Three Types of Depolarization-Activated Potassium Currents in Acutely Isolated Mouse Vestibular Neurons

C. Chabbert,* J. M. Chambard,* A. Sans, and G. Desmadryl

Institut National de la Santé et de la Recherche Médicale U432, Neurobiologie et Développement du Système Vestibulaire, 34095 Montpellier cedex 5, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Chabbert, C., J. M. Chambard, A. Sans, and G. Desmadryl. Three Types of Depolarization-Activated Potassium Currents in Acutely Isolated Mouse Vestibular Neurons. J. Neurophysiol. 85: 1017-1026, 2001. The nature and electrophysiological properties of Ca2+-independent depolarization-activated potassium currents were investigated in vestibular primary neurons acutely isolated from postnatal mice using the whole cell configuration of the patch-clamp technique. Three types of currents were identified. The first current, sensitive to TEA (ITEA) and insensitive to 4-aminopyridine (4-AP), activated at -40 mV and exhibited slow activation (tau ac, 38.4 ± 7.8 ms at -30 mV, mean ± SD). ITEA had a half activation potential [Vac(1/2)] of -14.5 ± 2.6 mV and was inactivated by up to 84.5 ± 5.7% by 10-s conditioning prepulses with a half inactivation potential [Vinac(1/2)] of -62.4 ± 0.2 mV. The second current, sensitive to 4-AP (maximum block around 0.5 mM) and to alpha -dendrotoxin (IDTX) appeared at -60 mV. Complete block of IDTX was achieved using either 20 nM alpha -DTX or 50 nM margatoxin. This current activated 10 times faster than ITEA (tau ac, 3.5 ± 0.8 ms at -50 mV) with Vac(1/2) of -51.2 ± 0.6 mV, and inactivated only slightly compared with ITEA (maximum inactivation, 19.7 ± 3.2%). The third current, also sensitive to 4-AP (maximum block at 2 mM), was selectively blocked by application of blood depressing substance (BDS-I; maximum block at 250 nM). The BDS-I-sensitive current (IBDS-I) activated around -60 mV. It displayed fast activation (tau ac, 2.3 ± 0.4 ms at -50 mV) and fast and complete voltage-dependent inactivation. IBDS-I had a Vac(1/2) of -31.3 ± 0.4 mV and Vinac(1/2) of -65.8 ± 0.3 mV. It displayed faster time-dependent inactivation and recovery from inactivation than ITEA. The three types of current were found in all the neurons investigated. Although ITEA was the major current, the proportion of IDTX and IBDS-I varied considerably between neurons. The ratio of the density of IBDS-I to that of IDTX ranged from 0.02 to 2.90 without correlation with the cell capacitances. In conclusion, vestibular primary neurons differ by the proportion rather than the type of the depolarization-activated potassium currents they express.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Vestibular primary neurons are involved in transmitting afferent information about accelerations from the inner ear vestibular mechanoreceptors to vestibular nuclei. Vestibular afferents are classified as regularly or irregularly discharging patterns (Smith and Goldberg 1986). Irregular afferents have phasic response dynamics, higher sensitivities to natural and to external galvanic stimulations, and larger axons than regular fibers. They also differ in their response to the activation of efferent pathway (Goldberg et al. 1984; Smith and Goldberg 1986). These authors suggest that discharge regularity as well as the other functional properties of vestibular primary afferents could be the consequence of differences in membrane conductances between each class of neurons. However, the intrinsic properties supporting these differences are unknown. We therefore developed a preparation of vestibular ganglion neurons acutely isolated from mice. Previous analysis of voltage-activated conductances showed the presence in all vestibular neurons of one sodium current (Chabbert et al. 1997) and five calcium currents (Chambard et al. 1999; Desmadryl et al. 1997), but potassium currents that might influence their discharge patterns were not investigated yet. Since a large variety of voltage-activated K+ currents have been described in various neuronal preparations, and since they have been reported to play an important role in shaping membrane electrical activity (Rudy 1988), we characterized these currents in vestibular primary neurons.

Over the last two decades many studies have been performed, characterizing the outward voltage-activated K+ conductances expressed in dorsal root ganglion (DRG) neurons (Everill et al. 1998; Gold et al. 1996; Kostyuk et al. 1981; Robertson and Taylor 1986), and other sensory neurons (Brew and Forsythe 1995; Garcia-Diaz 1999; Locke and Nerbonne 1997a; Manis and Marx 1991; McFarlane and Cooper 1991; Oyelese and Kocsis 1996; Rathouz and Trussell 1998; Stansfeld and Feltz 1988). Identification of the different current types according to their activation and inactivation properties, which often overlap, remains difficult (Gold et al. 1996; McFarlane and Cooper 1991). Pharmacological agents such as tetraethylammonium (TEA) and 4-aminopyridine (4-AP) have been widely used to separate different classes of voltage-activated K+ currents. However, separation of the different types of 4-AP-sensitive currents has often been impaired both by the overlap in the doses required to block each current (Gold et al. 1996; Hoshi and Aldrich 1988; McFarlane and Cooper 1991) and by the voltage sensitivity of the blocking effect (Yeh et al. 1976). Conversely, several peptide toxins that selectively block each type of 4-AP-sensitive K+ current are now available. alpha -Dendrotoxin (alpha -DTX), a toxin purified from snake venom, has been reported to selectively block a fast activating and partially inactivating 4-AP-sensitive K+ current (Stansfeld and Feltz 1988). More recently margatoxin (MgTX), a toxin purified from scorpion venom, has been shown to block a 4-AP-sensitive K+ current in human peripheral T-lymphocytes (Garcia-Calvo et al. 1993). Another peptide purified from sea anemone, blood depressing substance (BDS-I), has been reported to block a fast activating and inactivating K+ current (Diochot et al. 1998).

The aim of the present study was to identify the different types of depolarization-activated K+ currents present in vestibular primary neurons using these new pharmacological tools, and to characterize their respective kinetic properties. Records were obtained using the whole cell configuration of the patch-clamp technique applied to primary neurons acutely isolated from postnatal mice. Based on their pharmacological and electrophysiological properties, we report here for the first time that three distinct outward Ca2+-independent depolarization-activated K+ conductances are present in vestibular primary neurons. These three conductances are expressed in various proportions between neurons.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture

Potassium currents were studied in neurons acutely isolated from the superior branch of the vestibular nerve innervating the utricular macula and the horizontal and lateral cristae using an isolation procedure previously described (Desmadryl et al. 1997). Ganglia were aseptically dissected from postnatal day 5 to 8 (P5 to P8) mice (CERJ, Le Genest, France) rapidly killed by decapitation (the day of birth was considered as postnatal day 0). About 20 ganglia for each experiment were collected in phosphate-buffered saline (PBS; Life Technologies). We tested different incubation times and trypsin concentrations without any difference in the amplitude or the shape of the recorded currents. We settled on protocols employing trypsin at 0.25% for 12 min at 37°C in PBS containing 0.25% EDTA-trypsin (Life Technologies). Ganglia were triturated with fire-polished Pasteur pipettes of three decreasing diameters in a cell culture medium containing Neurobasal medium (Life Technologies), 10% B27 (Life Technologies), 25 µM glutamate, and 0.25 mM glutamine. Neurons were plated onto 35-mm culture dishes (Nunc) coated with 10 µg/ml poly-D-ornithine (Sigma) in cell culture medium. Cells were used between 1 and 4 h after dissociation. Under phase contrast microscopy, dissociated neurons had a spherical shape and birefringent cytoplasm as previously reported (Desmadryl et al. 1997). Cell diameters ranged between 12.5 and 25 µm. Only isolated neurons exhibiting no processes were chosen for the electrophysiological studies. Their capacitances ranged from 9 to 32 pF (18.6 ± 4.4 pF, mean ± SD) in a sample of 80 neurons.

Electrophysiological recordings

Whole cell recordings of voltage-dependent potassium currents were obtained at room temperature (25°C) under conditions optimized to ensure their complete isolation from other voltage-dependent currents. Tetrodotoxin (1 µM, Sigma) was used to block Na+ currents, and extracellular Na+ was replaced by choline. Calcium was omitted from the extracellular medium, and 2 mM EGTA was added to block voltage-activated Ca2+ currents, as well as Ca2+-activated K+ currents. The standard extracellular solution contained (in mM) 135 cholineCl, 5 KCl, 10 HEPES, 10 glucose, 1 MgCl2, 2 EGTA, and 0.001 TTX. For extracellular TEA solutions, cholineCl was replaced by equimolar TEACl. The pH of the recording solutions was adjusted to 7.35 and osmolality set at 300 mOsm/l. Recording pipettes pulled from hematocrit tubes (Modulohm I/S, Herlev, Denmark) were coated with ski wax to reduce capacitive transients. Pipettes 2-3 MOmega were filled with the following intracellular solution (in mM): 135 KCl, 10 EGTA, 25 HEPES, and 10 glucose. The pH was adjusted to 7.35 and the osmolality set at 300 mOsm/l. Whole cell currents were recorded using a Axopatch 200B (Axon Instruments, Foster City, CA) patch-clamp amplifier. After seal formation and membrane disruption, cell capacitance and series resistance were estimated from the decay of the capacitance transient induced by a ±10-mV pulse from a holding potential (HP) of -100 mV. Series resistance was in the range of 5-9 MOmega , and the membrane capacitance could be charged with a time constant of 100 µs. Series resistances were 85% compensated after cancellation of the capacitive transients. No linear leakage compensation was performed. Voltage errors resulting from uncompensated series resistances were corrected only for the high-threshold TEA-sensitive current when maximum voltage error exceeded 5 mV. The liquid junction potential between the internal and the extracellular solution, measured according to Neher (1992), was -6.8 mV at 25°C for control extracellular medium. Data presented are not corrected for junction potential unless specified. Current signals were filtered at 5 kHz, digitized, and stored.

Drugs

4-AP and TEA were obtained from Sigma-Aldrich and were dissolved directly in the extracellular medium. alpha -DTX (Latoxan), MgTX (Bachem), and BDS-I (a generous gift from S. Diochot, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France) were dissolved as stock solutions at 10-4 M in bidistilled water and stored at -80°C. Drugs and peptides were applied to the bathing medium in the vicinity of the cell by a fast gravity perfusion system.

Analysis

All experimental parameters, such as the holding and test potentials, were controlled with an IBM PC equipped with a Tecmar Labmaster analog interface (Axon Instruments). Cell stimulation, data acquisition, and analysis were performed using Pclamp software (v 5.5 and v 6, Axon Instruments). The mean chord conductances were calculated, assuming a potassium equilibrium potential of -71 mV. For each component of the whole cell K+ current, activation and inactivation curves of pooled data were best fitted with single Boltzmann function of the form G/Gmax = 1/{1 + exp[(V - V1/2)/k]}, where V1/2 was half activation or inactivation potential [Vac(1/2), Vinac(1/2)] and k the limiting slope. Time constants (tau ) were best fitted with single exponential function of the form A · exp(-t/tau ), where A is the current peak and tau  the exponential time constant. Except when specified, pooled data are given as means ± SD. Statistical significances were examined using ANOVA, corrected for unequal variances when necessary.


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Whole cell voltage-activated outward K+ currents

K+ currents were evoked by depolarizing pulses at various voltages from HP -100 mV. Figure 1 illustrates activation (Fig. 1A) and inactivation (Fig. 1B) of currents recorded in representative neurons in control external solution. Examination of their general shape suggested that they were composed of at least two distinct components with an early transient component and a sustained component in most neurons (Fig. 1Aa, -10 mV trace). Analysis of the current-voltage (I-V) relationship for the sustained component confirmed the presence of two distinct currents. The first component of the sustained current activated at potentials just positive to -60 mV, and the second above -30 mV. This can be seen as an increase in the slope of the I-V curve (Fig. 1Ab, arrow). The presence of two components in the sustained current was further supported by plotting relative activation, expressed as G/Gmax, versus test potential in a sample of four neurons (Fig. 1Ac). The curve was composed of two Boltzmann components (dotted lines) with Vac(1/2) of -47 and -12 mV, which contributed to 45 and 55% of the total current, respectively, in this cell. These two Vac(1/2) values matched those estimated below for the alpha -DTX and TEA-sensitive components.



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Fig. 1. Whole cell depolarization-activated K+ currents in vestibular primary neurons. Aa: representative whole cell K+ currents elicited in control external solution by 400-ms depolarizing pulses ranging from -80 to +30 mV in 10-mV increments from holding potential (HP) -100 mV. Ab: plot of current amplitudes taken either 5 ms after the beginning (open circle ) or before the end () of the test pulse, as a function of the test potential for the neuron shown in Aa. Dotted lines emphasize the increase in the slope of the current-voltage relation (I-V) for sustained component (arrow). Ac: plot of relative activation of the sustained component of whole cell K+ current as a function of test potentials for a sample of 4 neurons. Dotted lines show the 2 Boltzmann components that compose the data curve. Ba: steady-state inactivation of whole cell K+ currents elicited by 400-ms depolarizing pulses to +20 mV preceded by 10-s conditioning prepulses from -110 to -20 mV from HP -100 mV. Bb: plot of relative currents against conditioning voltages for the neuron shown in Ba. Note that in this neuron whole cell currents were inactivated by up to 60%. Bc: plot of relative inactivation of whole cell K+ currents as a function of test potentials for a sample of 5 neurons. Currents were measured either 5 ms after the beginning (open circle ) or before the end () of the test pulses.

Steady-state inactivation of the whole cell K+ current confirmed that it was composed of different components. Figure 1Ba illustrates inactivation elicited at +20 mV by 10-s conditioning prepulses to various voltages. Plotting current amplitudes normalized to maximum current as a function of conditioning voltages revealed that a large part of the whole cell K+ current did not inactivate even for long conditioning prepulses (Fig. 1Bb), whereas the inactivating portion exhibited distinct voltage dependence when measured at the beginning (open circle ), or the end () of the test pulse. Plotting relative inactivation against conditioning voltages in a sample of five neurons indicated that the early transient and sustained components inactivated with Vinac(1/2) of -76 and -61 mV, respectively.

To determine that depolarization-activated whole cell currents are carried by K+, the reversal potential was estimated by stepping the HP from -100 to -20 mV for 20 ms to activate the whole cell K+ current, followed by 100-ms depolarizations from -100 to +20 mV in 10 mV increments. Reversal potential Erev was estimated by measuring tail currents 2 ms after the end of the depolarizing pulses in external solutions containing 5 or 30 mM K+. This procedure gave a mean Erev of -71.2 ± 1.4 mV (n = 8) in standard external solution and -32.7 ± 1.2 mV (n = 8) in 30 mM K+ external solution (including the junction potential values). The calculated equilibrium potential in our recording conditions using the Nernst equation gave -83.2 and -38 mV, respectively. From our data it appears that the Erev of the whole cell K+ current was dependent on the K+ gradient, but less than that expected for an absolute K+ current, suggesting that K+ channels in our preparation are not absolutely selective to K+. Alternatively, these discrepancies could be the consequence of K+ accumulation in the extracellular space, as reported by Rathouz and Trussell (1998) in neurons of the avian nucleus magnocellularis.

Pharmacological identification of the different components of the whole cell K+ current

We first studied the effect of external applications of increasing concentrations of 4-AP and TEA on the whole cell K+ current evoked in primary vestibular neurons. Figure 2 illustrates the separation into three distinct components of whole cell K+ current elicited in a representative neuron by depolarizing pulses to -10 mV (Fig. 2A) and on the corresponding I-V relations (Fig. 2B). Applications of 4-AP at concentrations below 0.5 mM (typically we used 0.1 mM) blocked a fast activating sustained component of the whole cell K+ current (Fig. 2, Aa and Ab, and C), that activated from -60 mV (Fig. 2, Ba and Bb). Above 0.5 mM, 4-AP also blocked a fast activating transient component with maximum effect around 2 mM (Fig. 2, Aa and Ac, and C). This component of the whole cell K+ current activated at -60 mV. Applications of TEA blocked a component of the whole cell K+ current with an effect from 1 mM, and maximum block around 40 mM (Fig. 2C). The current sensitive to TEA (ITEA) exhibited slow activation and higher threshold of activation than the 4-AP-sensitive currents (around -40 mV; Fig. 2, Ba and Bb). In most studies, 4-AP did not completely block one component of the whole cell K+ current without affecting the other. We therefore studied the effect of alpha -DTX, MgTX, and BDS-I, three toxins reported to selectively block distinct 4-AP-sensitive K+ currents. Figure 3 illustrates representative effects of these toxins on single traces of whole cell K+ currents. Application of alpha -DTX blocked a fast activating sustained component of the whole cell K+ current with noticeable effect at 2 nM and maximum blocking effect around 20 nM (Fig. 3A). MgTX blocked a component of the whole cell K+ current with an effect from 10 nM and maximum blocking effect around 50 nM (Fig. 3B). Applications of nanomolar concentrations of BDS-I blocked a fast activating transient component of the whole cell K+ current with noticeable effect from 20 nM and maximum blocking effect around 250 nM (Fig. 3C). When used at its maximum blocking concentration, alpha -DTX impeded the effect of subsequent application of 0.1 mM 4-AP (Fig. 3D). A similar result was obtained with MgTX (Fig. 3E). Each of the two toxins also prevented the effect of the other without preventing the effect of 2 mM 4-AP on the fast activating transient component (Fig. 3F). Presence of 250 nM BDS-I in the external solution did not prevent the blocking effect of alpha -DTX or TEA, whereas it prevented those of 2 mM of 4-AP (Fig. 3G).



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Fig. 2. Pharmacological separation of the different components of the whole cell K+ current using 4-aminopyridine (4-AP) and tetraethylammonium (TEA). Effect of subsequent external applications of 4-AP and TEA on whole cell K+ currents. A: representative traces elicited by depolarizing pulses to -10 mV from HP -100 mV (a) in control solution (1), presence of 0.1 mM 4-AP (2), 2 mM 4-AP (3), and 40 mM TEA (4) in the same cell. Digital subtraction reveals the 2 currents sensitive to 4-AP (b and c), and the current sensitive to TEA (d). B: corresponding I-V relationship of whole cell K+ currents evoked by 300-ms depolarizing pulses from -70 to 0 mV from HP -100 mV showing whole cell K+ current ( and ), currents sensitive to 0.1 and 2 mM 4-AP (open circle ,  and triangle , black-triangle, respectively), and currents sensitive to 40 mM TEA (diamond  and black-lozenge ). Current amplitudes were measured either 5 ms after the beginning (, open circle , triangle , and diamond ; Ba) or 5 ms before the end (, , black-lozenge , and black-triangle; Bb) of the test pulse. C: effect of subsequent applications of increasing concentrations of TEA (as indicated), followed by applications of 0.1 and 2 mM 4-AP on current traces evoked by 200 ms pulses to +20 mV.



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Fig. 3. Effects alpha -dendrotoxin (alpha -DTX), margatoxin (MgTX), and blood depressing substance (BDS-I) on whole cell K+ currents. A: effect of subsequent applications of 2 and 20 nM of alpha -DTX on current traces evoked by 400-ms pulses to +20 mV. B: effect of subsequent applications of 10 and 50 nM of MgTX on current traces evoked by 700-ms pulses to +20 mV. C: effect of subsequent applications of 20 and 250 nM of BDS-I on current traces evoked by 200-ms depolarizing pulses to -10 mV. Note the complete block of the transient component, whereas the sustained one was not affected. D: effect of 0.1 mM of 4-AP following application of 20 nM of alpha -DTX on current traces evoked by 400-ms pulses to -10 mV. E: effect of 0.1 mM of 4-AP following application of 50 nM of MgTX on current traces evoked by 700-ms pulses to -10 mV. Note that the transient component was not affected. F: effect of subsequent applications of 20 nM alpha -DTX, 50 nM MgTX, and 2 mM 4-AP on current traces evoked by 200-ms pulses to +20 mV. Note that 4-AP blocks a transient component without affecting the sustained one. G: effect of subsequent applications of 20 nM alpha -DTX, 2 mM 4-AP, and 40 mM TEA on current traces evoked by 400-ms pulses to -10 mV in solution containing 250 nM BDS-I. All experiments were conducted from HP -100 mV.

Figure 4 illustrates the isolation of the three components of whole cell K+ current using alpha -DTX, MgTX, or BDS-I on depolarizing pulses to -10 mV (Fig. 4, Aa-Ca) and on the corresponding I-V relations (Fig. 4, Ab-Cb). Pharmacological properties of the toxins reported in Fig. 3, and the similarity in the I-V relations shown in Fig. 4 indicate that alpha -DTX, MgTX, and 4-AP (below 0.5 mM) affect the fast activating sustained component of the whole cell K+ current, thereafter referred as IDTX, whereas BDS-I and 4-AP (at millimolar concentrations) affect its transient component, thereafter referred as IBDS-I.



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Fig. 4. Pharmacological separation of the different components of the whole cell K+ currents using alpha -DTX, MgTX, and BDS-I. Aa: effect of external application of 20 nM alpha -DTX on whole cell K+ currents evoked by 150-ms depolarizing pulses to -20 mV from HP -100 mV. Representative traces elicited in control solution (1), and in presence of alpha -DTX (2). Digital subtraction (1) - (2) reveals currents sensitive to alpha -DTX (IDTX). Ab: corresponding I-V relationship from HP -100 mV of whole cell K+ currents evoked by 150-ms pulses ( and ), and of IDTX (open circle  and ) measured 5 ms either after the beginning ( and open circle ) or before the end ( and ) of the test pulse. Ba: effect of external application of 50 nM MgTX on whole cell K+ currents evoked in similar conditions as in A. Bb: corresponding I-V relation showing the MgTX-sensitive current. Ca: effect of external application of 250 nM BDS-I on whole cell K+ currents evoked in control solution by 250-ms pulses at -10 mV following 150-ms prepulses to -100 mV from HP -50 mV. This protocol was used to reduce the large TEA-sensitive current. Cb: corresponding I-V relations showing the BDS-I-sensitive current.

Characterization of ITEA

The high-threshold slow activating and inactivating TEA-sensitive current was isolated by subtracting traces evoked in 40 mM TEA external solution from those elicited in standard external solution (Fig. 5). Steady-state voltage-dependent activation of ITEA was studied using depolarizing pulses at various voltages from HP -100 mV. ITEA appeared from a threshold between -50 and -40 mV and exhibited slow activation and slow time-dependent inactivation (Fig. 5Aa). This current was fully activated around +20 mV. Figure 5Ab illustrates the plot of relative activation of ITEA expressed as G/Gmax as a function of the test potential in a sample of nine neurons. Values of current amplitudes were taken 5 ms before the end of the test pulse. The data curve was best fitted with single Boltzmann function with Vac(1/2) of -14.5 ± 2.6 mV and k of 10.2 ± 0.6 mV (means ± SE). The time constant for activation (tau ac) of ITEA was voltage dependent and ranged from 38.4 ± 7.8 ms at -30 mV, to 5.1 ± 1.7 ms at +25 mV for a sample of seven neurons.



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Fig. 5. Electrophysiological characterization of the high-threshold, slow activating and inactivating TEA-sensitive K+ current. Traces were obtained by subtracting those elicited in TEA 40 mM external solution from those elicited in standard external solution. Aa: family of ITEA elicited by 150-ms depolarizing pulses ranging from -80 to +30 mV in 10-mV increments from HP -100 mV in a representative neuron. Ab: plot of relative activation of ITEA as a function of the test potential in a sample of 9 neurons. Ba: steady-state inactivation of ITEA elicited by 400-ms depolarizing pulses to +20 mV preceded by a 10-s conditioning prepulses from -120 to -20 mV from HP -100 mV. Bb: plot of relative inactivation of ITEA as a function of the test potential in a sample of 6 neurons. Ca: steady-state time-dependent inactivation of ITEA elicited by 250-ms test pulses to +20 mV preceded by a conditioning prepulse to -20 mV from HP -100 mV. Cb: plot of the relative time-dependent inactivation of ITEA as a function of prepulse duration in a sample of 7 neurons. Da: recovery from inactivation of K+ currents elicited in the same neuron as in Ca using similar protocol, except that HP and conditioning prepulses were changed to -20 and -100 mV, respectively. Db: plot of the relative time-dependent recovery from inactivation of ITEA as a function of prepulse durations in a sample of 6 neurons. Data points were fitted with single exponential function (solid line). In Ab, Bb, Cb, and Db, current amplitudes were measured 5 ms before the end of the test pulses.

The inactivation, not visible within the 150-ms pulse, was evident for longer depolarizations. Steady-state voltage-dependent inactivation of ITEA was studied on currents elicited at +20 mV following 10-s prepulse protocol (Fig. 5Ba). Current amplitudes measured 5 ms before the end of the test pulse were normalized to maximum current and expressed as I/Imax. In a sample of six neurons, the mean maximum inactivation of ITEA was 84.5 ± 5.7%. Plot of relative inactivation as a function of the prepulse voltages was best fitted with single Boltzmann function (Fig. 5Bb), with Vinac(1/2) of -62.4 ± 0.2 mV, with k of 11.2 ± 0.3 mV (means ± SE).

Steady-state time-dependent inactivation and recovery from inactivation of ITEA were studied using two-step protocols. Inactivation of ITEA elicited by depolarizing pulses to +20 mV was achieved by increasing the duration of conditioning prepulses to -20 mV (Fig. 5Ca). Recovery of ITEA previously inactivated by holding the neuron at -20 mV for several minutes was achieved by increasing the duration of conditioning prepulses to -100 mV (Fig. 5Da). Relative time-dependent inactivation (Fig. 5Cb) and recovery from inactivation (Fig. 5Db) of ITEA were plotted as a function of prepulse duration in a sample of seven neurons. Data points were fitted with single exponential functions of the form A exp(-t/tau ), with time constants of 6.7 ± 1.0 s and 254.4 ± 14.1 ms (solid lines).

Steady-state voltage-dependent deactivation of ITEA was determined using a protocol in which a 20-ms pulse to +10 mV was applied to fully activate the current, followed by a range of more negative potentials from -45 to -70 mV to shut it (not shown). The fit of tail currents revealed that they decayed with a single exponential time course. The mean time constant of deactivation (tau deac) at -50 mV was 26.4 ± 3.6 ms for a sample of four neurons. The presence of 20 nM alpha -DTX, 50 nM MgTX, or 2 mM 4-AP in the external solution did not change either the maximum amplitudes at +20 mV (Imax) nor the kinetic characteristics of ITEA (Table 1).


                              
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Table 1. Comparison of amplitudes elicited by +20-mV depolarization pulses, and kinetic characteristics of ITEA, IDTX, and IBDS-I in presence of various blockers

Characterization of IDTX

The low-threshold, fast activating, sustained alpha -DTX-sensitive current was isolated by subtracting traces evoked in the presence of 20 nM alpha -DTX from those elicited in control solution (Fig. 6). From HP -100 mV, IDTX activates at a threshold between -70 and -60 mV, and was fully activated at around -30 mV (Fig. 6Aa). It exhibited fast activation and very little time-dependent inactivation even for 10-s depolarizing pulses. Plot of relative activation of IDTX as a function of the test potential in a sample of seven neurons (Fig. 6Ab, ) was best fitted with single Boltzmann function with Vac(1/2) of -51.2 ± 0.6 mV, and k of 4.0 ± 0.6 mV (means ± SE). The rise time of activation was voltage dependent and displayed very fast kinetics. On a sample of seven neurons, tau ac ranged from 3.5 ± 0.8 ms at -50 mV to 1.4 ± 0.3 ms at -10 mV. The current showed weak voltage-dependent inactivation (Fig. 6Ba). A 10-s prepulse protocol inactivated the current evoked at -10 mV by 19.7 ± 3.2% (n = 5; Fig. 6Bb). Increasing prepulse duration up to 30 s produced a maximum inactivation of about 25%.



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Fig. 6. Electrophysiological characterization of the low-threshold, fast activating alpha -DTX- and MgTX-sensitive K+ current. Traces for voltage-dependent activation (A) and inactivation (B) were obtained by subtracting traces elicited in presence of 20 nM alpha -DTX () or 50 nM MgTX (open circle ) in control external solution from those elicited in their absence. Aa: family of IDTX elicited by 150-ms depolarizing pulses ranging from -80 to -10 mV in 10-mV increments from HP -100 mV. Ab: plot of relative activation of IDTX and MgTX-sensitive current as a function of the test potential in a sample of 7 and 9 neurons, respectively. Ba: steady-state inactivation of IDTX elicited by 200-ms depolarizing pulses to -10 mV preceded by a 10-s conditioning prepulse from -120 to -20 mV from HP -100 mV. Bb: plot of current amplitudes normalized to maximum current and expressed as I/Imax for IDTX and MgTX-sensitive current as a function of the test potential in a sample of 5 and 4 neurons, respectively. In Ab and Bb, current amplitudes were measured 5 ms before the end of the test pulses.

Steady-state voltage-dependent deactivation was studied by applying a 10-ms pulse to -30 mV to fully activate IDTX, then stepping down to more negative potentials from -45 to -70 mV. In a sample of five neurons, the mean tau deac was 15.6 ± 3.1 ms at -50 mV.

The Imax values, and kinetic characteristics of IDTX, did not significantly differ when MgTX was substituted for alpha -DTX (Table 1 and Fig. 6, Ab and Bb, open circle ).

Characterization of IBDS-I

The low-threshold, fast activating and fast inactivating BDS-I-sensitive current was isolated either by subtracting traces evoked in the presence of 250 nM BDS-I from control, or taking advantage of its fast and complete time-dependent inactivation property (see below and Fig. 7). Results obtained by the two procedures did not differ significantly.



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Fig. 7. Electrophysiological characterization of the low-threshold, fast activating and inactivating BDS-I-sensitive K+ current. Aa: family of IBDS-I elicited in control solution by 125-ms depolarizing pulses ranging from -80 to +10 mV in 10-mV increments, preceded by a 200-ms prepulse to -100 mV from HP -50 mV. Ab: plot of relative activation of IBDS-I estimated at peak current as a function of the test potential in a sample of 15 neurons. Ba: steady-state inactivation of IBDS-I elicited using the following inactivating protocol. From HP -50 mV, a 200-ms pulse to -100 mV was applied, followed by 100-ms conditioning steps between -120 and -10 mV, before a 200-ms test pulse to +10 mV. Bb: plot of relative inactivation of IBDS-I as a function of conditioning voltage in a sample of 7 neurons. Ca: family of K+ currents elicited using the following 2-step protocols. From HP -100 mV, a conditioning prepulse to -50 mV was applied, followed by 250-ms test pulses to 0 mV. The duration of the conditioning prepulse was increased in 20-ms steps. Cb: plot of the relative peak currents measured isochronously (5 ms after the beginning of the test pulse) and expressed as I/Imax as a function of prepulse duration for a sample of 8 neurons. Data points were well fitted with single exponential function of the form Ae(-t/tau; solid line). Da: family of K+ currents elicited in the same neuron as in Ca using similar protocol, except that HP and conditioning prepulses were changed to -50 and -100 mV, respectively, and conditioning prepulse was increased in 50-ms steps. Db: plot of the relative peak currents as a function of prepulse duration. Data points were well fitted with a single exponential function (solid line). In C and D, 20 nM alpha -DTX and 20 mM TEA were added in the bathing solution.

Steady-state voltage-dependent activation of IBDS-I was studied by subtracting traces elicited by 125-ms test pulses ranging from -80 to +10 mV from HP -50 mV, from those obtained with a 200-ms prepulse to -100 mV. IBDS-I was activated at a threshold between -60 and -50 mV, with a fast activation and fast inactivation kinetics as shown for a representative neuron (Fig. 7Aa). IBDS-I was fully activated at 0 mV and almost totally inactivated after 150 ms.

Activation of IBDS-I estimated at the peak of the current in a sample of 15 neurons (Fig. 7Ab) gave Vac(1/2) of -31.3 ± 0.4 mV and k of 8.5 ± 0.4 mV (means ± SE). The rise time of activation was of the same order as that of IDTX. tau ac and decay time (tau inac) were voltage dependent, ranging from 2.3 ± 0.4 ms at -50 mV to 0.8 ± 0.1 ms at +10 mV, and 53.5 ± 11.7 ms at -50 mV to 12.2 ± 3.1 ms at +10 mV, respectively.

Steady-state voltage-dependent inactivation of IBDS-I was studied using a 200-ms pulse to -100 mV from HP -50 mV, followed by 100-ms conditioning steps between -120 and -10 mV, before a 200-ms test pulse to +10 mV. Traces for a representative neuron and the relative inactivation for a sample of seven neurons are shown in Fig. 7, Ba and Bb, respectively. IBDS-I had Vinac(1/2) of -65.84 ± 0.3 mV, and k of 5.51 ± 0.25 mV (means ± SE).

Time dependence of inactivation and recovery from inactivation of IBDS-I were studied using two step protocols in the presence of 20 nM alpha -DTX and 20 mM TEA to reduce TEA- and alpha -DTX-sensitive currents. Under these experimental conditions, the Imax values and the kinetic characteristics of IBDS-I did not change (Table 1). IBDS-I evoked by 250-ms test pulses to 0 mV from HP -100 mV were progressively inactivated by increasing the duration of a conditioning prepulse to -50 mV (Fig. 7Ca). After complete inactivation was achieved by holding the membrane potential to -50 mV, IBDS-I progressively recovered when the duration of a conditioning prepulse to -100 mV was increased (Fig. 7Da). Time-dependent inactivation and recovery from inactivation of IBDS-I estimated in a sample of eight neurons had mean time constants of 25.4 ± 0.7 ms (Fig. 7Cb) and 95.8 ± 4.0 ms (Fig. 7Db), respectively.

Distribution of K+ currents among vestibular primary neurons

The three types of depolarization-activated K+ current were found in all neurons investigated, with substantial variations in their relative amplitudes, densities, and distributions (Table 2). ITEA was the predominant current making up to 56% of the total current. There was a large variability in the relative expression of the two fast activating currents. The ratio of the density of IBDS-I and IDTX varied from 0.02 to 2.90 (mean 0.90 ± 1.20; n = 38). No correlation between the size or the capacitance of the recorded neurons and the relative distribution of each current was noticed (data not shown).


                              
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Table 2. Comparison of the amplitude, density, and relative proportion of the three depolarization-activated K+ currents in vestibular neurons


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By using a combination of pharmacological and electrophysiological approaches, we identified and characterized for the first time, three different types of Ca2+-independent depolarization-activated K+ currents in vestibular primary neurons acutely isolated from postnatal mouse. Experimental conditions, previously used to study voltage-activated Na+ and Ca2+ conductances on the soma of neurons enzymatically dissociated from the vestibular ganglion (Chabbert et al. 1997; Chambard et al. 1999; Desmadryl et al. 1997), were adapted to the study of voltage-activated K+ currents. The first current was isolated on the basis of its sensitivity to TEA and was termed ITEA. Two fast activating currents sensitive to 4-AP were isolated according to their sensitivity to two toxins, alpha -DTX and BDS-I, and were termed IDTX and IBDS, respectively.

ITEA

ITEA is characterized by its sensitivity to TEA and its insensitivity to 4-AP in the same way that IK described in a majority of DRG neurons (Gold et al. 1996; Kostyuk et al. 1981), in other sensory neurons (Garcia-Diaz 1999; Manis and Marx 1991; McFarlanne and Cooper 1991), and in a number of cortical neurons (for review see Locke and Nerbonne 1997a), ITEA exhibits a high threshold of activation (between -30 and -40 mV), displays voltage-dependent activation, with a time course of activation 10 times slower than those of the 4-AP-sensitive currents. Although its half activation potential (-15 mV) is comparable to those found in adult rat DRG neurons (IKlt in Gold et al. 1996) and in neurons from the auditory brain stem nuclei (Rathouz and Trussel 1998), it differs notably from several other sensory neurons. For example, its half activation potential is 30 mV more negative that in neonatal rat DRG neurons (McFarlane and Cooper 1991), and 20 mV more negative than in several cortical neurons (see Locke and Nerbonne 1997a). Such a property is of importance regarding its putative function in limiting firing by holding the membrane potential near Ek with the consequence of inducing rapid accommodation during sustained depolarization (Oyelese and Kocsis 1996). Although the majority of IK in other sensory neurons (Kostyuk et al. 1981; McFarlane and Cooper 1991; Rathouz and Trussel 1998), and other cell types (see Rudy 1988) are usually described as noninactivating currents, ITEA displays voltage-dependent inactivation with a half inactivation potential around -65 mV. Similar observations were previously reported in rat visual cortical neurons (Locke and Nerbonne 1997a), and in adult rat DRG neurons (Gold et al. 1996). Gold et al. (1996) showed that two out of the three types of IK found in these cells (termed Iki and Iklt) displayed such properties. In most respects, ITEA found in vestibular neurons is identical to Iklt described in those cells.

IDTX

This current shares most of the properties that define ID (a alpha -DTX-sensitive current) in sensory neurons (Everill et al. 1998; Stansfeld and Feltz 1988; Stansfeld et al. 1986, 1987), namely its low-voltage threshold, fast time course of activation and deactivation, voltage-dependent activation, and partial steady-state inactivation. K+ currents possessing these properties have been described in a wide variety of neurons (Brew and Forsythe 1995; Foehring and Surmeier 1993; Rathouz and Trussel 1998; Reid et al. 1999; Southan and Robertson 1998; Storm 1988). In some preparations, discrepancies can be noticed in the voltage dependence for activation (Locke and Nerbonne 1997a; McFarlane and Cooper 1991; Wu and Barish 1992) and in the sensitivity to alpha -DTX (Everill et al. 1998; Wu and Barish 1992). Such discrepancies could be the consequence of a lack of selectivity of alpha -DTX, when used over nanomolar concentration range, since other types of K+ channels have been reported to be sensitive to alpha -DTX. For example, hippocampal neurons express a population of IA channel (Ficker and Heinemann 1992) that possess a binding site for alpha -DTX (Halliwell et al. 1986), whereas IA channels are usually reported to be insensitive to alpha -DTX. In our experimental conditions, no effect of alpha -DTX on the kinetics or the voltage dependence of IBDS was noticed. Similarly an alpha -DTX sensitivity has been described for IK in guinea pig DRG neurons (Penner et al. 1986). This was not the case in the present study. Another explanation for the variability is that several types of alpha -DTX-sensitive K+ currents, distinct from IA or IK are expressed in different neurons, and sometimes within a single neuron as reported in human peripheral myelinated neurons (Reid et al. 1999). To determine whether a single type or several populations of IDTX are present in vestibular primary neurons, it will be important to determine which alpha -subunits form the IDTX K+ channels. The coupled sensitivities of IDTX to alpha -DTX and MgTX we described here is a first step on this way. Only three types of alpha -subunit, Kv1.1, Kv1.2, and Kv1.6, have been reported to be sensitive to alpha -DTX, and only Kv1.2 is insensitive to TEA (Grissmer et al. 1994). Although this observation suggests that Kv1.2 is one of the alpha -subunits that form the IDTX channel in vestibular neurons, it cannot be excluded that Kv1.1 and Kv1.6 may also be involved in the structure of this channel, since they have been described as heteromeric structures with Kv1.2 in K+ channels of the rat cerebellum (Koch et al. 1997). The involvement of IDTX in limiting repetitive firing both in sensory (Stansfeld et al. 1986) and cortical neurons (Brew and Forsythe 1995; Wu and Barish 1992) is widely accepted. However, the precise role of ID in shaping a single action potential remains somewhat uncertain. By directly measuring the action potential-evoked Ca2+ rise in basket cell terminals, Tan and Llano (1999) indicated that 4-AP-sensitive K+ channels are involved in depolarizing the action potential, but not the alpha -DTX-sensitive ones. Opposite observations were made in rat visual cortical neurons, where ID plays an important role in action potential depolarization (Locke and Nerbonne 1997b). Other authors suggest that ID might be a large component of the afterhyperpolarization of action potential in CA1 pyramidal neurons (Golding et al. 1999), or rather be involved in the control of resting membrane potential in basket cells terminals (Robertson and Southan 1999).

IBDS

IBDS shares most of the properties previously reported for IA in a wide variety of neurons (Rudy 1988). IA is thought to modulate the timing of repetitive action potential generation, the repolarization of single action potential, and the time required to reach the threshold to fire an action potential (Storm 1988; Tan and Llano 1999; Wu and Barish 1992). A threshold for activation around -60 mV, an absence of sensitivity to alpha -DTX, and time constants for activation and inactivation in the same range to those of IBDS-I have been described for IA (rise time <0.5 ms; decay time 10-25 ms at 0 mV) in DRG neurons (Stansfeld et al. 1987). Kinetics and voltage dependence for activation and inactivation in the same range as those found for IBDS-I were reported for IA in cochlear ganglion neurons (Garcia-Diaz 1999) [tau ac at 0 mV <0.2 ms; Vac(1/2) -38.4 mV; Vinac(1/2) = -75 mV], and in nodose neurons (McFarlane and Cooper 1991) [tau ac at -10 mV = 1-1.5 ms and decay time 10-30 ms; Vac(1/2) = -21 mV; Vinac(1/2) = -73 mV]. IBDS-I found in vestibular neurons displays a kinetic of recovery from inactivation slower than those reported in dorsal cochlear nucleus pyramidal cells (Kanold and Manis 1999), and in basal ganglia and basal forebrain neurons (Tkatch et al. 2000), but faster than those reported in hippocampal neurons (Martina et al. 1998; Wu and Barish 1992), and in striatal neurons (Song et al. 1998). Attempts to identify the subunits that form IA-type channels in other neurons revealed that Kv 1.4, Kv 3.3 and Kv 3.4, Kv 4.2 and Kv 4.3 alpha -subunits underlie the transient K+ current (Diochot et al. 1998; Ohya et al. 1997; Rudy et al. 1999). Analysis of the biophysical properties of the current indicated that the time course of inactivation of IA tends to be voltage independent when carried by various Kv 4 channels, and highly voltage dependent, as is the case for IBDS-I, when carried by other subunits (Everill et al. 1998). An interesting observation is the fact that in our preparation, IBDS-I displays a sensitivity to nanomolar concentrations of BDS-I. This result is of interest first because it constitutes the first demonstration of a BDS-I sensitivity of a neuronal IA, and second because it suggests the identity of at least one of the subunits that form the channel, since the toxin isolated from sea anemone is a selective blocker of the K+ channels form with Kv3.4 alpha -subunit (Diochot et al. 1998). Diochot et al. (1998) also reported a small blocking effect of BDS-I on the Kv1.2 containing K+ channel expressed in COS cells. It is unlikely, however, that IBDS-I is in fact an inactivating component of the ID current since the concentration of BDS-I we used was 40 times smaller than those used in the COS-transfected cells. Again, alternative experimental approaches will be needed to determine which alpha -subunits form the IBDS-I channels present in vestibular neurons, and whether they are assembled in heteromeric structures.

Our results demonstrate that the distribution of the three distinct depolarization-activated K+ currents in vestibular neurons is heterogeneous. Although ITEA is the major current, the proportion of IDTX and IBDS-I varies considerably from one neuron to another. Whether these observations reflect different functional populations of neurons remains uncertain since the current density ratios of IBDS-I:IDTX within neurons are distributed as a range rather than a distribution into distinct groups. Moreover, there was no obvious correlation between the size or the capacitance of the recorded cells and the respective densities of the two 4-AP-sensitive currents. Similar observations have been reported in rat DRG neurons (Everill et al. 1998), where IK, ID, and IA are expressed with a large variability between cells, and as a range rather than in distinct sub-groups.

The role that each of the three K+ currents plays in determining the electrophysiological properties of vestibular neurons now needs to be correlated with the different firing patterns (regular and irregular), exhibited by the primary afferent (Goldberg et al. 1984). The presence of the three types of K+ currents within all the neurons investigated and the large variability in the expression of the two fast activating currents suggest that, if they are involved in shaping action potential, a variation in their relative expression would be sufficient to account for the diversity in the electrical activity recorded in these neurons. The use of specific toxins should permit determination of the involvement of each of the voltage-activated K+ currents in the electrical activity of the vestibular neurons. However, such investigations should be carefully conducted, since these compounds could interact with other voltage-activated ionic conductances (e.g., BDS-I has a slight effect of on sodium current) (Diochot et al. 1998).


    ACKNOWLEDGMENTS

The authors thank Prof. J. M. Goldberg and Dr. J. Ashmore for carefully reading the manuscript. We are grateful to G. Dayanithy, M. Desarménien, F. Scamps, J. Valmier, and G. Lennan for helpful discussions and comments on the manuscript. We also thank S. Diochot and L. Béress (Klinikum der Christian-Albrechts-Universität zu Kiel, Kiel, Germany) for providing BDS-I.

This work was supported by Division de la Recherche et des Etudes Doctorales---Université Montpellier 2 to C. Chabbert and Centre National des Etudes Spatiales Grant 99-793.


    FOOTNOTES

* C. Chabbert and J. M. Chambard contributed equally to this project.

Address for reprint requests: C. Chabbert, INSERM U432, Neurobiologie et Développement du Système Vestibulaire, UM2, cp 089 place E. Bataillon, 34095 Montpellier cedex 5, France (E-mail: chabbert{at}univ-montp2.fr).

Received 20 July 2000; accepted in final form 20 November 2000.


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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society