Alphaxalone Activates a Cl
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
 |
ABSTRACT |
Valeyev, Alexander Y.,
John C. Hackman,
Alice M. Holohean,
Patrick M. Wood,
Jennifer L. Katz, and
Robert A. Davidoff.
Alphaxalone Activates a Cl
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
(5
-pregnane-3
-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.
 |
INTRODUCTION |
There is already persuasive evidence that neurosteroids work at
specific allosteric modulatory sites on GABAA
receptor/Cl
channel complexes. In particular,
the anesthetic alphaxalone (5
-pregnane-3
-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
).
 |
METHODS |
The procedures and techniques used are identical to those
described in the preceding paper (Valeyev et al. 1999
).
 |
RESULTS |
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 (
1) and fast (
2)
time constants. The mean open times
1 and
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. , 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.
|
|
 |
DISCUSSION |
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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