patch pipette in the presence of Tris-Hepes (circles) and 80 Cl (triangles) extracellular solutions before (filled symbols) and after (open symbols) turgor pressure increases in the initially cylindrical OHC (V_hold= 0 mV, step from +60 to −100 mV). Currents are substantially increased and pressure sensitive in Cl⁻-containing solution. Inset: C–V functions, corresponding to the treatments in A, reflect a significant increase in membrane displacement charge and are shifted in the hyperpolarizing direction in Cl⁻-containing solution. The additional charge increase and C_m shift in the pressurized state (▿) is due to increased Cl⁻ flux, because in Cl⁻-free solutions (^), as well as in Cl⁻-saturated solutions (Kakehata & Santos-Sacchi, 1995), the C_m functions are shifted to depolarizing potentials with positive pressure. B, non-linear charge (Q_max), determined from C–V function fits (n = 3) does not increase in initially collapsed cells in Cl⁻-containing extracellular solution (140 Cl) until Cl⁻ flux is restored by cell re-inflation.

Figure 12. Whole-cell current responses evoked by fluid-jet stimulation of the lateral membrane

A, traces depict an intensity series (6 dB increments in the voltage that drives the piezoelectric fluid-jet) at 488 Hz. Note AC and DC components. DC components grow with stimulus level indicating that they do not arise from bottoming out of the piezoelectric driver. Zero current level is indicated by horizontal line on left of top trace. Traces are separated vertically for clarity. Tris-Hepes intracellular vs. 80 Cl extracellular. B, fundamental (f₀) and first harmonic (f₁) are present in FFT transform from whole-cell currents generated by OHC in response to 1 kHz fluid-jet mechanical stimulation against the lateral wall of the cell. I_m, membrane current plotted on a decibel scale. C, I_f0 is blocked by collapsing the OHC with negative pipette pressure; re-inflation restores the current. Niflumic acid (250 μm)-treated OHC. D, removal of extracellular Cl⁻, in the absence of intracellular Cl⁻ reduces the fluid-jet evoked current. E, the reduction of non-linear peak capacitance after 250 μm niflumic acid (NiA) treatment (16.6 pF (V_pkcm: −12.7 mV) vs. 8.63 pF (V_pkcm: 20.3 mV) derived from C–V functions) produced a reduction of the capacitive component and shift in the current's voltage dependence to the depolarized levels. Consequently, characteristics of the ionic component are revealed as I_f0 magnitude displays a minimum where an abrupt phase reversal occurs. Tris-Hepes-based intracellular solution (10 mm[Cl⁻]); extracellular solution 80 Cl.

Figure 13. Effects of control anions on OHC non-linear capacitance and current

A, the OHC was perfused with solutions of minimal composition in order to evaluate the effects of control anions. The intracellular solution was maleate-based (mm: 120 maleate, 250 Tris, 2 EGTA). The initial extracellular maleate-based solution (mm: 120 maleate, 250 Tris) was sequentially substituted by Tris-Hepes (mm: 220 Hepes, 60 Tris), Tris-sulfate (mm: 120 SO₄²⁻, 250 Tris) and Tris-Cl (mm: 155 Cl⁻, 165 Tris) solutions. Capacitance measures and corresponding fits (smooth lines) to eqn (1) provide Boltzmann parameters for the tested anions: (maleate:V_pkcm, 91 mV; z, 0.82; Q_max, 0.23 pC; C_lin, 27.96 pF; Hepes: V_pkcm, 60 mV; z, 0.99; Q_max, 0.30 pC; C_lin, 27.89 pF; sulfate: V_pkcm, 75 mV; z, 0.92; Q_max, 0.65 pC; C_lin, 28.28 pF; Cl⁻: V_pkcm, 58 mV; z, 0.91; Q_max, 1.16 pC; C_lin, 28.0 pF. B, corresponding ionic currents generated by downward voltage ramps (+150 to −100 mV, 125 ms; corrected for ramp-induced passive capacitive offset current component – 52 pA for this cell). Note increasing outward (inward anionic) currents associated with the different anions, and the corresponding increases in non-linear capacitance.