Alphaxalone Activates a Clminus Conductance Independent of GABAA Receptors in Cultured Embryonic Human Dorsal Root Ganglion Neurons

Alexander Y. Valeyev,1 John C. Hackman,1,2 Alice M. Holohean,1,2 Patrick M. Wood,3 Jennifer L. Katz,3 and Robert A. Davidoff1,2

 1Neurophysiology and Spinal Cord Pharmacology Laboratories, Veterans Affairs Medical Center; and  2Department of Neurology and  3The Miami Project to Cure Paralysis, University of Miami School of Medicine, Miami, Florida 33101


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Valeyev, Alexander Y., John C. Hackman, Alice M. Holohean, Patrick M. Wood, Jennifer L. Katz, and Robert A. Davidoff. Alphaxalone Activates a Clminus Conductance Independent of GABAA Receptors in Cultured Embryonic Human Dorsal Root Ganglion Neurons. J. Neurophysiol. 82: 10-15, 1999. Whole cell and cell-attached patch-clamp techniques characterized the neurosteroid anesthetic alphaxalone's (5alpha -pregnane-3alpha -ol-11,20-dione) effects on GABAA receptors and on Cl- currents in cultured embryonic (5- to 8-wk old) human dorsal root ganglion neurons. Alphaxalone applied by pressure pulses from closely positioned micropipettes failed to potentiate the inward Cl- currents produced by application of GABA. In the absence of GABA, alphaxalone (0.1-5.0 µM) directly evoked inward currents in all dorsal root ganglion neurons voltage-clamped at negative membrane potentials. The amplitude of the current was directly proportional to the concentration of alphaxalone (Hill coefficient 1.3 ± 0.15). The alphaxalone-induced whole cell current was carried largely by Cl- ions. Its reversal potential was close to the theoretical Cl- equilibrium potential, changing with a shift in the external Cl- concentration as predicted by the Nernst equation for Cl- ions. And because the alphaxalone-current was not suppressed by the competitive GABAA receptor antagonist bicuculline or by the channel blockers picrotoxin and t-butylbicyclophosphorothionate (TBPS; all at 100 µM), it did not appear to result from activation of GABAA receptors. In contrast to GABA-currents in the same neurons, the whole cell current-voltage curves produced in the presence of alphaxalone demonstrated strong inward rectification with nearly symmetrical bath and pipette Cl- concentrations. Fluctuation analysis of the membrane current variance produced by 1.0 µM alphaxalone showed that the power density spectra were best fitted to double Lorentzian functions. The elementary conductance for alphaxalone-activated Cl- channels determined by the relationship between mean amplitude of whole cell current and variance was 30 pS. Single-channel currents in cell-attached patches when the pipette solution contained 10 µM alphaxalone revealed a single conductance state with a chord conductance of ~29 pS. No subconductance states were seen. The current-voltage determinations for the single-channels activated by alphaxalone demonstrated a linear relationship. Mean open and shut times of single alphaxalone-activated channels were described by two exponential decay functions. Taken together, the results indicate that in embryonic human DRG neurons, micromolar concentrations of alphaxalone directly activate Cl- channels whose electrophysiological and pharmacological properties are distinct from those of Cl- channels associated with GABAA receptors.


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There is already persuasive evidence that neurosteroids work at specific allosteric modulatory sites on GABAA receptor/Cl- channel complexes. In particular, the anesthetic alphaxalone (5alpha -pregnane-3alpha -ol-11,20-dione), a synthetic steroid whose structure is related closely to some naturally occurring pregnane steroids, modulates and directly activates GABAA receptors in a number of neuronal preparations (Lambert et al. 1995; Majewska 1992; Olsen and Sapp 1995; Paul and Purdy 1992). The present study stands in sharp contrast: we have found that in cultured embryonic human dorsal root ganglion (DRG) neurons, alphaxalone activates a Cl- conductance independent of the GABAA receptor.

In previous studies, low concentrations of both alphaxalone and pregnane steroids potently enhanced GABAA-receptor-mediated membrane responses (Barker et al. 1987; Callachan et al. 1987; Cottrell et al. 1987; Gee et al. 1988; Harrison and Simmonds 1984) by a process involving prolongation of the mean open time of the associated GABA-activated Cl- channels (Barker et al. 1987; Mistry and Cottrell 1990; Twyman and Macdonald 1992). In the absence of GABA, higher concentrations of alphaxalone and pregnane steroids are reported to directly open the Cl- channels of GABAA receptors (Barker et al. 1987; Callachan et al. 1987; Cottrell et al. 1987; Peters et al. 1988). The basis of this neurosteroid interaction with GABAA receptors is unknown, although it is believed that both modulating and activating effects of neurosteroids are mediated by sites that are distinct from the binding sites on the GABAA receptor/Cl- channel complex for GABA, barbiturates, benzodiazepines, and picrotoxin (Cottrell et al. 1987; Gee et al. 1988).

The present studies focus on how alphaxalone affects cultured, embryonic human DRG neurons, the same neurons described in the preceding paper (Valeyev et al. 1999). In contrast to findings at other, nonhuman GABAA receptors, we found that micromolar concentrations of alphaxalone did not enhance GABAA-receptor-mediated membrane responses but did directly activate a Cl- current. Moreover the alphaxalone-induced Cl- current had biophysical and pharmacological properties that differed from those of GABA-activated Cl- currents in the same neurons. The data suggest that in cultured, embryonic human DRG neurons, alphaxalone does not bind to the previously described allosteric modulatory site for neurosteroids located on the GABAA-receptor Cl- channel complex. A preliminary account of this work has appeared (Valeyev et al. 1996).


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The procedures and techniques used are identical to those described in the preceding paper (Valeyev et al. 1999).


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In contrast to effects at GABAA receptors on other types of neurons, alphaxalone did not potentiate the inward currents produced by GABA under whole cell, voltage-clamp conditions (not illustrated). However, when voltage-clamped at negative membrane potentials, embryonic human DRG neurons responded with inward currents to micromolar applications of alphaxalone (concentrations ranging from 1.0 to 5.0 µM; Fig. 1A) in the absence of exogenous GABA in the medium. This occurred in essentially all neurons. As seen in Fig. 1A, the alphaxalone-induced currents were dependent on the concentration of alphaxalone applied. The Hill coefficient was 1.3 ± 0.15 (mean ± SE; Fig. 1B). When the intrapipette [Cl-] was 134 mM, the mean peak current activated by alphaxalone (1.0 µM) measured 28.0 ± 5.5 pA (n = 12) (holding potential, Vh, of -60 mV). The currents required <1.0 s to reach peak amplitudes after exposure to alphaxalone, did not attenuate during prolonged applications (Fig. 4A), and returned quickly to baseline after removal. Activation of the Cl- current by alphaxalone (10 µM) was not prevented by internal perfusion of the neurons with EGTA concentrations of 10.0 mM (n = 6).



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Fig. 1. Alphaxalone concentration-responses in cultured embryonic human dorsal root ganglion neurons. A: whole cell current traces produced by applications of increasing concentrations of alphaxalone in the absence of GABA in the medium. B: alphaxalone concentration-response relationship. Alphaxalone responses are plotted as means ± SE of 3-5 independent determinations of the peak current normalized to the peak current induced by 1.0 µM alphaxalone.

Currents directly activated by alphaxalone are carried by Cl- ions

Current-voltage (I-V) curves show that when Cl- was the main intracellular anion ([Cl-]o/[Cl-]i = 151 mM/140 mM), the reversal potential for the alphaxalone-induced response was close to 0 mV (-4.4 ± 2.1 mV, n = 6). Moreover, as can be seen in Fig. 2, alphaxalone-induced currents displayed strong inward rectification at positive holding potentials (i.e., outward currents were smaller than inward currents at equivalent potentials). In addition, reduction of [Cl-]i (to 90 mM by isotonic replacement of CsCl by CsF) shifted the reversal potential to -33.3 ± 5.6 mV (n = 6), a value that conformed to the expected change in the theoretical Nernstian Cl- equilibrium potential for a Cl--current (theoretical value: -30.0 mV).



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Fig. 2. Whole cell current-voltage (I-V) relationship for alphaxalone-activated Cl- current. Peak amplitudes of currents induced by alphaxalone (2.5 µM) applied by 1- to 5-s pressure pulses plotted against membrane potential for 2 levels of [Cl-]i (n = 6 neurons for each Cl- level). Inward rectification was seen at positive membrane potentials. Brackets indicate SE of mean.

Alphaxalone-induced currents are not blocked by GABAA receptor antagonists

If the Cl- currents activated by alphaxalone are mediated by GABAA receptors, these currents should be blocked by antagonist compounds that bind to various sites on the GABAA receptor/channel complex (Barker et al. 1987; Cottrell et al. 1987; Ong et al. 1988). However, as seen in Fig. 3, alphaxalone-induced whole cell currents were found to be insensitive both to the specific, competitive GABAA receptor antagonist bicuculline (100 µM, n = 6), and to the channel blockers picrotoxin (100 µM, n = 6) and t-butylbicyclophosphorothionate (TBPS, 100 µM, n = 6).



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Fig. 3. Alphaxalone-activated currents are unaffected by GABAA antagonists. Downward deflections in whole cell patch-clamp recordings represent inward Cl- currents. Left: currents activated by alphaxalone (2.5 µM) applied by pressure injection. Right: currents activated by alphaxalone in the presence of bicuculline methochloride (BCC, 100 µM), picrotoxin (PTX, 100 µM), and t-butylbicyclophosphorothionate (TBPS, 100 µM). All neurons were voltage clamped at -60 mV. Applications of alphaxalone represented by bars above current traces.

Fluctuation analysis of alphaxalone-evoked current noise

Current fluctuation analysis was carried out on neurons voltage-clamped at -60 mV. Alphaxalone applied in a concentration of 1.0 µM evoked nondesensitizing responses lasting 60-90 s (Fig. 4A). Between three and seven spectra were obtained from each neuron studied. Figure 4B shows an example of the power density spectrum of alphaxalone-induced Cl- current fluctuations. The spectrum in all neurons was well described by the sum of two Lorentzian components with slow (tau 1) and fast (tau 2) time constants. The mean open times tau 1 and tau 2 were 67.2 ± 11.7 and 3.2 ± 2.1 ms (n = 12), and the elementary conductance determined by the relationship between mean amplitude of whole cell current and variance was 30 ± 3.4 pS (n = 12).



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Fig. 4. Fluctuation analysis of alphaxalone-activated Cl- current under whole cell clamp. A, top: low-gain DC record; bottom: high-gain AC record of currents evoked by alphaxalone (1.0 µM) in a neuron voltage-clamped at -60 mV. Application of alphaxalone represented by bar above current traces. B: spectral density plot of current fluctuations during alphaxalone application. Difference spectrum fit by a double Lorentzian function. down-arrow , corner frequencies ( fc) of 20.2 and 34.6 Hz corresponding to estimated apparent mean open times for alphaxalone-activated Cl- channels of 78.8 and 4.6 ms.

Single alphaxalone-activated channel currents in cell-attached patches

On-cell recordings from 21 intact neurons were used to evaluate directly the elementary properties of alphaxalone-activated Cl- channels. Without alphaxalone in the intrapipette solution, no spontaneous channel activity was recorded. Single-channel currents, seen in all patches when pipette solutions contained alphaxalone (10 µM), were composed of small-amplitude, brief-duration openings that usually occurred as single events or as brief bursts of openings and closings (see Fig. 5 for sample records). By measuring their amplitudes, it was possible to produce histograms for which the distribution of amplitudes was best fitted with one Gaussian function (Fig. 6). These amplitude distributions revealed no indication of subconductance states. Alphaxalone-induced single channel currents demonstrated a mean current amplitude of 0.32 ± 0.04 pA (Vp = -57 mV). Figure 7 illustrates that the amplitude of single-channel currents varied linearly with the patch potential (Vp). The calculated chord conductance derived from single-channel I-V relationships was 29.0 ± 3.1 pS.



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Fig. 5. Single-channel currents evoked by alphaxalone. Representative single-channel currents recorded in cell-attached configuration at different patch potentials. Alphaxalone (10 µM) was present in the pipette solution. Patch potential (Vp) indicated for each trace. Upward deflections show outward current corresponding to inward flow of Cl- ions.



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Fig. 6. Alphaxalone-activated single-channel current histogram. Channel currents were recorded in a cell-attached patch with alphaxalone (10 µM) in the recording pipette. Distribution of amplitudes was best fit by a single Gaussian function with a peak amplitude of -0.32 ± 0.04 pA. Vp was -57 mV.



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Fig. 7. I-V relationship of alphaxalone-activated single channel currents. Single-channel currents induced by alphaxalone (10 µM) in cell-attached patches from 4 neurons plotted against Vp. Each point represents the mean of a Gaussian distribution of channel amplitudes.

To evaluate the kinetic properties of the single-channel conductance state activated by 10 µM alphaxalone in on-cell patches, both open and closed dwell-time distributions were determined. Figure 8A shows that the distribution of open-channel events for a patch held at Vp = +40 mV was best fitted with the sum of two exponential components with mean time constants of 2.7 ± 1.9 and 65.3 ± 23.5 ms. In open-time histograms from six patches, a best fit also was obtained with double exponential functions. These data suggest that alphaxalone-activated channels have at least two open states. Similarly, kinetic analysis of the closed-time distributions for a patch held at Vp = +40 showed two exponential components with mean time constants of 17.9 ± 9.5 and 147.2 ± 26.8 ms (Fig. 8B). The best fits of the distributions of closed times from six patches was obtained with two exponential functions with mean time constants of 17.9 ± 9.5 and 147.2 ± 26.8 ms.



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Fig. 8. Kinetics of alphaxalone-gated channels. Data refer to single-channel on-cell recordings obtained with alphaxalone (10 µM) in the recording pipette. Vp = +40 mV. A: open-time distribution for single channel currents best fit by the sum of 2 exponentials having decay time constants of 2.7 ± 1.9 and 65.3 ± 23.5 ms. B: closed-time distribution best fit by the sum of 2 exponentials having time constants of 17.9 ± 9.5 and 147.2 ± 26.8 ms.


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Alphaxalone is reported to activate a Cl- conductance via the GABAA receptor/Cl- channel complex in a number of neuron types (Lambert et al. 1995; Majewska 1992; Olsen and Sapp 1995; Paul and Purdy 1992). Of interest in the present results are the unique findings in cultured embryonic human DRG neurons: namely that micromolar concentrations of alphaxalone directly activate a Cl- current with electrophysiological and pharmacological properties distinct from the GABA-activated Cl- current in the same neurons. In other words, the actions of alphaxalone in these experiments do not appear to be mediated either by allosteric modulation of the GABAA receptor complex or by direct allosteric activation of the Cl- ion conductance associated with GABAA receptors.

This view is supported by several different lines of evidence. First of all, micromolar concentrations of alphaxalone did not potentiate Cl- currents produced by GABA. Nor did the compounds direct actions require the presence of GABA. We found alphaxalone to directly activate Cl- channels in cell-attached patches in the absence of GABA in the recording pipette. Alphaxalone also activated whole cell Cl- currents when exogenous GABA was not added to the medium.

The reversal potential for the alphaxalone-induced response was close to the theoretical Nernstian Cl- equilibrium potential calculated from the given extra- and intracellular Cl- concentrations both when Cl- was the main intracellular anion and when [Cl-]i was reduced. And because nonspecific cation currents demonstrate negligible anion permeability, it is doubtful that the alphaxalone-activated current represented a nonspecific cation current (Colquhoun et al. 1981; Yellen 1982). Similarly, the finding that alphaxalone-induced currents were not affected by the presence of EGTA in the internal solution presumably indicates that a Ca2+-dependent Cl- conductance cannot account for the Cl- current investigated here (Korn and Weight 1987; Mayer 1985).

Patterns of rectification set the alphaxalone-induced Cl- current apart from the GABA-induced Cl- current in embryonic human DRG neurons and in nonhuman neurons. In various preparations, both a lack of rectification and the presence of outward rectification---but never inward rectification---of GABA-generated whole cell Cl- currents have been reported (Allen and Albuquerque 1987; Curmi et al. 1993; Fatima-Shad and Barry 1992; Gray and Johnston 1985; Hamill et al. 1983; Smith et al. 1989; Valeyev et al. 1999; Weiss et al. 1988). In sharp contrast, alphaxalone-induced whole cell currents displayed strong inward rectification at positive holding potentials. In our cultured embryonic human DRG neurons, GABA-activated whole cell Cl- currents demonstrated outward rectification (Valeyev et al. 1999). It should be noted that the alphaxalone-induced current also differs from the inwardly rectifying Cl- conductance described in frog oocytes, Aplysia neurons, and rat hippocampal neurons (Chesnoy-Marchais 1983; Mager et al. 1995; Parker and Miledi 1988; Staley 1994). The latter current is voltage dependent, whereas the amplitude of single-channel currents activated by alphaxalone varies linearly with the patch potential. With regard to their single-channel I-V relationships, however, results from both alphaxalone- and GABA-activated single-channel studies were similar to one another, showing a lack of rectification (Valeyev et al. 1999). Such a linear I-V relationship favors a voltage-dependent change in channel gating as the mechanism underlying the display of rectification in whole cell recordings.

Nearly all reports indicate that subconductance states are generated by GABA at GABAA receptors (Bormann and Clapham 1985; Hamill et al. 1983; Macdonald et al. 1989; Mistry and Hablitz 1990; Ozawa and Yuzaki 1984; Taleb et al. 1987; Weiss et al. 1988). Likewise, in cultured embryonic human DRG neurons, GABA activates a subconductance state (Valeyev et al. 1999). Significantly, recordings of single-channel activity produced by exposure to alphaxalone failed to reveal a subconductance state. And finally, alphaxalone-induced Cl- currents were not blocked by bicuculline, picrotoxin, or TBPS when these compounds were applied in concentrations that block GABA responses both in cultured embryonic human DRG cells (Valeyev et al. 1999) and in noncultured, adult DRG neurons from a number of nonhuman species (Akaike et al. 1985; Dunlap 1984; Gallagher et al. 1978; Inoue and Akaike 1988). Bicuculline, picrotoxin, and TBPS appear to act at different sites of the GABAA receptor/Cl- channel complex. Bicuculline is established firmly as a specific, competitive antagonist of the GABAA recognition site (Akaike et al. 1985, 1987); picrotoxin and some bicyclic cage compounds such as TBPS behave as noncompetitive Cl- channel blockers at GABAA receptors (Akaike et al. 1985; Newland and Cull-Candy 1992). That none of these compounds blocked alphaxalone-induced currents is further evidence of its independence from the GABAA receptor. In sum, the Cl- currents activated by alphaxalone and by GABA in human embryonic DRG neurons have disparate characteristics.

An association between the effects of general anesthetics and synaptic inhibition has been contemplated for many years (Eccles et al. 1963). It has been suggested that direct GABAA receptor activation is the mechanism by which anesthetics induce general anesthesia (Schulz and Macdonald 1981). We propose that direct activation of a Cl- conductance that does not involve GABAA receptors in neuronal membranes also may be a factor in the anesthetic actions of alphaxalone. This proposal is plausible because the concentrations used in the present experiments are equivalent to those that have been measured in the plasma during surgical anesthesia with alphaxalone in humans (Sear and Prys-Roberts 1979).


    ACKNOWLEDGMENTS

This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-30600 and NS-37946 and the Office of Research and Development, Medical Research Service, Department of Veteran Affairs (VA).


    FOOTNOTES

Address for reprint requests: A. Y. Valeyev, Dept. of Neurology (D4-5), P. O. Box 016960, University of Miami School of Medicine, Miami, FL 33101.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 14 January 1998; accepted in final form 3 March 1999.


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