1Department of Physiology, Tulane University Medical School, New Orleans, Louisiana 70112; and 2Department of Pharmacology, Chonbuk University Dental School, Chonju, Korea 561-756
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ABSTRACT |
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Lee, Hye Kyung, Lian Liu, and Keith S. Elmslie. Effect of High Ba2+ on Norepinephrine-Induced Inhibition of N-Type Calcium Current in Bullfrog Sympathetic Neurons. J. Neurophysiol. 83: 791-795, 2000. The voltage-dependent inhibition of N-type calcium current by neurotransmitters is the best-understood example of neuronal calcium channel inhibition. One of the mechanisms by which this pathway is thought to inhibit the calcium current is by reducing the permeation of divalent cations through the channel. In this study one prediction of this hypothesis was examined, that high concentrations of divalent cations reduce the maximum neurotransmitter-induced inhibition. Norepinephrine (NE)-induced inhibition was compared in external solutions containing either 2 or 100 mM Ba2+. Initially, NE dose-response curves were generated by averaging data from many neurons, and it was found that the relationship was right shifted in the high-Ba2+ external solution without an effect on maximum inhibition. The IC50 was 0.6 and 3 µM in 2 and 100 mM Ba2+, respectively. This shift was verified by comparing the effect of NE on single neurons exposed to both 2 and 100 mM Ba2+. The inhibition induced by 1 µM NE was reduced in 100 mM Ba2+ compared with that in 2 mM Ba2+. However, the response to 100 µM NE was identical between high and low Ba2+. Thus, divalent cations appear to act as a competitive inhibitor of NE binding, which likely results from these ions' interacting with negatively charged amino acids that are important for catecholamine binding to adrenergic receptors. Because the maximum inhibition induced by NE was similar in low and high Ba2+, the effect of inhibition on single N-type calcium channels was not altered by the divalent cation concentration.
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INTRODUCTION |
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Many neurotransmitters have been shown to inhibit
N-type calcium channels via a voltage-dependent and membrane delimited
pathway (Hille 1994; Jones and Elmslie
1997
). The inhibition appears to be mediated intracellularly by
G protein
subunits (Herlitze et al. 1996
;
Ikeda 1996
), which are thought to bind directly to the
N-type calcium channel. The voltage dependence appears to arise from
the transient dissociation of the
subunit from the N-channel
(Ehrlich and Elmslie 1995
; Elmslie and Jones
1994
; Golard and Siegelbaum 1993
). This
inhibition has been hypothesized to place the N-channel into a
"reluctant" gating mode (Bean 1989
; Elmslie
et al. 1990
). Based on whole-cell recordings, this reluctant gating mode is thought to possess a lower open probability
(Po), shorter open times (Elmslie et al.
1990
), and a smaller single-channel current amplitude
(Kuo and Bean 1993
) than the normal "willing" mode.
If this is true, N-channel inhibition results from changes in both
permeation and gating. Initial reports of single N-channel gating in
the presence of neurotransmitter failed to observe reluctant gating
(Carabelli et al. 1996
; Patil et al.
1996
). However, support for reluctant gating comes from
whole-cell recordings showing that tail currents during inhibition are
faster than control (Boland and Bean 1993
;
Elmslie et al. 1990
). In addition, two laboratories have
presented preliminary data showing that putative reluctant events can
be observed at voltages depolarized to those used previously (Colecraft et al. 1999
; Lee and Elmslie
1997
;). As predicted by the willing-reluctant model, these
putative reluctant events have low Po and brief
openings compared with willing events.
It is clear that recordings from single N-channels are key to
determining the effect of inhibition on the channel. However, Kuo and Bean (1993) presented data suggesting that the
isotonic Ba2+ typically used to record single
calcium channels may obscure the effect of inhibition on N-channel
permeation. Using bullfrog sympathetic neurons they showed that
N-channel inhibition by GTP
S (a direct G protein activator) was
reduced in high Ba2+ (Kuo and Bean
1993
). They postulated that this reduction resulted from a loss
of the permeation component of inhibition. Thus inhibition in high
Ba2+ may result primarily from changes in gating,
whereas inhibition in low Ba2+ results from
changes in both gating and permeation.
Since Kuo and Bean (1993) published their finding, it
has been demonstrated in bullfrog sympathetic neurons that whole-cell recordings using isotonic Ba2+ are contaminated
by a previously unrecognized calcium current, which we have called
novel current or, more recently, Ef-current (Elmslie et al. 1994
; Elmslie 1997
). This
current is insensitive to dihydropyridines,
-conotoxin GVIA, and NE
(Elmslie et al. 1994
), and we have estimated that it
comprises 30-50% of the total whole-cell calcium current in external
solutions containing 100 mM Ba2+. However,
Ef-current has not been observed in recordings using a 2-mM Ba2+ external solution (Elmslie et
al. 1992
; Elmslie et al. 1994
). The presence of
the neurotransmitter-insensitive Ef-current could be
responsible for the apparent reduction of calcium current inhibition in
high Ba2+ observed by Kuo and Bean
(1993)
. Therefore, we compared the NE-induced inhibition of
N-type calcium current in low and high Ba2+ to
determine whether high Ba2+ alters inhibition. We
found that, with Ef-current removed, the maximum
inhibition of N-current by NE was the same in both low and high
Ba2+. If gating and permeation are altered by
inhibition in low Ba2+, then both are altered in
high Ba2+. Thus, single N-channel recordings can
be used to test the prediction that inhibition alters N-channel
permeation. Part of this work has been published in abstract form.
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METHODS |
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Neurons were dissociated from paravertebral sympathetic ganglia
of adult bullfrogs (Rana catesbeiana), as described
previously (Elmslie 1992). Isolated neurons were
maintained at 4°C until use. Whole-cell currents were recorded at
room temperature.
The internal pipette solution consisted of (in mM) 82.6 NMG (N-methyl-D-glucamine) · Cl, 2.5 NMG · HEPES, 10 NMG2 · EGTA, 5 Tris2 · ATP, 6 MgCl2, and 0.3 Li2 · GTP, titrated to pH 7.2 with NMG base. The low-Ba2+ external solution contained (in mM) 2 BaCl2, 117.5 NMG · Cl, and 2.5 NMG · HEPES (pH 7.2). The high-Ba2+ external contained (in mM) 100 BaCl2, 10 NMG · HEPES, 5 MnCl2, and 10 tetraethylammonium · Cl, pH 7.2.
The electrodes were fabricated from Corning 7052 glass (1.5 mm OD, 0.86 mm ID; A-M Systems, Everett, WA), and the series resistance ranged from
1 to 2 M. Series resistance compensation was set to 95% using the
circuitry of the Axopatch 200A amplifier (Axon Instruments, Foster
City, CA). The experiment was controlled by a Macintosh II computer
running S3 data acquisition software written by Dr. Stephen Ikeda
(Guthrie Research Institute, Sayre, PA). Records were leak subtracted
using averaged and scaled hyperpolarizing steps of one-quarter
amplitude. As previously described, currents were measured as the
average between 2.5 and 5 ms into the voltage step (Elmslie
1992
). The facilitation ratio was calculated by dividing the
postpulse current (after strong depolarization) by the prepulse current
(preceding strong depolarization). In control and recovery, this value
was ~1. During NE, this value was positively correlated with the
magnitude of inhibition.
In our first set of experiments, we exposed single neurons to NE concentrations of 1, 3, 10, 30, and 100 µM to obtain a dose-response relationship (Fig. 1). Only neurons exposed to at least three NE concentrations were analyzed. For these experiments each cell was exposed to a single external solution (i.e., either 2 or 100 mM Ba2+). The different NE concentrations were applied in a random order across cells to minimize the effects of desensitization on the averaged data.
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However, NE responses can be highly variable when compared across cells. In our second set of experiments (Fig. 2), we tested a single concentration of NE on the same cell in both external solutions. This allowed us to make within-cell comparisons, but the multiple NE applications induced desensitization. We used the following procedure to compensate for the desensitization. For each cell, one Ba2+ solution was selected as the "control" external and the other as the "test" external. The Ba2+ external solution selected as control was varied across cells. NE responses were measured in each external solution with responses in the control solution bracketing the NE response elicited in the test solution. The two NE responses in the control Ba2+ solution were averaged to compensate for desensitization during the experiment and compared with the NE response in the test external solution.
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The third set of experiments in this study examined the effect of
holding potential on the NE response in high external
Ba2+ (Fig. 3). In
these experiments, applications of NE were made on each cell as the
holding potential was varied between 80 mV and
40 mV. One holding
potential was selected as control and the other as test. NE responses
during the control holding potential bracketed that during the test
holding potential, and the two control responses were averaged for
comparison with the test response. The control holding potential was
varied across cells.
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RESULTS |
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Changing the external Ba2+ concentration
from 2 to 100 mM had several effects on the calcium current. The
current amplitude increased approximately fourfold, voltage-dependent
activation and inactivation shifted ~40 mV to the right, and an
-conotoxin GVIA-insensitive calcium current was revealed
(Elmslie et al. 1994
). We were interested in the effect
of high Ba2+ on the NE-induced inhibition of
N-type calcium current and therefore made several adjustments to
compensate for the other effects of high Ba2+ on
the current. To compensate for the shift in voltage-dependent properties, the holding potential and step voltages were generally depolarized by 40 mV when testing in high Ba2+
(see Fig. 2). The depolarized holding potential had the additional benefit of almost completely inactivating Ef-current
(Elmslie et al. 1994
), so that the NE effect was not
altered by the presence of this NE-insensitive current. Finally,
Mn2+ was added to the 100-mM
Ba2+ external solution to reduce the amplitude of
the current to a level that could be well voltage clamped. Previously,
we have demonstrated that observations made in
high-Ba2+-containing external solutions are not
altered by the presence of Mn2+ (Elmslie
et al. 1994
).
The effect of high Ba2+ on the NE response was
initially examined by comparing dose-response relationships generated
by exposing single neurons to a range of NE concentrations (1-100
µM) in a single external Ba2+ solution (either
2 or 100 mM). If the effect of high Ba2+ is to
block the permeation component of inhibition, we predicted that
inhibition would be reduced at all NE concentrations in high Ba2+ compared with low
Ba2+. Alternatively, if
Ba2+ was acting as a competitive inhibitor of NE
binding to its receptor, N-current inhibition would be reduced at low
NE concentrations, but high concentrations of NE would overcome the
Ba2+ block. The data show that the NE
dose-response relation was right shifted in
high-Ba2+ external solution with a calculated
IC50 of 3 µM in 100 mM
Ba2+ and a maximum inhibition of 30% (Fig. 1).
In the low-Ba2+ external, the
IC50 was 0.6 µM and the maximum inhibition was 30% (Fig. 1). Although we did not test NE concentrations below 1 µM,
the IC50 compares well with values previously
published using 2 mM Ba2+ (Elmslie
1992; IC50 = 0.6 µM, maximum inhibition
48%). Thus, the high external Ba2+ solution
shifted the NE dose-response relationship to the right without altering
the maximum inhibition, as though Ba2+ were
acting as a competitive inhibitor of the adrenergic receptor.
We have found large variations in NE responses measured from different cells. Therefore, we wanted to verify these results in a completely separate set of experiments in which we compared the effect of a single NE concentration in both high and low Ba2+ within the same neuron (Fig. 2). The high-Ba2+ external solution reduced the inhibition induced by 1 µM NE to an average of 18 ± 8% (mean ± SD, n = 6 cells) when compared with the average inhibition of 52 ± 14% in 2-mM Ba2+ solution (Fig. 2B). This difference was not a result of desensitization, because smaller responses were observed when NE was applied in high Ba2+ before testing the response in low Ba2+. However, high Ba2+ had less effect on the NE-induced inhibition as the concentration of NE increased and became negligible at 100 µM NE (Fig. 2, C and D). Note in Fig. 2 that the average inhibition in 1 µM NE is larger than that in 100 µM NE. This was caused by the variability in the NE response that can result from comparing across cells (Fig. 2). Different neuronal preparations were used to test each NE concentration. The neurons used to examine 1 µM NE responded more strongly than those used to examine 100 µM NE. This demonstrates the importance of the within-cell comparison to verify our conclusion from the dose-response relationships that were generated by averaging responses from many cells.
The previous two sets of experiments have demonstrated that the NE dose-response relationship is right-shifted in our high-Ba2+ external solution when compared with the low-Ba2+ external solution. Mn2+ was added to the high-Ba2+ external solution to reduce the amplitude of the N-current so that we could more easily maintain voltage control in the 100-mM Ba2+ solution. Because Mn2+ was absent from the low-Ba2+ external, we wanted to test whether 100 mM Ba2+ alone could alter the NE inhibition. Using the same procedures as in the previous experiment, we examined the effect of 1 µM NE in both 2- and 100-mM Ba2+ external solutions (without Mn2+) in the same neuron. One micromolar NE was chosen for this test, because the largest difference in inhibition between low- and high-Ba2+ external solutions was observed at this concentration. In five cells examined the average inhibition was 38.7 ± 12.5% in 2 mM Ba2+ and 25.6 ± 11.8% in 100 mM Ba2+. The high-Ba2+ external solution (without Mn2+) decreased the NE-induced inhibition by 33%. However, this reduction was smaller than that observed with the 100-mM Ba2+ + 5-mM Mn2+ external solution. In that solution the inhibition induced by 1 µM NE was reduced by ~65% (for data in both Figs. 1 and 2). Thus 100 mM Ba2+ alone can alter the NE inhibition. The smaller effect of high Ba2+ without Mn2+ could have resulted from poor voltage control during the large calcium currents. On the other hand, both Mn2+ and Ba2+ may have affected the NE response.
Our results show that the maximum NE response is not affected by the
concentration of external Ba2+. However,
Kuo and Bean (1993) showed a reduced maximal inhibition (induced by GTP
S) in high Ba2+ compared with
low Ba2+. One difference is that our
high-Ba2+ recordings were obtained from a holding
potential of
40 mV to inactivate Ef-current,
whereas Kuo and Bean (1993)
used a holding potential of
90 mV. The consequence of the hyperpolarized holding potential
in 100 mM Ba2+ is that
Ef-current comprises a substantial portion of the
total current (30-50%). However, it does not significantly contribute to the whole-cell current when using low-Ba2+
external (Elmslie et al. 1992
; Elmslie et al.
1994
). To test whether Ef-current reduces
the magnitude of the NE response, we compared the effect of 30 µM NE
in high Ba2+ from holding potentials of
80 mV
and
40 mV (Fig. 3). It has been demonstrated that N-current is not
inactivated by the
40-mV holding potential in isotonic
Ba2+ (Elmslie et al. 1994
;
Elmslie 1997
). At a holding potential of
80 mV, the
NE-induced inhibition was significantly reduced, compared with that
measured from a holding potential of
40 mV in the same cell. The
average inhibition was 32 ± 3% and 39 ± 7% for holding potentials of
80 mV and
40 mV, respectively (n = 5). This difference cannot be explained by desensitization, because the
NE response was reduced even when NE was tested from the
80-mV
holding potential before it was tested from the
40-mV holding
potential. The difference in the NE responses amounted to an 18%
reduction in the inhibition at
80 mV when compared with
40 mV,
which is similar to the reduction observed by Kuo and Bean
(1993)
in the GTP
S effect when comparing low versus high
external Ba2+ solutions. Thus, the presence of
the neurotransmitter-insensitive Ef-current reduces
the magnitude of the calcium current inhibition.
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DISCUSSION |
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We have shown that a high-Ba2+ external
solution alters the NE response by shifting the dose-response
relationship to the right, but that the maximum NE inhibition is not
affected. Our results differ from those of Kuo and Bean
(1993), who found that the maximum inhibition induced by
GTP
S was reduced by high external Ba2+. It is
unlikely that the differences between the two studies result from the
methods used to inhibit the current, because GTP
S and NE induce
inhibitions with identical characteristics (Elmslie 1992
). The most likely explanation is that the
high-Ba2+ recordings of Kuo and Bean
(1993)
were contaminated by Ef-current. In the majority of our experiments, we used a holding potential of
40
mV when recording in high-Ba2+ external solution
to inactivate Ef-current. When we used a
hyperpolarized holding potential, similar to Kuo and Bean
(1993)
, the NE response was significantly reduced.
The effect of our high-Ba2+ external solution on
the NE response is consistent with a competitive block of the
adrenergic receptor. Because the concentrations of both
Ba2+ and Mn2+ were altered
between the low- and high-Ba2+ external
solutions, both could be blockers of the receptor. When we used a
high-Ba2+ external without
Mn2+, the reduction in NE inhibition was smaller
than when Mn2+ was present. Thus, it is possible
that both Mn2+ and Ba2+
participate in the apparent competitive block of the adrenergic receptor. Effects of divalent cations on the binding of agonists and
antagonists to adrenergic receptors have been previously investigated by examining the binding of radioactive compounds to membrane preparations. At concentrations in the 0.1-10-mM range, divalent cations were found to facilitation agonist binding and to inhibit antagonist binding to adrenergic receptors (Asakura et al.
1984; Jarrott et al. 1982
; Loftus et al.
1984
; Nomura et al. 1984
; Rouot et al.
1982
). The effect on agonist binding primarily resulted from an
increased number of binding sites, which was thought to result from
interactions between divalent cations and G proteins to switch the
receptor from a low-affinity to a high-affinity state (Asakura
et al. 1984
). Unlike these binding studies, the applied
divalent cations in our study did not have access to the intracellular
face of the membrane. In addition, our internal solution contained 10 mM EGTA to prevent increases in the internal concentration of divalent
cations. Thus, we should not observe these effects. In the binding
studies, concentrations of divalent cations higher than ~10 mM
resulted in lower agonist binding (Asakura et al. 1984
;
Loftus et al. 1984
; Nomura et al. 1984
).
However, this effect of divalent cations was not examined in detail;
thus, no explanation was proposed for this effect. Since these binding experiments were conducted, much information has been revealed about
the composition of the catecholamine-binding site on the adrenergic
receptors, which provides clues about where divalent cations may be
exerting their blocking effect. It is known that one or more aspartic
acid residues are crucial for high-affinity binding of catecholamines
to adrenergic receptors (Strader et al. 1994
). The
negatively charged acidic group on the aspartate residue is thought to
interact with the positively charged amine of the catecholamine.
Divalent cations may be interacting with the negatively charged amino
acids in the binding site. Such an interaction could interfere with the
binding of NE to produce a competitive block of the adrenergic receptor
that we observe.
The conclusion of Kuo and Bean (1993) that
voltage-dependent inhibition alters N-channel permeation was primarily
supported by reduced inhibition observed when monovalent cations were
permeating the N-channel. Their observation of a reduction of
inhibition by isotonic Ba2+ was interpreted
within this framework as evidence that the permeation component of
inhibition was reduced by the high Ba2+
concentration. If this were true, the high Ba2+
typically used in single-channel recordings would mask the effect of
neurotransmitters on permeation. Our results show that after adjusting
for the right-shift in the dose-response relationship, the NE response
was not reduced by high Ba2+. Therefore, if
inhibition results in changes in N-channel permeation, these changes
should be observed in single-channel studies using isotonic
Ba2+. Previous single N-channel studies have
observed a neurotransmitter-induced increase in the latency-to-first
channel opening, but these studies have failed to observe changes in
open times and single-channel current amplitude (Carabelli et
al. 1996
; Patil et al. 1996
). These studies
focused on N-channel activity at voltages less than +40 mV. Data from
two recent preliminary reports show that reluctant openings can be
observed only at voltages of +40 mV or more (Colecroft et al.
1999
; Lee and Elmslie 1997
). A detailed analysis
of these openings will determine whether both gating and permeation are altered during neurotransmitter inhibition of N-type calcium current. We conclude that if permeation is a mechanism of N-channel inhibition, it should be observed in single-channel recordings using high external
Ba2+ as the charge carrier.
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ACKNOWLEDGMENTS |
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We thank Drs. Geoffrey G. Schofield and Yong Sook Goo and H. Liang for helpful comments on the manuscript.
This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-33671.
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FOOTNOTES |
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Address for reprint requests: K. S. Elmslie, Dept. of Physiology, SL-39, Tulane University Medical School, 1430 Tulane Ave., New Orleans, LA 70112.
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 13 July 1999; accepted in final form 29 September 1999.
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REFERENCES |
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