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Action of 2,3-butanedione monoxime on capacitance and electromotility of guinea-pig cochlear outer hair cells

Gregory I. Frolenkov, Fabio Mammano * and Bechara Kachar

	ABSTRACT

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1. Whole-cell patch-clamp recordings were obtained from isolated cochlear outer hair cells (OHCs) while applying 2,3-butanedione monoxime (BDM) by pressure. BDM (5 mM) shifted the range of voltage sensitivity of membrane capacitance and cell length in the hyperpolarised direction by -49.6 ± 4.0 mV (n = 12; mean ± S.E.M.), without appreciable effects on membrane conductance. The shift was completely reversible and dose dependent, with a Hill coefficient of 1.8 ± 0.4 and a half-maximal dose of 3.0 ± 0.8 mM (values ± S.D).

2. The shift of the capacitance curve was also reproducible in cells whose natural turgor had been removed. BDM had no detectable effect on the capacitance of Deiters' cells, a non-sensory cell type of the organ of Corti.

3. The effect of BDM on membrane capacitance was faster than that of salicylate. At similar saturating concentrations (20 mM), the time constant of the capacitance changes was 1.8 ± 0.3 s (n = 3) for salicylate and 0.75 ± 0.06 s (n = 3) for BDM. The recovery periods were 13 ± 1 s and 1.7 ± 0.4 s, respectively (means ± S.E.M.).

4. The effect of BDM, a known inorganic phosphatase, was compared to the effects of okadaic acid, trifluoperazine and W-7, which are commonly used in studies of protein phosphorylation. Incubation of OHCs with okadaic acid (1 M, 30-60 min) shifted the voltage sensitivity of the membrane capacitance in the hyperpolarised direction. Incubation with trifluoperazine (30 M) and W-7 (150 M) shifted it in the opposite, depolarised direction. BDM induced hyperpolarising shifts even in the presence of W-7.

5. Simultaneous measurement of membrane capacitance and intracellular free Ca²⁺ concentration ([Ca²⁺]_i) showed that BDM action on OHC voltage-dependent capacitance and electromotility is not mediated by changes of [Ca²⁺]_i.

6. Our results suggest that: (a) the effects of BDM are unrelated to its inorganic phosphatase properties, cell turgor conditions or Ca²⁺ release from intracellular stores; and (b) BDM may target directly the voltage sensor of the OHC membrane motor protein.

	INTRODUCTION

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The membrane capacitance of OHCs is a function of transmembrane voltage (Ashmore, 1989). Although the detailed molecular mechanism remains to be established, this is thought to depend on aggregates of 'motor' proteins residing in the plasma membrane (reviewed in Frolenkov et al. 1998). Membrane motors have been postulated to possess a voltage sensor that generates fast asymmetric current transients at the onset and offset of voltage steps applied across the plasma membrane (Gale & Ashmore, 1997). The charge displacement associated with operation of the motors' voltage sensor imparts a characteristic bell-shaped dependence of membrane capacitance on transmembrane potential (Santos-Sacchi, 1991). Similar properties are displayed by gating charges in voltage-dependent ion channels (Armstrong, 1992), but the faster rate of charge translocation in OHCs suggests that the membrane motors are not modified ion channels (Géléoc et al. 1999). Rather than controlling ion flow, the voltage-driven conformation changes of the membrane motor proteins are thought to apply a stress to the plasma membrane changing the cell resting length, a phenomenon known as electromotility. Surface forces associated with local area changes in the plasma membrane (Kalinec et al. 1992) are presumably transferred to the underlying cortical cytoskeleton that contributes to the orientation of force output along the longitudinal axis of the cell (Holley et al. 1992; Kalinec et al. 1992; Tolomeo et al. 1996).

Only a few compounds, namely salicylate (Dieler et al. 1991; Tunstall et al. 1995), lanthanides (Santos-Sacchi, 1991; Kakehata & Santos-Sacchi, 1996) and sulfhydryl reagents (Kalinec & Kachar, 1993; Frolenkov et al. 1997) are presently known to block electromotility. Salicylate and lanthanides were additionally shown to eliminate the voltage-dependent fraction of the membrane capacitance (Santos-Sacchi, 1991; Tunstall et al. 1995; Kakehata & Santos-Sacchi, 1996). The operating range of the voltage-dependent capacitance is affected by a number of chemical reagents (Wu & Santos-Sacchi, 1998), none of which is likely to target selectively the putative membrane motors of the OHC.

In this paper we report the effects of an inorganic phosphatase, 2,3-butanedione monoxime (BDM), on OHC electromotility. BDM is capable of dephosphorylating acetylcholinesterases (Wilson & Ginsburg, 1955), as well as various smooth muscle proteins (Waurick et al. 1999). It also interferes with acto-myosin function through the myosin adenosine triphosphatase (ATPase) reaction shifting the equilibrium between two acto-myosin states towards the more weakly (pre-stroke) bound form (McKillop et al. 1994). In addition to its effect on the contractile apparatus, BDM promotes Ca²⁺ release from ryanodine-operated intracellular stores (Adams et al. 1998) and modulates ion (Lee et al. 1995; Ye & McArdle, 1995) and gating currents (Ferreira et al. 1997). We found that BDM shifts the operating range of the OHC voltage-dependent capacitance in an extremely rapid and reversible manner, more than any other chemical or physical manipulation reported so far.

	METHODS

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Cell preparation

Adult guinea-pigs (200-400 g) were killed by exposure to a rising concentration of carbon dioxide and decapitated, according to NIH guidelines for animal use. The temporal bones were removed from the skull and placed in a modified Leibowitz cell culture medium (L-15) containing (mM): NaCl (137), KCl (5.4), CaCl₂ (1.3), MgCl₂ (1.0), Na₂HPO₄ (1.0), KH₂PO₄ (0.44), MgSO₄ (0.81). The osmolarity was adjusted to 325 ± 2 mosmol l^-1 with D-glucose and the pH was adjusted to 7.35 with NaOH. To isolate OHCs, the bulla was opened to expose the cochlea and the otic capsule was chipped away with a surgical blade starting from the base. Strips of the organ of Corti were dissected from the modiolus with a fine needle, transferred with a glass pipette to a 100 l drop of medium containing 1 mg ml^-1 of collagenase type IV (Life Technologies, Rockville, MD, USA), and kept there for 15-20 min. In some experiments, the strips were pre-incubated (30-60 min at 37°C) with drugs affecting protein phosphorylation: okadaic acid, trifluoperazine, and W-7 (Calbiochem, San Diego, CA, USA). As controls for these experiments, cells were maintained in standard medium for the same amount of time. After incubation, cells were dissociated by gentle reflux of the tissue through the needle of a Hamilton syringe (N. 705, 22 gauge) and allowed to settle on the slide for 5-10 min. OHCs were placed in a laminar flow bath (100 l), in which solutions could be changed (about 5 ml h^-1) by means of a pressurised perfusion system (BPS-4; ALA Scientific Instruments, Westbury, NY, USA), and maintained at room temperature (22-24°C) throughout the experiments.

Patch-clamp recordings

OHCs were visualised under the microscope and the following morphological features were used to determine viability: uniform cylindrical shape, basal location of the nucleus, membrane birefringence and intact stereocilia. In most experiments, patch-clamp pipettes were filled with a CsCl-based intracellular solution containing (mM): CsCl (140), MgCl₂ (2.0), EGTA (5.0), Hepes (5), adjusted to pH 7.2 with CsOH and brought to 325 mosmol l^-1 with D-glucose. In some experiments involving the pressure application of BDM and sodium salicylate, the intracellular solution contained (mM): KCl (144), MgCl₂ (2.0), EGTA (0.5), Na₂HPO₄ (8.0), NaH₂PO₄ (2.0), Mg-ATP (2.0), Na-GTP (0.2), adjusted to pH 7.2 with KOH and brought to 325 mosmol l^-1 with D-glucose. For Ca²⁺ imaging experiments, this solution was supplemented with 0.1 mM Oregon Green 488 BAPTA-1 (Molecular Probes).

Patch-clamp recordings were performed using an Axopatch-1D amplifier (Axon Instruments). Pipettes for conventional whole-cell recordings were formed on a programmable puller (P87, Sutter Instruments) from 1.0 mm o.d. borosilicate glass (no. 30-30-0, FHC, Bowdoinham, ME, USA). Current and voltage were sampled at 100 kHz using a standard laboratory interface (Digidata 1200A, Axon Instruments) controlled by pCLAMP 7.0 software (Axon Instruments). The uncompensated pipette resistance was typically 3-5 M when measured in the bath and the access resistance did not exceed 15 M under whole-cell patch-clamp conditions. Potentials were corrected off-line for the error due to the access resistance. Junction potentials were -4.2 mV for the KCl-based solution and -4.9 mV for the CsCl-based solution, as computed by the pCLAMP 7.0 software using the given solution composition. These values were very similar and rather small, and therefore no correction was applied to the data for liquid junction potentials.

Drug delivery

A puff pipette, prepared similarly to the patch pipette, was filled with BDM, sodium salicylate or ionomycin (Sigma), dissolved in the extracellular solution. It was placed near the basolateral wall of the OHC and pressure (10-15 kPa) was applied to its back by a pneumatic injection system (PLI-100, Medical Systems Corp., Greenvale, NY, USA) gated under software control. Typical drug delivery time was 30 ms, as determined by positioning the patch pipette in front of the puff pipette and monitoring the change in junction potential.

Capacitance measurement

Measurements of membrane capacitance were derived from those of asymmetric currents evoked by pre-stepping the cell potential to large hyperpolarised values (V_pre), around -160 mV, for about 1 ms from a holding potential (V_h) of -60 mV, followed by depolarising steps of variable amplitude and 2-3 ms duration. The potential was then returned to V_pre for 2-3 ms before resetting it to V_h, preparing the cell for the next step (Fig. 1, right inset). Charge movement Q was estimated by time integration of the asymmetric currents at the step offset, when the cell was temporarily returned to V_pre, i.e. under constant driving force conditions. As the time constant of the patch-clamp recording was in the range 0.1-0.3 ms, more than 99 % of the current had settled within 2 ms. Leakage currents were estimated and subtracted off-line by assuming that asymmetric currents had completely decayed at the end of the eliciting pulse. This procedure was found to introduce less noise than the standard P/4 technique (Armstrong & Bezanilla, 1977). In most cases, ionic currents were not activated appreciably during the brief voltage commands applied.

Alternatively, measurements of membrane capacitance were performed using the 'membrane test' feature of the pCLAMP 7.0 acquisition software, which continuously delivered a test square wave of period T = 4 ms to the cell, through the patch-clamp amplifier. This produced transient currents that decayed exponentially with a (voltage-dependent) time constant . The software was designed for the simultaneous on-line measurement of , the total resistance, R_t, recorded by the amplifier, and the electrical charge delivered to the membrane (membrane capacitance), C_m. Unfortunately the pCLAMP software accurately estimates parameters R_m (cell membrane resistance), R_a (pipette access resistance) and C_m only if R_m >> R_a, a condition which was not always met. To circumvent this problem, we reversed the pCLAMP algorithm off-line to recover the original values for the time integral of the transient current, Q and R_t. We then re-computed R_m, R_a and C_m according to the equations shown below. The voltage step V elicited a whole-cell current:

(1)

The charge delivered to the equivalent circuit by the transient current:

(2)

and the total resistance is:

Rt = Rm + Ra.

(3)

Solving simultaneously eqns (1), (2) and (3) yields:

(4)

The patch parameters were continuously monitored, at a resolution of 25 Hz, by averaging the responses to 10 positive and 10 negative consecutive test steps. The series resistance and linear capacitance compensation circuitry of the patch-clamp amplifier was not used. Instead, to obtain the voltage dependence of C_m we applied triangular voltage ramps, swinging the cell potential from V_h = -100 mV to V_h = +160 mV in 6 s (Fig. 1, left inset). Before the C_m calculation, the values of total resistance R_t,0, estimated by pCLAMP 7.0, were corrected for the slope of the ramp as follows:

Rt = V/ [(V/Rt,0) - I ]

(5)

where I is the increment of the whole-cell current produced by voltage ramp in T/2 = 2 ms.

To test the accuracy of the C_m determination, we performed measurements of C_m on a model electronic circuit in which we varied R_m from 500 to 5 M keeping R_a = 10 M constant and C_m equal to one of three values: 10, 20 or 30 pF. The values of C_m, calculated according to the above procedure, differed from their nominal values by no more than 2 pF, provided that R_m/R_t > 0.6. Under these conditions, the estimate of C_m did not vary significantly when the voltage was commanded to follow a ramp. Large errors in the C_m estimate occurred at R_m/R_t < 0.6 because the amplitude of the exponentially decaying transient current was less than the steady-state current response to the test step. The pCLAMP algorithm was then unable to 'lock' the exponential decay and to calculate its time constant. Therefore, all data points obtained when R_m/R_t > 0.6 have been excluded from the analysis. Measurements of the membrane capacitance during test ramps were fitted with:

(6)

which is the derivative of a Boltzmann function. C₀ is the linear (voltage-independent) capacitance, C_non-lin is the non-linear (voltage-dependent) capacitance, C_max is the maximum voltage-dependent capacitance, V_p is the potential at the peak of C_m(V) or mid-point potential and W = k_BT/ze is a constant that is a measure of the sensitivity of the non-linear charge displacement to potential. W is expressed in terms of a charge of valency z moving from the inner to the outer aspect of the plasma membrane. k_B is Boltzmann's constant, T is absolute temperature and e is the electron's charge.

The voltage-independent fraction of the membrane capacitance scales linearly with the overall surface area of the cell, whereas its voltage-dependent fraction is proportional to the area of the lateral membrane surface, where the putative motor elements are located (Huang & Santos-Sacchi, 1993). Therefore, in order to compare the data obtained from different cells, the voltage-dependent capacitance was divided by the area of the lateral plasma membrane as follows:

m(V) = (Cm(V) - C0)/[(C0 - Cap - Cbas)/lb],

(7)

where _m(V) is the specific voltage-dependent capacitance of the lateral plasma membrane (in F cm^-2). C_ap (4.38 pF) and C_bas (1.85 pF) are the capacitances of the apical and basal parts of OHC, devoid of motor proteins (Huang & Santos-Sacchi, 1993). Therefore the difference formula C₀ - C_ap - C_bas gives the linear voltage-independent capacitance of the lateral plasma membrane. _lb (1 F cm^-2) is the specific capacitance of the lipid bilayer.

The two methods of capacitance measurement described above produced similar results (Fig. 1). In general, however, the capacitance curve obtained by the step method was shifted in the depolarised direction. This is a consequence of pre-stepping the holding potential to highly hyperpolarised values. Pre-pulse delivery is known to affect the voltage at peak capacitance: depolarisation shifts V_p in the hyperpolarising direction, and hyperpolarisation does the opposite (Santos-Sacchi et al. 1998). All experiments using drug application by pressure were performed using the more accurate membrane test method. However, to compute the statistical results shown in Fig. 3B the faster step method was adopted. Data obtained with different methods were never combined.

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Figure 1 Measurements of voltage-dependent capacitance of isolated outer hair cell , capacitance values derived by the membrane test method during the application of a ramp protocol (left inset); , values derived by applying the step protocol (right inset) to the same cell. Data were fitted to the derivative of a Boltzmann function (continuous and dashed lines, respectively). Parameters of the fits: Cmax = 18.7 ± 0.2 pF, Vp = -45.8 ± 0.3 mV, W = 32.2 ± 0.3 mV (membrane test method); Cmax = 18.2 ± 0.3 pF, Vp = -33.7 ± 0.6 mV, W = 31.5 ± 0.5 mV (step protocol). The patch pipette was filled with the CsCl-based intracellular solution (see Methods).

Motility measurements

Motility measurements were performed as described in Frolenkov et al. (1997). Briefly, OHC movements were recorded with a video camera interfacing with an inverted microscope equipped with differential interference contrast optics to an optical disk recorder (Panasonic TQ-3031F). Digitised images were analysed off-line with the image-processing system Image 1 (Universal Imaging, West Chester, PA, USA). For movement quantification, a measuring rectangle ranging in length from 5 to 20 m and composed of 3-15 rows of pixels was positioned across the moving edge of the cell. The average intensity profile across the edge of the cell was calculated and the number of points in the profile was increased 10 times by cubic spline interpolation. Movement of the cell edge was calculated from the frame-by-frame shift (computed by a least-squares procedure) in the interpolated intensity profiles. The sensitivity of the measurement was ~0.02 m, as previously determined (Frolenkov et al. 1997). Data obtained in this way were fitted by a scaled Boltzmann function:

(8)

Here L₀ is the length of the cell at the holding potential V_h , whereas L_max is the maximum voltage-dependent length change. V_p and W have the same meaning as in the expression for the voltage-dependent capacitance.

Ca²⁺ fluorescence imaging

Light from a 175 W stabilised xenon arc source (Lambda DG-4, Sutter Instruments) was coupled via a liquid light guide to the epifluorescence section of an Axiomat microscope (Carl Zeiss), which was equipped with an Omega Optical XF100 filter-block optimised for the Ca²⁺-selective dye Oregon Green 488 BAPTA-1. The illumination intensity was attenuated with a neutral density filter to avoid phototoxicity by reducing dye photo-bleaching rates to < 0.1 % s^-1. Fluorescence images were formed on a scientific grade cooled CCD sensor (Micromax 1300Y, Princeton Instruments) using an oil-immersion objective (X100, NA 1.40; PlanApo, Carl Zeiss). The sensor's output was binned 3 X 3 and digitised at 12 bits per pixel to produce 400 X 330 pixel images that were recorded to a host PC controlled by the Axon Imaging Workbench software (Axon Instruments) and analysed off-line. For each image pixel, fluorescence signals were computed as ratios F/F = [F(t) - F(0)]/F(0), where t is time, F(t) is fluorescence following a stimulus that causes Ca²⁺ elevation within the cell, and F(0) is pre-stimulus fluorescence computed by averaging 10-20 images. Both F(t) and F(0) were corrected for mean background fluorescence computed from a 20 X 20 pixel rectangle devoid of obvious cellular structures.

All values are given as means ± S.E.M. unless otherwise stated. Statistical significance was estimated using Student's t test at the P < 0.05 level. Curves generated by text equations were fitted to data by a Levenberg-Marquardt algorithm using Origin 6.0 software (Microcal Software, Northampton, MA, USA). This software also estimated the standard errors of fitting parameters.

	RESULTS

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Effect of BDM on the voltage-dependent capacitance and length change

Pressure application of BDM to isolated OHCs had no effect on the whole-cell current at holding potentials, V_h, between -70 mV and -50 mV. However, BDM induced rapid and substantial drops of the membrane capacitance at V_h (Fig. 2A). The latency of this effect did not exceed 30 ms, which corresponded to the typical drug delivery time in the present experiments (see Methods). Following the application of BDM, the current-voltage relationship, determined by subjecting the membrane potential to a voltage ramp from -110 mV to 90 mV in 6 s, showed minor and reversible changes only at voltages above -20 mV (Fig. 2B). Instead, the voltage dependence of the cell length (Fig. 2C) and membrane capacitance (Fig. 2D) shifted in the hyperpolarised direction without any visible change in cell morphology. The shift was completely reversible and dose dependent (Fig. 3A), with a Hill coefficient of 1.8 ± 0.4 and a half-maximal dose of 3.0 ± 0.8 mM. The main parameter affected by BDM was the mid-point potential, V_p, of the voltage-dependent capacitance and length change (Fig. 3B and C). The membrane capacitance of Deiters' cells, a non-sensory cell type of the organ of Corti, was unaffected by the application of BDM (0.2-20 mM; data not shown).

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Figure 2 Effect of BDM on the voltage-dependent capacitance and electromotile responses A, voltage stimuli applied by the patch-clamp amplifier (V), whole cell current (Im) and membrane capacitance (Cm) responses. Holding potential (Vh) = -50 mV. Im is clipped during voltage ramps to show the absence of the BDM effect on the baseline current. Note rapid and reversible drop of Cm during BDM application (20 mM, 35 s; filled bar). B-D, current-voltage relationships (B), percentage length change (C) and voltage-dependent fraction of the capacitance (D), measured before (), during () and after () the application of BDM. Parameters of the fits for the electromotility data in (C): Lmax/L0 = 3.9 ± 0.1 %, Vp = -33 ± 1 mV, W = 37 ± 1 mV (control); Lmax/L0 = 4.9 ± 0.5 %, Vp = -114 ± 11 mV, W = 57 ± 4 mV (BDM); Lmax/L0 = 3.7 ± 0.1 %, Vp = -31 ± 1 mV, W = 38 ± 1 mV (washout). Parameters of the fits for the capacitance data in (D): Cmax = 19.2 ± 0.3 pF, Vp = -46.7 ± 0.9 mV, W = 31.8 ± 0.7 mV (control); Cmax = 17.9 ± 0.4 pF, Vp = -105 ± 4 mV, W = 37 ± 2 mV (BDM); Cmax = 18.7 ± 0.3 pF, Vp = -48.4 ± 0.8 mV, W = 31.2 ± 0.4 mV (washout). Data in A-D are from the same cell. The patch pipette was filled with the KCl-based intracellular solution.

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Figure 3 Pooled data showing BDM effects on capacitance and motility A, dose-response curve showing the change in the mid-point (Vp) of the voltage dependence of the membrane capacitance vs. BDM concentration. Each point is the mean ± S.E.M. of the indicated number of cells. Continuous line through data is a non-linear fit obtained from the generalised logistic function y = [BDM]h/([BDM]h + KAh), where the exponent h is the Hill coefficient and KA is the half-saturating concentration of BDM. Parameters of the fit: h = 1.8 ± 0.4, KA = 3.0 ± 0.8 mM. B, Boltzmann parameters describing the dependence of cell capacitance on membrane potential in the control cell group (open bars; n = 7) and in cells treated with 5 mM BDM (filled bars; n = 8): Vp, mid-point potential; W, voltage sensitivity; and max, maximum voltage-dependent capacitance per square centimetre of lateral plasma membrane. C, Boltzmann parameters of the electromotility responses measured in the same groups: Vp and W as above; Lmax, maximum motile response as a percentage of the length of the cell at holding potential. ***P < 0.001. Data were obtained using the CsCl-based intracellular solution.

Effect of intracellular pressure removal on the capacitance response to BDM

Cell turgor (intracellular pressure) is an important factor in the control of OHC electromotility (Shehata et al. 1991; Chertoff & Brownell, 1994) and voltage-dependent capacitance (Iwasa, 1993; Kakehata & Santos-Sacchi, 1995). To eliminate the potentially confounding effects of turgor changes, we tested the effect of BDM on OHCs whose turgor had been reduced by applying negative pressure to the back of the patch pipette. The change in the mid-point potential, V_p = -47.5 ± 5.2 mV (n = 5), produced by BDM (5 mM) in these collapsed cells was statistically significant (P < 0.001) and was similar to that of the control group (Fig. 3B), indicating that BDM can affect the voltage dependence of the capacitance also in the absence of turgor changes.

Comparing the effects of BDM and salicylate

Acetylsalicylic acid has a number of reversible effects on the auditory system and, when applied to OHCs in vitro, it produces a remarkable reduction of electromotility and associated voltage-dependent capacitance (Tunstall et al. 1995). The time course of the sodium salicylate effect on the membrane capacitance is shown in Fig. 4A, for comparison with that of BDM (Fig. 2A). At equally saturating concentrations (20 mM), the onset time constant of the capacitance changes obtained by a single exponential fit was 1.8 ± 0.3 s (n = 3) for salicylate and 0.75 ± 0.06 s (n = 3) for BDM. The difference is statistically significant (P < 0.05). The recovery period following the application of salicylate (13 ± 1 s) was significantly longer (P < 0.0001) than the recovery period following the application of BDM (1.7 ± 0.4 s). Salicylate reduced the peak value of the capacitance by more than 90 % (Fig. 4B) without major shifts in V_p, indicating that the mechanisms of action of the two drugs are different (compare Fig. 2D).

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Figure 4 Effect of salicylate on the voltage-dependent capacitance. A, membrane capacitance measured before, during (filled bar) and after the pressure application of sodium salicylate (20 mM). B, voltage dependence of the OHC capacitance derived from the responses to the voltage ramps (numbered from 1 to 4 in A) before (), during () and after () the application of salicylate. Parameters of the fits: C0 = 25.0 ± 0.3 pF, Cmax = 23.6 ± 0.3 pF, Vp = -52.7 ± 0.7 mV, W = 39.3 ± 0.9 mV (control); C0 = 21 ± 24 pF, Cmax = 9 ± 24 pF, Vp = -5 ± 6 mV, W = 96 ± 158 mV (salicylate); C0 = 25.3 ± 0.3 pF, Cmax = 25.9 ± 0.3 pF, Vp = -61.5 ± 0.7 mV, W = 34.7 ± 0.8 mV (washout). The patch pipette was filled with the KCl-based intracellular solution. Compare Fig. 2A and D.

Comparing the effects of BDM and protein phosphorylation

As an inorganic phosphatase, BDM has been used to modify the phosphorylation state of many cellular proteins (Eisfeld et al. 1997). In Fig. 5A the effect of BDM on the voltage-dependent capacitance of OHCs is compared to that of other drugs commonly used in studies of protein phosphorylation. Following incubation in okadaic acid (1 M, 30-60 min at 37°C), a powerful inhibitor of the protein phosphates-1 and -2A that promote phosphorylation of a wide range of proteins in vivo (Haystead et al. 1989), V_p shifted in the hyperpolarised direction. Incubation for 30-60 min with the specific calmodulin inhibitors trifluoperazine (30 M) and W-7 (150 M) (Johnson & Wittenauer, 1983) shifted V_p in the opposite, depolarised direction. The effects of these reagents did not depend on the intracellular pressure and were reproducible in both the cells with normal turgor and the cells collapsed by a gentle suction through the patch pipette. Furthermore, the experiment in Fig. 5B and C shows that the ability of BDM to shift V_p in the hyperpolarised direction was unaffected by the blockade of Ca²⁺-calmodulin-dependent phosphorylation obtained with 150 M W-7, a saturating concentration (Hidaka et al. 1981; Itoh & Hidaka, 1984). Since the voltage dependence of C_m was significantly shifted in the depolarising direction in the presence of W-7, BDM produced a transient increase of C_m baseline (Fig. 5B) in contrast to the typical decrease of C_m observed in control conditions (Fig. 2A). However, the shifts of V_p induced by 5 mM BDM in the presence of W-7 (V_p = -29 ± 2 mV, n = 3) and in the control conditions (Fig. 5C) were comparable. These results suggest that the effect of BDM on the OHC capacitance is not related to its activity as an inorganic phosphatase.

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Figure 5 Comparison of the effects of BDM and protein phosphorylation on the voltage-dependent capacitance A, changes in the mid-point potential (Vp; means ± S.E.M.) following the application of BDM (5 mM, n = 12), okadaic acid (1 M, n = 6, a phosphorylation promoter), and two inhibitors of Ca2+-calmodulin-dependent protein phosphorylation, trifluoperazine (TFP; 30 M, n = 5) and W-7 (150 M, n = 7). All changes were significant (P < 0.001). B, OHC capacitance responses to the application of BDM (5 mM, filled bar) in the presence of W-7 (150 M). C, voltage dependence of the membrane capacitance, derived from the raw ramp responses shown in B (control, and ; BDM, ; washout, ). Parameters of the fits: Cmax = 39.3 ± 0.4 pF and 38.4 ± 0.5 pF, Vp = -3.2 ± 0.6 mV and -1.9 ± 0.7 mV, W = 19.4 ± 0.4 mV and 20.2 ± 0.4 mV (control); Cmax = 34.9 ± 0.3 pF, Vp = -29.1 ± 0.5 mV, W = 23.2 ± 0.3 mV (BDM); Cmax = 39.9 ± 0.3 pF, Vp = -2.7 ± 0.3 mV, W = 21.9 ± 0.2 mV (washout). Data were obtained using the KCl-based intracellular solution.

Effect of BDM on the intracellular Ca²⁺ concentration

To determine whether intracellular free Ca²⁺ plays any role in the action of BDM on membrane capacitance, we used fluorescence microfluorimetry and monitored cytoplasmic Ca²⁺ levels in OHCs loaded with Oregon Green 488 BAPTA-1 (100 M), a single wave-length fluorescent probe highly selective for this divalent cation. As shown in Fig. 6A and B, no fluorescence change was detected either during or following the application of BDM (5 M, n = 5), although sizeable capacitance changes were evoked (Fig. 6C). The Ca²⁺ ionophore ionomycin was subsequently applied to the cell through a second puff pipette. This drug is known to induce a generalised, transient increase of [Ca²⁺]_i by making the plasma membrane, as well as the membranes of intracellular Ca²⁺ stores, permeable to Ca²⁺. As expected, ionomycin elevated intracellular Ca²⁺ (n = 3) even after the application of BDM (Fig. 6A, right; Fig. 6B, right), indicating that the absence of Ca²⁺ responses to BDM was not a consequence of a high basal level of the [Ca²⁺]_i or low sensitivity of the measuring technique. In fact, a second application of ionomycin produced a distinguishable, albeit small, increase of [Ca²⁺]_i suggesting that the basal level of [Ca²⁺]_i in the OHCs was well below the saturating level for the Oregon Green (300-400 nM).

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Figure 6 The effect of BDM is not mediated by an increase of intracellular free Ca2+ concentration A, fluorescence images of an OHC loaded with 100 M Oregon Green 488 BAPTA-1 through the patch pipette before (left), immediately after the application of BDM (5 mM; middle), and after the subsequent application of ionomycin (10 M; right). Scale bar: 10 m. B, percentage fluorescence responses, relative to the pre-stimulus condition, of the cell in A to the consecutive applications of BDM (middle, filled bar) and ionomycin (right, open bar). Vh = -60 mV. The fluorescence intensity trace was obtained by averaging pixel values over the whole cell body. C, voltage dependence of the membrane capacitance of the same OHC measured before (), during (), and after () the application of BDM shown in A and B. Parameters of the fits: C0 = 24 ± 1 pF, Cmax = 21.4 ± 0.9 pF, Vp = -26.2 ± 0.7 mV, W = 37 ± 2 mV (control); C0 = 23.6 ± 0.5 pF, Cmax = 19.8 ± 0.4 pF, Vp = -54.0 ± 0.7 mV, W = 40 ± 1 mV (BDM); C0 = 24.3 ± 0.9 pF, Cmax = 20.1 ± 0.8 pF, Vp = -27.6 ± 0.7 mV, W = 36 ± 2 mV (washout). The patch pipette was filled with the KCl-based intracellular solution.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

BDM is a clinically used drug that is known to exert multiple and complex effects on cell physiology (see Introduction). We found that it rapidly and reversibly affects the operating range of the voltage-dependent capacitance and length changes of OHCs, shifting both of them in the hyperpolarised direction more than any other drug or manipulation (Kakehata & Santos-Sacchi, 1995; Santos-Sacchi et al. 1998; Santos-Sacchi & Huang, 1998) reported so far. The action of BDM on the OHC capacitance and electromotility is entirely reversible and extremely rapid both at the onset and at the offset of the drug application (Fig. 2).

The operating range of the voltage-dependent capacitance and length change has been reported to shift in the hyperpolarised direction following the decrease in intracellular pressure (cell turgor), which alters the tension in the plasma membrane (Iwasa, 1993; Gale & Ashmore, 1994; Kakehata & Santos-Sacchi, 1995). To eliminate these potentially confounding effects, we applied BDM to cells that had been collapsed by applying gentle suction through the patch pipette. The shifts of the operating range of the voltage-dependent capacitance observed under these conditions were similar to the controls, indicating that BDM action is unlikely to be mediated by turgor changes altering membrane tension.

Protein dephosphorylation and voltage-dependent capacitance

As an inorganic phosphatase, BDM may dephosphorylate a number of different proteins (Green & Saville, 1956; Coulombe et al. 1990). In our experiments BDM shifted the operating range of OHC electromotility and voltage-dependent capacitance in the hyperpolarised direction. In contrast, drugs that promote protein dephosphorylation, W-7 and trifluoperazine (Johnson & Wittenauer, 1983), induced depolarising shifts. Hyperpolarising shifts were observed after exposure to okadaic acid, which promotes the phosphorylation of a wide range of proteins in vivo (Haystead et al. 1989). In fact, when BDM was applied to cells incubated and bathed in W-7, its effects were remarkably similar to those produced under control conditions. We conclude that the effects of BDM on OHCs cannot be explained by its action as a phosphatase.

Role of intracellular Ca²⁺

A system of flattened, membrane-bound intracellular compartments, the subsurface cisternae, is found in the closest proximity to the electromotility machinery, at nanometre distances below the cortical lattice (Holley et al. 1992). The preferential distribution of Ca²⁺-ATPase near the innermost layer of the cisternae, in strict apposition to linearly arranged mitochondria (Schulte, 1993; Ikeda & Takasaka, 1993), supports a role for these structures as intracellular stores of Ca²⁺. The increase of free Ca²⁺ concentration has been shown to induce circumferential contraction and longitudinal elongation of the OHC (Dulon et al. 1990). Inhibition of these effects by calmodulin antagonists (Dulon et al. 1990) and antagonists of calmodulin-dependent kinases (Puschner & Schacht, 1997; Coling et al. 1998) suggests the involvement of Ca²⁺-calmodulin-dependent protein phosphorylation. BDM has been reported to promote the release of Ca²⁺ from the sarcoplasmic reticulum of skeletal and cardiac muscle (Tripathy et al. 1999) by modulating ryanodine receptors (Adams et al. 1998). However, fluorescence imaging experiments like the one shown in Fig. 6 indicate that the action of BDM on OHC membrane capacitance does not appear to involve mobilisation of intracellular Ca²⁺. We still cannot rule out the possibility that localised sub-membrane changes of [Ca²⁺]_i occur and pass undetected by our fluorescence imaging. On the other hand, the fast recovery of the capacitance after BDM and the inability of the ionomycin-induced increase of [Ca²⁺]_i to modulate the capacitance (Frolenkov et al. 2000), argue against the possibility that the BDM effect on OHC is mediated by [Ca²⁺]_i.

We did not observe any morphological changes in OHCs either during or after the application of BDM, suggesting that it does not affect (directly or indirectly) the cytoskeleton of the OHC. Therefore, it is unlikely that BDM action is mediated by changes in membrane tension associated with modification of the OHC cytoskeletal structure, such as those produced by BDM in muscle cells (McKillop et al. 1994).

Mechanism of action of BDM

It has been proposed that BDM exerts its inhibitory action on K_ATP channels (Smith et al. 1994) as well as L-type Ca²⁺ channels (Eisfeld et al. 1997; Allen et al. 1998) by mechanisms unrelated to protein dephosphorylation. Consistent with these findings, our results indicate that the effects of BDM on OHCs are not related to its inorganic phosphatase properties. Instead, the time course, reversibility, lack of dependence on intracellular Ca²⁺, together with the magnitude and the direction of the shift of the voltage-dependent capacitance induced by BDM suggest that BDM may directly target the voltage sensor of the OHC putative membrane motors. Interestingly, the gating charge movement of the L-type Ca²⁺ channel has been shown to be reduced after the application of BDM (Ferreira et al. 1997). Thus, the unique characteristics of BDM might be useful to the study of the mechanisms by which the recently proposed candidates for OHC motor protein, the sugar transporter GLUT5 (Géléoc et al. 1999) and a protein called 'prestin' (Zheng et al. 2000), could act as sensors of transmembrane potential.

Finally, BDM is used clinically for its protective actions on human myocardial force production (Perreault et al. 1992). Another class of drugs widely used in clinical practice, salicylates, are known to target OHC electromotility (Tunstall et al. 1995) and have ototoxic side effects (Stypulkowski, 1990). The distinct and powerful action of BDM on OHC electromotility indicates that this compound should be evaluated for its potential effects on hearing.

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Acknowledgements

This work was supported by the National Institutes on Deafness and other Communication Disorders (Intramural research project Z01 DC 00002-11) and in part by grants from Istituto Nazionale di Fisisca della Materia (Progetto di Ricerca Avanzata CADY) and Ministero della Ricerca Scientifica to F.M. We thank Kuni Iwasa and Richard Chadwick for critical comments and helpful discussions.

Corresponding author

B. Kachar: NIDCD-NIH, Section on Structural Cell Biology, 9000 Rockville Pike, Bldg 36/5D15, Bethesda, MD 20892-4163, USA.

Email: kacharb{at}nidcd.nih.gov

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