Department of Physiology and Pharmacology, State University of New York, Health Science Center at Brooklyn, Brooklyn, New York 11203-2098
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ABSTRACT |
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Chen, Yuan and
Nicholas J. Penington.
Competition Between Internal AlF4 and
Receptor-Mediated Stimulation of Dorsal Raphe Neuron G-Proteins Coupled
to Calcium Current Inhibition.
J. Neurophysiol. 83: 1273-1282, 2000.
Intracellular aluminum
fluoride (AlF4
), placed in a patch pipette, activated
a G-protein, resulting in a "tonic" inhibition of the
Ca2+ current of isolated serotonergic neurons of the rat
dorsal raphe nucleus. Serotonin (5-HT) also inhibits the
Ca2+ current of these cells. After external bath
application and quick removal of 5-HT to an AlF4
containing cell, there was a reversal or transient disinhibition (TD)
of the inhibitory effect of AlF4
on Ca2+
current. A short predepolarization of the membrane potential to +70 mV,
a condition that is known to reverse G-protein-mediated inhibition,
reversed the inhibitory effect of AlF4
on
Ca2+ current and brought the Ca2+ current to
the same level as that seen at the peak of the TD current. With
AlF4
in the pipette, the TD phenomenon could be
eliminated by lowering pipette MgATP, or by totally chelating pipette
Al3+. In the presence of AlF4
, but with
either lowered MgATP or extreme efforts to eliminate pipette
Al3+, the rate of recovery from 5-HT on wash was slowed, a
condition opposite to that where a TD occurred. The putative complex of AlF4
-bound G-protein
(G
·GDP·AlF4
) appeared to free
G-
-subunits, mimicking the effect on Ca2+ channels of
the G·GTP complex. The ON-rate of the inhibition of
Ca2+ current, after a depolarizing pulse, by
-subunits released by AlF4
in the pipette was
significantly slower than that of the agonist-activated G-protein. The
OFF-rate of the AlF4
-mediated inhibition
in response to a depolarizing pulse, a measure of the affinity of the
free G-
-subunit for the Ca2+ channel, was slightly
slower than that of the agonist stimulated G-protein. In summary,
AlF4
modified the OFF-rate kinetics of
G-protein activation by agonists, but had little effect on the kinetics
of the interaction of the
-subunit with Ca2+
channels. Agonist application temporarily reversed the effects of
AlF4
, making it a complementary tool to GTP-
-S for
the study of G-protein interactions.
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INTRODUCTION |
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Aluminum fluoride
(AlF4) binds to the
-subunit of
heterotrimeric G-proteins (Sternweis and Gilman 1982
) by
forming a complex with G·GDP (guanosine diphosphate) thus
mimicking the terminal
-phosphate of guanosine triphosphate
(GTP) (Bigay et al. 1987
; Chabre 1990
).
The G
·GDP·AlF4
complex resembles that of the
GTP-bound form of the G-protein, and it allows the
- and
-subunits to dissociate and interact with effectors downstream,
although questions have been raised about the generality of this
mechanism (Yatani and Brown 1991
). We reasoned that
AlF4
might be a suitable tool to directly, but
reversibly, activate G-proteins in an intact cell and investigate the
effect of intracellular AlF4
on G-protein coupling to
Ca2+ currents.
The -subunits of G-proteins contain a helical domain that is
critical for their GTPase activity. The
G
·GDP·AlF4
complex of several G
-subunits is
reportedly a stabilized transition state that results in increased
GTPase activity (Scheffzek et al. 1997
), but in some
systems Al3+ or even guanine nucleotide is not required
(Vincent et al. 1998
). This increase in GTPase activity
should be reflected in a faster ON and OFF rate
of agonist stimulation. In addition, a new class of GTPase-activating
proteins (GAPs) has been discovered to modulate the ON- and
OFF-rate of G-protein-mediated effects on K+
channels and other effectors of G
i and G
o
proteins (Koelle 1997
; Saitoh et al.
1997
). These GAPs also called RGS proteins (regulators of
G-protein signaling) bind with a higher affinity to the
G
·GDP·AlF4
complex of G
-subunits than to
the GTP-
-S bound state (Berman et al. 1996
),
indicating that they also stabilize the GTPase active conformation of
G-proteins. These findings suggest that the kinetics of activation and
inactivation of the G-protein(s) involved in Ca2+ current
modulation, in the presence of AlF4
, may differ from
those observed with agonist stimulation.
Our early observations revealed that AlF4 caused a
weak stimulation of the G-protein responsible for the modulation of
calcium channel gating, but the nature of the stimulation differed
markedly from agonist stimulation by activating a 5-HT1A receptor
(Penington and Kelly 1990
). In addition, serotonin
(5-HT) could further inhibit the calcium current, and, unexpectedly, a
pronounced transient disinhibition (TD) of the current was observed on
removing the agonist. The occurrence of the TD was reminiscent of a
prominent rebound facilitation of calcium current, on wash out of an
inhibitory agonist from NG108-15 cells grown in conditions that
promoted a state of tonic G-protein stimulation (Kasai
1991
). In keeping with these observations, muscarinic agonists
can also generate a similar rebound inactivation of a
G-protein-mediated opening of K+ channels in the heart,
when that G-protein is in a state of weak tonic activation by low
concentrations of an intracellular GTP analogue, guanylyl
imidodiphosphate (Otero et al. 1991
). We have tested two
hypotheses concerning the actions of AlF4
in the
inhibitory effect of G-proteins on calcium channel activity. 1) There can be a competition between the receptor and
AlF4
stimulated G-protein activity, resulting in the
short-term removal of tonic G-protein stimulation by
AlF4
. 2) We investigated whether the
interaction occurs at the level of the G-protein, or the interaction of
the G-
-subunit with calcium channels.
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METHODS |
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These results were obtained from acutely isolated serotonergic dorsal raphe (DR) neurons that exhibited stable Ba2+ currents in 5 mM Ba2+.
Cell preparation
Male Sprague Dawley rats (200-250 g) were anesthetized with
halothane and then decapitated. Three coronal slices (500 µm) through
the brain stem at the level of the dorsal raphe nucleus were prepared
using a Vibroslice (Campden Instruments) in a manner that has
previously been described (Penington and Kelly 1990; Penington et al. 1991
). The slices were placed in cold
artificial cerebrospinal fluid (ACSF) containing (in mM) 119 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 26 NaHCO3, 1.2 NaH2PO4, and 11 glucose, pH
7.3-7.4 when bubbled with 95% O2-5%
CO2. The slices were placed on an agar base, and
a piece of gray matter 2 × 2 mm was cut from immediately below
the cerebral aqueduct containing the dorsal raphe nucleus. The pieces
of tissue were then incubated in a PIPES buffer solution containing
0.07% trypsin (Sigma Type XI) under pure oxygen for 90 min according
to the method of Kay and Wong (1986)
. The pieces of
tissue were then triturated in Dulbecco's modified Eagle's medium and
the isolated cells allowed to settle on a glass coverslip coated with
concanavalin A. Within 5 min of plating, the cells were firmly anchored
to the coated coverslip.
Recording
The extracellular solution was continually perfused at a rate of
~2 ml/min into a bath containing ~1 ml of recording solution. Neurons with truncated dendrites and a cell soma with one dimension of
at least 20 µm were voltage-clamped using an Axopatch 200A (Axon
Instruments, Foster City, CA) patch-clamp amplifier in the whole cell
configuration. Electrodes were coated with silicone elastomer
(Sylgard), and they ranged in resistance from 1.5 to 2.5 M. Leak and
capacitance were subtracted from the Ca2+ current
records. Leak sweeps consisted of 16 hyperpolarizing steps of 10 mV
that were then averaged. The leak sweep currents were scaled to the
appropriate size and then subtracted from the individual current
records except where noted. Leak sweeps were obtained at regular
periods during the experiment. The voltage-clamp data were filtered at
2 kHz and then digitized at 100 µs per point. Voltage protocols were
generated and analyzed by an IBM PC Pentium clone using the Axobasic 1 patch-clamp software, and the resultant data were written to disk for
analysis off-line. Recordings from neurons acutely dissociated from the
adult rat brain were carried out at room temperature (24°C). The
measurements of Ca2+ current are expressed as
means ± SE, and in some cases the means were tested for equality
using a Student's t-test or paired t-test. Where
multiple comparisons of means were attempted a random effects, ANOVA
was performed followed by a Student-Newman-Keuls test. Estimates of
free Mg2+ and Mg2ATP were obtained using the
program WinMaxC version 2.0 (Bers.ccm), which can be found on the
internet (Patton 1999
).
Solutions
The control pipette solution used in experiments that measured Ca2+ current was 130 mM CsCl, 10 mM HEPES, 10 mM EGTA, 4 mM MgATP, 4 (or 1) mM MgCl2, 300 µM GTP, and 14 mM phosphocreatine (with or without deferoxamine, 500 µM; ±AlCl3, 10 µM; where noted in text), pH 7.3 CsOH. The CsF solution contained 130 mM CsF, 10 mM HEPES, 10 mM EGTA, 2 mM MgATP, 2 mM TrisATP, 1 mM MgCl2, and 300 µM GTP, 14 mM phosphocreatine, ±deferoxamine (500 µM), ±AlCl3 (10 µM), pH 7.3 CsOH. AlCl3 was only used in early experiments because trace Al3+ was found to be sufficient. When deferoxamine was used to chelate Al3+, AlCl3 was not added to the pipette solution. We routinely used the following solution to establish seals for whole cell recording: this contained (in mM) 134 NaCl, 20 HEPES, 10 glucose, 2 CaCl2, 2.5 KCl, and 2 MgCl2. The external recording solution, designed to isolate calcium channel currents (carried by Ba2+), contained 160 TEACl, 5 BaCl2, 10 HEPES, and 20 sucrose, pH 7.3 with TEAOH.
5-HT creatine sulfate, GTP--S tetra lithium salt, and GmpPNP were
obtained from Sigma Chemical (St. Louis, MO). 5-HT was added to the
bath near the cell using an electric valve-controlled, flow pipe, fast
application system.
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RESULTS |
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Aluminum fluoride caused a transient disinhibition of Ca2+ current after washing off 5-HT
AlF4 in the pipette solution was examined for
its ability to stimulate the G-protein of dorsal raphe neurons
directly. When serotonin was applied, a large TD of
Ca2+ current occurred as the serotonin was washed
off. Once the response had stabilized, the maximum size of the TD was
measured and expressed as a percentage increase, comparing the peak of
the TD current to the peak current before the application of
5-HT (Fig. 1A, n = 6, 27.4 ± 1.9%, mean ± SE). With the use of the
CsF pipette solution, the current before the application of 5-HT
usually activated more slowly [Fig. 1B (a) compared
with CsCl solution Fig. 1D (a)]; slow activation is
indicative of G-protein stimulation. The rate of activation of
Ca2+ current was not quantified in this study because when
activated with a square-wave step it is a hybrid of the rate of
activation and inactivation (Jones and Elmslie 1997
).
The TD did not occur when the pipette contained CsCl solution and 10 µM AlCl3 as shown in Fig. 1C, and the
baseline Ca2+ current was quite constant when recorded
using CsCl. In comparison, the current before and after 5-HT attained
the same baseline level. To investigate whether the TD phenomenon was
specific for 5-HT, ATP (10 µM) was applied, which inhibited the
Ca2+ current and produced a TD similar to that caused by
5-HT (n = 5, not shown).
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ATP and free Mg2+ ions are cofactors of the transient disinhibition
The possibility that ATP, or MgATP, was important in the
generation of this phenomena was investigated. Two conditions were examined. In the first, control (n = 7) and CsF pipette
solutions (n = 6) contained 2 mM MgATP and 4 mM
MgCl2; this amount of MgATP did not allow the
occurrence of the TD in the CsF condition (Fig. 2A, maximum current after wash
of 5-HT was 1.4 ± 5.7% of pre-5-HT level). Using these
conditions in recordings from CsF containing cells slowed the recovery
from 5-HT because the time to one-half recovery
(t1/2) was 35.5 ± 5 s
(n = 6, Fig. 2A). In the control group
(CsCl) the t1/2 of recovery was
significantly faster (18.9 ± 1 s; P < 0.01, n = 7). In another group of cells, 2 mM TrisATP was
added back to the solution used in Fig. 2A. A TD measuring 13.4 ± 1.8% occurred (n = 8, Fig.
2B). Taken together, these data suggest that MgATP is
required for this phenomenon because our estimate of free MgATP was
doubled by adding 2 mM TrisATP when free Mg2+ was
halved (Table 1).
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The Mg2+, ATP, or MgATP dependence of this phenomenon was investigated further by providing the same total concentration of ATP (4 mM) and Mg2+ (4 mM) but in three different forms; all other constituents of the pipette solution were identical. 1) 4 mM MgATP and 0 mM MgCl2; 2) 1 mM KATP, 3 mM MgATP, and 1 mM MgCl2; and 3) 4 mM TrisATP and 4 mM MgCl2. Table 1 shows estimates of free Mg2+ and MgATP obtained using the program WinMaxC. It is notable that all currently available programs would predict identical free Mg2+ and MgATP for these three solutions because they do not take into account the effect of the salts with which they form a complex. In the first case when no MgCl2 was added to the intracellular solution, the TD was not observed (Fig. 2C, n = 4), and the peak calcium current was 3.3 ± 5% smaller on average after 5-HT was washed off. In the second condition the TD failed to occur in three of four recordings, and one cell showed a small TD (average of 4 cells 1.6 ± 1.8%). In the third condition, 4 mM TrisATP could replace 4 mM MgATP to produce the TD (mean, +13.6% TD; n = 3, not shown); however, calcium current rundown was accelerated. It is possible that the presence of Tris changed the free Mg2+ or MgATP concentrations so that each condition is not equivalent. Further, free Mg2+ is required for the TD, or the response to 5-HT would not occur, and some form of ATP is also implicated (see Fig. 4). From this point on, the standard pipette solution contained 1 mM MgCl2 unless otherwise stated.
Role of Al3+
To investigate whether F or a
complex of Al3+ and F
caused the TD, an attempt was made to reduce Al3+
in the pipette solution. Deferoxamine (500 µM), a chelator of Al3+, was included in the pipette solution (2 mM MgATP, 2 mM TrisATP, and 4 mM MgCl2), and no
Al3+ was added to the intracellular solution. Unexpectedly,
this solution allowed a TD to occur after application of 5-HT
(n = 3). These cells generated TDs that averaged a
17.4% increase over pre-5-HT baseline. In another group, 2 mM MgATP,
0 mM Tris ATP, and 1 mM MgCl2 plus deferoxamine was used.
Under these conditions a large TD developed (19.3 ± 2.2%,
n = 5, Fig.
3A). We had previously established that when adding only 2 mM MgATP in the pipette solution there was no TD (Fig. 2A). In summary, when the
Al3+ level was lowered with deferoxamine, but presumably
not abolished, the recordings all revealed a TD after 5-HT application.
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Subsequently an attempt was made to abolish the Al3+ ion
from the pipette solution altogether. To do this, the solution,
containing deferoxamine (500 µM), was made up in a plastic beaker,
and custom patch pipette glass (Warner Instruments No. PG150T-7.5) was
used that contains no Al3+. Using these conditions the TD
was not observed (Fig. 3B) (TD = 1.5 ± 1.8%,
n = 5), and the recovery from 5-HT was slowed
(t1/2 = 36 ± 6.7 s, slower
than control at the P < 0.01 level). This result
suggests that trace Al3+ in combination with
F is sufficient to produce the TD.
5-HT-mediated phosphorylation and dephosphorylation did not appear to mediate the TD
Because ATP appeared necessary for this response, the role of phosphorylation in the effect was investigated. 5'-Adenylylimido-diphosphate (AmpPNP) is an analogue of ATP that cannot donate a high-energy phosphate in enzyme reactions, and it is not a substrate for kinases. In a group of cells (n = 3) 4 mM AmpPNP was used in place of 4 mM MgATP, and the Ca2+ current ran down quickly obscuring the interpretation (Fig. 4A). To investigate whether AmPNP was able to substitute for ATP to support the development of the TD or alternatively block the effect of ATP at its binding site, 2 mM AmpPNP was added to the 2 mM MgATP solution with deferoxamine (500 µM). Without the AmpPNP, this solution allowed the cell to exhibit a TD (Fig. 3A). As can be seen in Fig. 4B, AmpPNP did not block the TD that occurred after 5-HT application; indeed the TD measured 15.4 ± 1.8% (n = 5). On two occasions, H-7 (100 µM), the potent nonselective kinase inhibitor, was added to the bath, and it failed to alter the TD (not shown).
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ATP--S is another ATP analogue that is less effective than ATP in
providing a high-energy phosphate, and a thio-phosphorylated product is
resistant to the action of phosphatases. With equal concentrations of
ATP-
-S and MgATP (2 mM) in the pipette solution, a large TD was
observed after 5-HT application that measured on average 26.4%
(n = 3). When 4 mM ATP-
-S, in place of all forms of
ATP, was added to the pipette, 5-HT had no effect on
Ca2+ current, although this current could be
greatly facilitated by a depolarizing prepulse. The calcium current
appeared to be fully modulated as if GTP-
-S only was present in the pipette.
Role of fluoride
Reducing the CsF concentration from 130 to 10 mM and substituting this with CsCl significantly reduced the size of the TD (Fig. 5A) compared with its size in CsF pipette solution (9.4 ± 2.7%, n = 5, Fig. 5A vs. 27.4 ± 1.9%, n = 6, P < 0.01, Fig. 1A).
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Disinhibition was G-protein dependent
AlF4 reportedly activates G-proteins by binding
to the GDP of G
·GDP, thus mimicking G
GTP. We attempted to
investigate this mechanism by adding GTP-
-S to the CsF pipette
solution at a concentration of 30 µM to activate the G-protein
directly. In four cells, the effect of GTP-
-S completely occluded
the TD (not shown).
GmpPNP can also directly activate G-proteins, but it has a lower
affinity for the GDP binding site than GTP--S (Otero et al.
1991
). Because it was suspected that weak G-protein activation plays a role in the TD, an attempt was made to reproduce the effect of
AlF4
with GmpPNP by adding 10 µM GmpPNP with 300 µM GTP to the control CsCl pipette solution. When 5-HT was applied
and washed off in two of four cells, a TD was observed similar to that
seen when using the CsF solution. In another three cells the GmpPNP was increased to 20 µM, and in two of three cells a TD was observed (Fig.
5B); in the remaining cell the calcium current was strongly inhibited by the GmpPNP, and this could not be reversed by 5-HT application. All the cells that responded with a TD in the presence of
GmpPNP had a similar sized TD that averaged 38.3 ± 7.3%
(n = 4).
5-HT displaced GDP-AlF4 from the G-protein
GTP-binding site
In the presence of AlF4 the G-protein was
partially activated; this is based on the finding that the
Ca2+ current was facilitated to a large degree by
a depolarizing prepulse to +70 mV (Fig.
6A) (Bean 1989
;
Elmslie et al. 1990
; Grassi and Lux 1989
;
Ikeda 1991
; Jones and Marks 1989
;
Scott and Dolphin 1990
). After 5-HT was applied and
washed off, and the TD current reached its peak, there was always a
negligible facilitation of the peak current by a depolarizing prepulse
to +70 mV, or in some cases prepulse depolarization inhibited the peak
Ca2+ current at the maximal TD (average
5 ± 0.02%, n = 14). Later in the recording, in a cell
representative of 14 that was previously exposed to 5-HT, a
depolarizing prepulse to +70 mV weakly facilitated the peak
Ca2+ current in the absence of 5-HT (Fig.
6A, right), suggesting that there was little
voltage-dependent G-protein stimulation at this time. Because the
degree of facilitation of the Ca2+ current, after
a depolarizing prepulse, represents the extent of voltage-dependent
interactions of the G-protein
-subunit with the
Ca2+ channel, we compared the decay of
facilitation of the Ca2+ current in the absence
of 5-HT using a CsF solution in cells that had not been exposed to 5-HT
and those that had been repeatedly exposed. Without applying 5-HT (see
Fig. 7A for protocol), the decay of the amount of facilitation of peak Ca2+
current was slow and linear (the slope was
1.2 ± 0.3 × 10
3 %/s, n = 6). When 5-HT was
applied every 200 s, the facilitation decreased more rapidly with
a time course that could be fit with a single exponential decay
(
= 468.2 s, n = 5, Fig. 6B).
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Repeated or prolonged application of 5-HT decreased the degree of
G-protein activation by AlF4 and promoted the
development of the TD
The TD required the prior application of 5-HT. However, because the size of the TD appeared to increase with the number of 5-HT applications or time into the experiment, its magnitude could be related to the time of the delay before the first application of 5-HT. To investigate this possibility, the Ca2+ current was elicited every 20 s for 900 s, and a regular test prepulse of 30 ms duration to +70 mV was applied every 60 s, to estimate the degree of G-protein stimulation. When 5-HT was applied for the first time 900 s into the recording, the TD failed to occur in five cells (Fig. 7A). In another experiment 5-HT was applied continuously for a period of 650 s from the beginning of the recording and then washed off. The removal of 5-HT was accompanied by a robust TD in three of four cells measuring 15.6 ± 9.5% (Fig. 7B). This result indicated that the TD does not depend on the extent of the delay before 5-HT was applied but rather appears to be related to the total duration of the application, although the duration of 5-HT application was not systematically varied.
Unlike recordings from cells containing CsCl, cells containing
AlF4 showed smaller Ca2+
currents and inhibitory responses to 5-HT. With time after going whole
cell, the peak baseline Ca2+ current grew larger,
as did the absolute response to 5-HT, expressed as a percent inhibition
of pre-5-HT baseline current. The relationship between the degree of
G-protein activation and the size of the TD was examined and compared
with the condition where there was little tonic G-protein activation,
e.g., recordings done with CsCl in the patch pipette. Early in the
recording, it was found that AlF4
appeared to
activate the G-protein, as it partially inhibited the calcium current.
Figure 7C shows that a graph the size of the baseline
Ca2+ current using CsF solution (1,977 ± 155 pA, n = 18) was significantly smaller than the size
of the peak baseline current using CsCl solution (3,019 ± 393 pA,
n = 12). There was little facilitation in control CsCl
solution (5.7 ± 0.8%, n = 12) (see also
Penington et al. 1991
). The average percentage
facilitation of the peak Ca2+ current with
AlF4
in the pipette after a conditioning step to +70
mV was 26.3 ± 2.1% (n = 13). When the previous
value was compared with the TD, also in AlF4
containing solution, expressed as a percentage of the pre-5-HT level
(27.4 ± 1.9%, n = 6, Fig. 6B), these
values were not statistically different from each other.
Figure 7D shows the relationship between the
development of the maximal inhibitory response to 5-HT and the
development of the maximal TD with time from the beginning of the
recording. The filled squares represent the normalized 5-HT inhibition
of the baseline current (as a fraction of the baseline before
application of the 5-HT). The open circles represent the normalized TD
(n = 6). The development of the degree of inhibition to
5-HT could be fit well by a single exponential function with a of
237.5 s. The development of the TD was clearly related to the
development of the maximum response to 5-HT, and these data were fit
with a single exponential. The
of the open circle data were 517.9 s
(5-HT 1 µM was applied every 200 s). The results indicate that as the inhibition to 5-HT increased, the TD also increased. The development of the TD with time (Fig. 7D) had a similar time
course to the decay of the facilitation current after multiple 5-HT
applications (Fig. 6B).
Decay of the TD may be a close approximation of the
ON-rate of AlF4 binding to G
GDP
When 5-HT was removed, the TD reached its peak and the G-protein
was presumably in the ground state (G·GDP). After the peak of the
TD, the current returned toward baseline, perhaps reflecting the onset
of a tonic reactivation of the G-protein. We reasoned that the rate of
return of the current to baseline may reflect the rebinding of
AlF4
to G
·GDP and measured the rate of this
relaxation. Because the rate of inhibition was very slow, we also
measured a functional index of the rate of interaction of the
G
-subunit freed by AlF4
with the
Ca2+ channel (the ON-rate of
inhibition) to ascertain whether this rate was slowed and could thus
account for the slow tonic inhibition. The time course of the decay of
the TD was fit well by a single exponential average
= 34.6 ± 4.2 s (n = 5; Fig.
8A). Despite the fact that the
decay was fit by a single exponential, it probably is comprised of at
least two steps. 1) AlF4
slowly binds to
G
·GDP, mimicking GTP and activating the G-protein (Chabre
1990
). 2) By comparison, the activated G-protein
-subunit binds to Ca2+ channels and
inhibits the Ca2+ current much faster
(Herlitze et al. 1996
; Ikeda 1996
). The
second step or "ON-rate of inhibition" was measured by
lengthening the interpulse interval between the depolarizing prepulse
voltage step of +70 mV that temporarily reverses the G-protein
stimulation by G
(Elmslie et al. 1990
;
Ikeda 1991
). The average of the time constants of the
ON-rate for G·GDP·AlF4
-mediated
inhibition (no agonist added) was 129.2 ± 27.8 ms
(n = 7, open triangle, Fig. 8B). The
ON-rate of the G·GDP·AlF4
-mediated
inhibition is likely to represent the second step (measured in ms), and
the first step: AlF4
binding to GDP appears to be
rate limiting and is measured in seconds. In Fig. 8B the
filled square is the ON-rate of 5-HT inhibition using a
CsCl pipette solution; the average of the
s was 66.8 ± 3.1 ms
(n = 5). The open square shows the ON-rate
of 5-HT-mediated inhibition using a CsF pipette solution; the
individual
s averaged 69.7 ± 14.3 ms (n = 6).
The presence of AlF4
did not change the
ON-rate of the 5-HT activated G-protein (
) subunit
binding to the Ca2+ channels.
|
AlF4 decreased the OFF-rate of
G-protein binding to the Ca2+ channel
AlF4 changed the OFF-rate of the
effect of 5-HT (Fig. 9A). The
OFF-rate is a measure of the affinity of the G-protein
(
) subunit binding to the Ca2+ channels. In
control (CsCl) solution, the average of the OFF-rates (
s) of the effect of 5-HT was 8.0 ± 0.3 ms (n = 9); this represents the OFF-rate of the GTP-bound
G-protein. The average OFF-rate of the effect of 5-HT using
CsF solution was 10.9 ± 0.5 ms (n = 7). Although
the difference between the rates is small, these values are
significantly slower (P < 0.05) than with CsCl. In addition, the average OFF-rate of the G-protein stimulating
effect of AlF4
alone (11.1 ± 1.3 ms,
n = 9) is not different from the OFF-rate of the effect of 5-HT using an AlF4
-containing
solution. These data indicate that the product of the
AlF4
bound G-protein (
) has a slightly higher
affinity for the Ca2+ channel than
the GTP-bound G-protein (
) in its interaction with
Ca2+ channels.
|
One could speculate that AlF4 changed the
affinity of the G-protein (subunit) for the Ca2+
channel and fluoride alone could do this. To address this question Al3+ was eliminated from the pipette solution by
preparing it in plastic, with deferoxamine (500 µM) using pipette
glass that did not contain Al3+. Under these
conditions the average OFF-rate for the effect of 5-HT
using a CsF pipette solution was not statistically different from that
using CsCl (Fig. 9B; CsF 9.6 ± 0.4 ms,
n = 5 vs. CsCl 8.0 ± 0.3 ms, P is not
significant). The presence of Al3+ slowed the
OFF-rate of the effect of 5-HT using a CsF pipette solution
(P = <0.05), suggesting that AlF4
and not F
increased the affinity of the
G-protein (
) subunit for the Ca2+ channel.
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DISCUSSION |
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The binding of an agonist to a receptor causes GDP dissociation
and GTP binding to the G-protein followed by the release of the G
from the G
-subunits (Gilman 1987
). Simultaneously
a conformational change occurs, leading to an increased GTPase activity
of the
-subunit. On washing off the agonist, the GTP-bound form
converts to the GDP-bound form, which is dependent on the rate of
GTPase activity. If the GTPase activity is high, this will be reflected in a faster recovery from the agonist. The main findings of the present
study were that intracellular AlF4
caused
approximately a one-third of maximum tonic stimulation of the G-protein
coupled to Ca2+ channels of DR neurons (Fig.
7C), consistent with a G·GDP·AlF4
complex resulting in a mimicry of the G·GTP complex (G-
release).
Based on the literature, a fairly parsimonious explanation of the
sequence of events occurring after agonist application to a DR cell
containing AlF4 is that after several applications of
5-HT, some G-proteins are in the basal state and some are activated
(
·GDP·
·GDP·AlF4
+
).
This should produce only partial channel inhibition, allowing further
inhibition by 5-HT. When 5-HT binds to its receptor, that enhances
release of GDP from
·GDP·
, and GTP binds, releasing more
. Perhaps by mass action (
·GDP·AlF4
·GDP
·GTP), or by direct enhancement of the
release of GDP·AlF4
, eventually most G-protein
-subunits are in the GTP bound state. On removal of 5-HT, GTPase
activity transiently returns most G-proteins to the basal
·GDP·
form, allowing maximal channel activity. Finally,
AlF4
rebinds. We assume that under these conditions
the steady state is a mixture of
·GDP·AlF4
+
, and
·GDP·
. A similar phenomenon has been reported
to occur with other weak activators of G-proteins on agonist
application (Cassel and Selinger 1977
;
Otero et al. 1991
). With repeated or prolonged
application of the agonist, the G-protein should gradually become
predominantly GTP bound in the presence of agonist, and GDP bound when
5-HT is removed. The observations of the present study can be
interpreted within the framework of the above hypothesis. In direct
support of this suggestion, we found that low concentrations of
GmpPNP, that weakly stimulated the G-protein, produced results similar
to AlF4
.
AlF4 bound to G
-subunits of G-proteins
produces a stable active transition state of the GTPase activity of
G
(Scheffzek et al. 1997
); this may contribute to the
relatively fast wash off of the effect of 5-HT (TD), because it would
speed the removal of GTP. It was puzzling at first to explain why
adding deferoxamine (a chelator of Al3+) did not prevent
the TD while presumably decreasing the amount of the
AlF4
complex. When steps were taken to remove
Al3+ completely, this reduced the rate of recovery from the
effect of 5-HT and the TD altogether; converting the rate of recovery to one that was much slower than that observed using CsCl in the pipette. In the absence of Al3+ (but in the presence of
F
) the GTPase activity appears to be greatly inhibited by
F
when the GTPase activating activity of the
G·GDP·AlF4
complex does not counteract it. A
possible explanation for this observation could be that decreasing the
amount of G·GDP·AlF4
to low levels by lowering
(but not abolishing) Al3+ may weaken the G-protein
stimulation by AlF4
and allow it to be more
effectively displaced by agonist application, permitting a TD to be
observed. Another possibility is that the [Al3+] is in
excess and as previously proposed it may compete with Mg2+
for several binding sites on G-proteins (Chabre 1990
),
perturbing them, and altering the binding of AlF4
to
G·GDP. In this fashion, lowering Al3+ may increase the
effect of the active species. The same argument has been put forward to
explain the effect of lowering Mg2+ on the effect of
AlF4
-stimulated G-proteins (Chabre
1990
).
ATP is required for the efficient coupling of the receptor to the
G-protein (Elmslie et al. 1993) and presumably for
GTPase activity. With AlF4
in the pipette, the rate
of recovery from 5-HT was dramatically slowed when added MgATP was
lowered from 4 to 2 mM. The fast OFF-response on washing
5-HT (TD) may be dependent on free Mg2+, which might be
explained by invoking the dependence of GTPase activity directly
(Bourne et al. 1991
) or indirectly on free
Mg2+. It is known that both agonist and
AlF4
-induced stimulation of the GTPase is
Mg2+ dependent, as is AlF4
binding to
G·GDP leading to GTPase activation (Chabre 1990
;
Gilman 1987
). Thus in the absence of sufficient ATP or
free Mg2+, F
but not Cl
may
inhibit the GTPase, and this may be prevented or reversed by adding
Al3+.
Our suggestion that receptor stimulation competes off the
GDP·AlF4 from the G-protein, replacing it with
G·GTP, is supported by the literature (Otero et al.
1991
) and is based on the following observation. When 5-HT is
washed off and the current has fully recovered from G-protein-mediated
inhibition (at the peak of the TD), no facilitation of the
Ca2+ current by depolarization to +70 mV was observed. This
is consistent with a complete lack of G-protein stimulation (once the
5-HT was removed), and presumably all the G·GTP has been replaced
with G·GDP. The peak Ca2+ current was occasionally
inhibited by a depolarizing prepulse delivered at the peak of the TD. A
reduction in N-channel peak current by depolarizing prepulses occurs
after complete removal of tonic G-protein inhibition with GDP-
-S
(Patil et al. 1998
), which supports the suggestion that
there is virtually no tonic G-protein activity at this time. The rate
of decline of the TD is consistent with the rate of rebinding of the
AlF4
to G·GDP (Bigay et al. 1987
);
but because the G·GDP bound form is required for
AlF4
binding (Chabre 1990
), the rate
of rebinding should ultimately depend on the rate of GTPase activity.
The slow ON-rate of AlF4
binding confirms
that the affinity of this interaction must be quite low (Chabre
1990
).
The nonhydrolyzable analogue AmpPNP was able to substitute for MgATP
and support the fast OFF-response of agonist removal in the
presence of AlF4, suggesting that phosphorylation is
not involved in this response. However, the finding that AmpPNP or
ATP-
-S can substitute for ATP could be explained by a substitution
for MgATP at certain binding sites that do not require ATP hydrolysis,
thus effectively freeing up the MgATP complex to take part in reactions
that require ATP hydrolysis. When ATP-
-S completely replaced ATP in
the pipette, the calcium current appeared to be fully modulated. A
possible explanation for this observation is that nucleosidediphosphate kinase produced GTP-
-S from ATP-
-S plus GTP (Elmslie et
al. 1993
).
The size of the TD appeared to be related to the concentration of
F in the pipette, but it would be difficult to construct
a dose response curve for the concentration of F
and the
size of the TD. The reason for this is that changing the concentration
of free F
will alter the concentration of
AlF4
(Chabre 1990
). Because it was
possible to eliminate the response by eliminating pipette
Al3+, our results suggest that the size of the response
appears to be related to the concentration of the
AlF4
, AlF3(OH)
or
AlF3
rather than the concentration of
F
.
The ON-rate of Ca2+ current inhibition by
receptor activation after a depolarizing prepulse appears to be related
to the concentration of external agonist, and the amount of activated
G-protein -subunit (Chen and Penington 1996
;
Elmslie and Jones 1994
; Herlitze et al.
1996
; Ikeda 1996
). The ON-rate of
Ca2+ current inhibition by AlF4
(with no
agonist application after a prepulse) was slower than the
ON-rate of 5-HT inhibition alone, suggesting that there
were fewer activated G·GDP·AlF4
complexes
yielding
interactions with Ca2+ channels compared
with the effect of 5-HT receptor stimulation. Under the conditions used
in the present study, AlF4
was not able to maximally
activate the G-protein in the manner of a full receptor agonist. After
5-HT was removed, AlF4
presumably bound again to the
GDP-bound G-protein and slowly released G-
subunits reinhibiting
the current. After an equilibrium in the cycle of agonist application
became stable, the TD was able to occur and reach a maximum size; this
was equal to the average baseline calcium current that would occur if a
CsCl pipette solution was used.
Another possible explanation for the TD could be advanced if the
affinity of the products of the G·GDP·AlF4
complex (
) for the Ca2+ channels was lower than
control in the presence of F
, and they dissociated more
quickly. In measuring the OFF-rate of Ca2+
current inhibition using CsF, it was revealed that the affinity of the
Ca2+ channel for G-
appeared to be a little higher
than that of the G·GTP complex. This finding appears to rule out as
an explanation an alteration in the kinetics of the interaction between
the Ca2+ channel and the
-subunits produced by
G·GTP and G·GDP·AlF4
complexes. Instead it
suggests that the action of AlF4
, which results in
the altered properties of G-protein effects on Ca2+
channels, resides with the kinetics of activation and inactivation of
the G-protein and the speed with which it releases
- and
-subunits.
Previous studies using intracellular F, presumably
made up in glass containing Al3+, found that high-threshold
L-type Ca2+ currents are eliminated by this intracellular
solution (Bertollini et al. 1994
). L-type current
components show very slow inactivation. Consequently inhibition of a
large L-type component of Ca2+ current would increase the
relative amplitude of a rapidly inactivating component when using a
F
-containing recording solution (Kay et al.
1986
). In addition, millimolar concentrations of Na or KF have
been shown to inhibit various phosphatases, phosphorylases, and ATPases
but to activate adenylyl cyclase (Murphy and Coll 1992
;
Murphy and Hoover 1992
; Rall and Sutherland
1958
). It has also been shown that phosphatase inhibition
produces an increase in N-type Ca2+ current inactivation
rate (Werz et al. 1993
); thus phosphatase inhibition may
partially explain the increased inactivation rate (Kay et al.
1986
). Serotonergic dorsal raphe neurons have a small L-type
Ca2+ current comprising <5% of the total current
(Penington et al. 1991
), and its abolition by
F
is not very apparent. We also occasionally observed an
increase in the rate of voltage-dependent Ca2+ current
inactivation, and a slower activation in comparison to recordings
obtained using a CsCl pipette solution. In raphe neurons the latter
effect appeared to be reversible and due to G-protein activation.
Usually the TD did not occur at the beginning of the recording, but it
developed after applying and washing 5-HT several times (see Figs.
3A, 4C, and 7, A,
B, and D). These results indicate that
the TD requires prior 5-HT application, although it appeared not to
depend on the duration of the recording before agonist application
(Fig. 7A) but was related to the total duration of the
application. It should be noted that the effects of varying the
duration of the 5-HT application on the development of the TD were not
systematically tested but only the extremes of repeated short pulses, a
continual long application, or finally a short application after
900 s of recording. Nevertheless, the data were consistent with
the hypothesis that 5-HT must displace AlF4 from the
G-protein before a TD can result.
There are three main characteristics of the effects of
AlF4 that require explanation. 1)
There is usually no TD at the beginning of the recording.
2) The stimulation of the G-protein by
AlF4
appeared to be relatively strong early in the
recording, as revealed by large amounts of Ca2+ current
facilitation induced by depolarizing prepulses, but becomes weaker with
time after several applications of 5-HT. 3) As the recording progressed, the baseline of the calcium current was enhanced.
These observations might be explained by supposing that at the
beginning of the recording the majority of the G-protein is bound with
AlF4
, leaving a small fraction in a high-affinity
state free to interact with the receptor. When the receptors are
activated, this would be likely to activate GTPase activity, so that
more of the G·GDP·AlF4
would be replaced by
G·GDP on washing the agonist. This could account for the gradual
increase in the size of the current and the gradual loss of the ability
of AlF4
to modulate the channels. When more
G
·GDP is available in the cell, G-
will bind to
G
-subunits more quickly, and recovery from 5-HT will be more rapid
(Wickman and Clapham 1995
). Presumably it takes ~1 min
for all of the AlF4
to rebind to G
·GDP (
= 33.6 s) (Chabre 1990
; this study). The percentage
of G-proteins bound to AlF4
, after an equilibrium
state with respect to agonist application frequency has been achieved,
would be less than at the start of the recording; this may explain the
reduction in tonic G-protein stimulation later in the experiment. These
findings clarify a number of questions about the modulation of
G-proteins and Ca2+ currents by AlF4
.
Further study will explore the potential usefulness of
AlF4
in studies of G-protein modulation by neurotransmitters.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Mental Health Grant MH-5504101 to N. J. Penington.
Present address of Y. Chen: Dept. of Pharmacology, Box 357280, Health Science Building, 1959 NE Pacific St., University of Washington, Seattle, WA 98195.
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FOOTNOTES |
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Address for reprint requests: N. J. Penington, Dept. of Physiology and Pharmacology, State University of New York, Health Science Center at Brooklyn, Box 29, 450 Clarkson Ave., Brooklyn, NY 11203-2098.
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 9 August 1999; accepted in final form 5 November 1999.
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REFERENCES |
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