Competition Between Internal AlF4minus and Receptor-Mediated Stimulation of Dorsal Raphe Neuron G-Proteins Coupled to Calcium Current Inhibition

Yuan Chen and Nicholas J. Penington

Department of Physiology and Pharmacology, State University of New York, Health Science Center at Brooklyn, Brooklyn, New York 11203-2098


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Chen, Yuan and Nicholas J. Penington. Competition Between Internal AlF4minus 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 (Galpha ·GDP·AlF4-) appeared to free G-beta gamma -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 beta gamma -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-beta gamma -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 beta gamma -subunit with Ca2+ channels. Agonist application temporarily reversed the effects of AlF4-, making it a complementary tool to GTP-gamma -S for the study of G-protein interactions.


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ABSTRACT
INTRODUCTION
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Aluminum fluoride (AlF4-) binds to the alpha -subunit of heterotrimeric G-proteins (Sternweis and Gilman 1982) by forming a complex with G·GDP (guanosine diphosphate) thus mimicking the terminal gamma -phosphate of guanosine triphosphate (GTP) (Bigay et al. 1987; Chabre 1990). The Galpha ·GDP·AlF4- complex resembles that of the GTP-bound form of the G-protein, and it allows the alpha - and beta gamma -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 alpha -subunits of G-proteins contain a helical domain that is critical for their GTPase activity. The Galpha ·GDP·AlF4- complex of several Galpha -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 Galpha i and Galpha 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 Galpha ·GDP·AlF4- complex of Galpha -subunits than to the GTP-gamma -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-beta gamma -subunit with calcium channels.


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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 MOmega . 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-gamma -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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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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Fig. 1. Aluminum fluoride (AlF4-) in the pipette solution inhibits barium current and allows a transient disinhibition to occur after application of serotonin (5-HT). A: with CsF in the pipette and (in mM) 4 MgATP and 1 MgCl2, the recovery current was larger than control after 5-HT (1 µM) was washed off. a) Control current. b) Current during inhibition by 5-HT. c) Current after washing off 5-HT. B: current passing through calcium channels of dorsal raphe (DR) neurons with a CsF pipette solution. The current in B exhibits a transient disinhibition (TD) as 5-HT was washed off. The current at points a, b, and c are equivalent in time to the currents in A, but these traces were not taken from the same cells. C: CsCl pipette solution containing the same concentration of nucleotides and MgCl2 along with 10 µM AlCl3 (control) shows a plot of peak Ca2+ current against time, the current returns to control after 5-HT was washed off. D: calcium current with a CsCl pipette solution shows no TD. In all figures time 0 is the time when the earliest recording could be established and is not coincident with going whole cell.

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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Fig. 2. ATP is required for transient disinhibition of Ba2+ current. A: halving the concentration of MgATP to 2 mM in the CsF pipette solution did not support a TD, and it dramatically slowed the recovery from 5-HT (1 µM). B: adding back 2 mM TrisATP to the pipette solution in A allowed the TD to occur. C: when no MgCl2 was added to the intracellular solution containing 4 mM MgATP, the TD was not observed, and the peak calcium current was smaller on average after 5-HT was washed off.


                              
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Table 1. Estimated free Mg2+ and Mg2ATP

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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Fig. 3. Al3+ is required with F- for the development of the TD, but high levels of AlF4- decreased the TD. Lowering free Al3+ in the pipette solution using the chelator deferoxamine (500 µM) did not prevent the TD. A: addition of deferoxamine allowed a TD to develop. In this experiment a solution containing 2 mM MgATP was used, which, without the deferoxamine, did not produce a TD (see Fig. 2A). B: Al3+ was reduced still further by preparing the solution in a plastic container instead of glass, using pipette glass without Al3+ and adding deferoxamine; using these conditions the disinhibition was not observed, and the effect of 5-HT recovered more slowly.

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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Fig. 4. Phosphorylation and dephosphorylation do not appear to be involved in TD generation by 5-HT (1 µM). Substituting 5'-adenylylimido-diphosphate (AmpPNP) for ATP does not prevent the TD; ATP-gamma -S also supports the TD. A: 4 mM MgATP in the pipette solution was replaced with 4 mM AmpPNP producing rapid Ca2+ current rundown (open circle ). When the peak current was corrected for rundown, there appeared to be a small TD. B: AmpPNP did not prevent the TD. The pipette contained 2 mM MgATP, 2 mM AmpPNP, plus deferoxamine in the CsF pipette solution. C: replacing 2 mM AmpPNP in the pipette solution used in B with 2 mM ATP-gamma -S did not prevent or prolong the TD.

ATP-gamma -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-gamma -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-gamma -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-gamma -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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Fig. 5. A: requirement for fluoride. Reducing the CsF concentration to 10 mM (120 mM CsF was replaced with CsCl), reduced the TD. B: disinhibition was G-protein dependent. GmpPNP (20 µM) with 300 µM GTP weakly activated the G-protein of DR neurons and permitted a TD to occur after rapid wash off of 5-HT (1 µM).

Disinhibition was G-protein dependent

AlF4- reportedly activates G-proteins by binding to the GDP of Galpha ·GDP, thus mimicking Galpha GTP. We attempted to investigate this mechanism by adding GTP-gamma -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-gamma -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-gamma -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 beta gamma -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 (tau  = 468.2 s, n = 5, Fig. 6B).



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Fig. 6. Repeated or prolonged application of 5-HT decreases the degree of G-protein activation by AlF4-. A: top figure shows the Ca2+ current facilitation by a conditioning step to +70 mV, shortly after going whole cell with AlF4- in the pipette, on the right the much diminished facilitation that occurs after several applications of 5-HT (1 µM) and the associated TDs. B: a depolarizing prepulse facilitated the AlF4--inhibited Ca2+ current in this cell, and the decline in this degree of facilitation was plotted against time. The filled square represents the percent facilitation in a cell that was not exposed to 5-HT (see Fig. 7A, open square, for protocol). The data points for this cell have been fit with a linear least-squares line, of decay 0.0149%/s. The open circles plot the percentage facilitation of baseline Ca2+ current, by a depolarizing step to +70 mV after the complete decay of the TD, from a cell containing CsF, which was treated with applications of 1 µM 5-HT every 200 s. The decay in the facilitation current was fit with a single exponential tau  of 468.2 s.



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Fig. 7. Relationship between the degree of G-protein activation and the size of the TD. A: time course of the action of 5-HT in reversing the effect of AlF4-. The cell contains CsF. In this experiment a prepulse was applied every 60 s (with no application of 5-HT) for 900 s; then one application of 5-HT (1 µM) produced no disinhibition (n = 5). B: in another group of cells, soon after going whole cell, 5-HT was applied for 650 s. When the 5-HT was washed off, there was a TD that occurred in all 4 cells. C: the graph on the left plots data from cells recorded with CsF or CsCl pipette solutions. The size of the baseline peak Ca2+ current and after a conditioning step to +70 mV measured as soon as possible after going whole cell is plotted. The size of the Ca2+ current baseline using a CsF solution is significantly smaller than the size of the peak baseline current using a CsCl solution. The size of the prepulse facilitated current using CsF was not significantly different from the size of the current using a CsCl solution. The graph on the right compares the % facilitation of the calcium current using CsF in the pipette at the beginning of the experiment with the % increase in peak calcium current over baseline at the peak of the TD after the TD effect had become maximal. D: with CsF as the pipette solution, both the percentage of the inhibition of baseline calcium current by 5-HT and the size of the TD grew exponentially. Filled squares represent inhibition by 5-HT expressed as a percentage of the baseline current just before the application of 5-HT. Open circles represent the size of the TD. As the inhibition to 5-HT increased, the size of the TD increased.

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 tau  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 tau  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 Galpha GDP

When 5-HT was removed, the TD reached its peak and the G-protein was presumably in the ground state (Galpha ·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 Galpha ·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 Gbeta gamma -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 tau  = 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 Galpha ·GDP, mimicking GTP and activating the G-protein (Chabre 1990). 2) By comparison, the activated G-protein beta gamma -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 Gbeta gamma (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 tau 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 tau 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 (beta gamma ) subunit binding to the Ca2+ channels.



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Fig. 8. A: the decay of the TD may be a close approximation to the rate of AlF4- binding to GDP. The tau  of the decay of the averaged data points was 33.6 s. The decay of the disinhibition is approx 500 times slower than the ON-rate of the AlF4- bound G-protein effect. B: ON-rate of the effect of AlF4- stimulated G-protein. Filled squares represent the ON-rate of 5-HT (1 µM) inhibition using a CsCl pipette solution. The tau  fitted to the averaged data were 67.1 ms. Open squares represent the ON-rate of the effect of 5-HT using a CsF pipette solution; tau  fitted to the averaged data were 66.7 ms. The averaged data were not significantly different in the 2 conditions. Open triangles represent the ON-rate of AlF4- inhibition of the Ca2+ current; tau  of the ON-rate of the averaged data were 132.8 ms.

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 (beta gamma ) subunit binding to the Ca2+ channels. In control (CsCl) solution, the average of the OFF-rates (tau 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 (beta gamma ) has a slightly higher affinity for the Ca2+ channel than the GTP-bound G-protein (beta gamma ) in its interaction with Ca2+ channels.



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Fig. 9. AlF4- changed the OFF-rate of G-protein binding to the Ca2+ channel. A: the OFF-rate of the G-protein stimulating effect of AlF4- alone (black-triangle, 10.9 ms, tau  fitted to averaged data, n = 9) was almost identical to the OFF-rate of the effect of 5-HT (1 µM) using the AlF4- containing solution (open circle , 10.85 ms); 2 curves are superimposed. Both of these curves are statistically slower than the control OFF-rate of the effect of 5-HT using a CsCl pipette solution (7.6 ms). B: Al3+ is responsible for these changes because Al3+ was eliminated from the pipette solution and the average OFF-rate for the effect of 5-HT using a CsF pipette solution was not statistically different from that using CsCl. CsF (tau  fitted to averaged data) 9.6 ms () vs. CsCl 7.6 ms (open circle ). The time between the test pulse and the prepulse was 5 ms.

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 (beta gamma ) subunit for the Ca2+ channel.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The binding of an agonist to a receptor causes GDP dissociation and GTP binding to the G-protein followed by the release of the Galpha from the Gbeta gamma -subunits (Gilman 1987). Simultaneously a conformational change occurs, leading to an increased GTPase activity of the alpha -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-beta gamma 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 (alpha ·GDP·beta gamma right-left-arrows alpha ·GDP·AlF4- + beta gamma ). 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 alpha ·GDP·beta gamma , and GTP binds, releasing more beta gamma . Perhaps by mass action (alpha ·GDP·AlF4- right-left-arrows alpha ·GDP right-arrow alpha  right-arrow alpha ·GTP), or by direct enhancement of the release of GDP·AlF4-, eventually most G-protein alpha -subunits are in the GTP bound state. On removal of 5-HT, GTPase activity transiently returns most G-proteins to the basal alpha ·GDP·beta gamma form, allowing maximal channel activity. Finally, AlF4- rebinds. We assume that under these conditions the steady state is a mixture of alpha ·GDP·AlF4- + beta gamma , and alpha ·GDP·beta gamma . 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 Galpha -subunits of G-proteins produces a stable active transition state of the GTPase activity of Galpha (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-beta -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-gamma -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-gamma -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-gamma -S from ATP-gamma -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 beta gamma -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 beta gamma 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-beta gamma 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 (beta gamma ) 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-beta gamma 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 beta gamma -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 alpha - and beta gamma -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 Galpha ·GDP is available in the cell, G-beta gamma will bind to Galpha -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 Galpha ·GDP (tau  = 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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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