Colchicine: a novel positive allosteric modulator of the human 5-hydroxytryptamine3A receptor.

The actions of colchicine were examined with the two-electrode voltage-clamp technique and radioligand binding assays in mouse and human 5-hydroxytryptamine(3A) receptors (5-HT(3A)Rs) expressed in Xenopus laevis oocytes. Colchicine inhibited 5-hydroxytryptamine (5-HT)-evoked currents in oocytes expressing mouse 5-HT(3A)Rs, with an IC(50) of 59.5 +/- 3 microM. In contrast to the mouse receptor, coapplication of colchicine with 5-HT (<1 microM) strongly enhanced 5-HT-evoked currents in oocytes expressing human 5-HT(3A)Rs. Colchicine applied alone did not induce a detectable current. In the presence of 0.5 microM 5-HT, the potentiation was concentration-dependent and reached the maximum (approximately 100%) when 750 microM colchicine was applied. However, colchicine-dependent inhibition can be observed at 5-HT concentrations > 1 microM. In oocyte membranes expressing mouse or human receptors, binding studies with colchicine (25 nM-1 mM) revealed no displacement of 1-methyl-N-((1R,3r,5S)-9-methyl-9 azabicyclo [3.3.1]nonan-3yl)-1H-indazole-3 carboxamide ([(3)H]BRL-43694), suggesting that actions of colchicine do not occur at the ligand binding domain. Functional effects of colchicine on both receptors occurred in the absence of preincubation and after cold temperature incubation, suggesting that the microtubule-depolymerizing effects of colchicine play no role in modulation of receptor function. Studies with interspecies chimeric receptors demonstrated that the distal one third of the N terminus is responsible for the bidirectional modulation by colchicine. Collectively, these results suggest that colchicine modulates receptor function through loops C and/or F through a gating mechanism.

The GABA(A) receptor antagonist picrotoxin inhibits 5-hydroxytryptamine type 3A receptors.

For a number of years it has been known that the CNS convulsant picrotoxin inhibits the GABA(A) receptor, an anion-selective member of the ligand-gated ion channel (LGIC) superfamily. PTX also inhibits other anion-selective LGIC members, such as GABA(C), glycine and glutamate-gated Cl(-) channels. In the present report, we tested the ability of picrotoxin to inhibit cation-selective 5-HT(3A) receptors. Murine 5-HT(3A) receptors were expressed in HEK293 cells, and functionally evaluated using whole-cell patch clamp recording. Picrotoxin inhibited 5-HT-gated currents in a concentration-dependent manner, with an IC(50) of approximately 30 microM. Moreover, the blockade by PTX was non-competitive and use-facilitated. Pentylenetetrazole and U-93631, ligands that act at a domain similar to that of picrotoxin in GABA(A) receptors, also inhibited the 5-HT(3A) receptor. For each ligand tested, its potency was 5-10 fold lower than typically observed in GABA(A) receptors. Our results demonstrate that, in addition to being a relatively non-selective inhibitor of anionic LGICs, picrotoxin also inhibits the cation-selective 5-HT(3A) receptor. Moreover, the fact that both PTZ and U-93631 similarly inhibit the 5-HT(3A) receptor is consistent with the suggestion that the site of picrotoxin action in this receptor may be comparable to that in anion-selective LGICs.

Benzylidene analogs of anabaseine display partial agonist and antagonist properties at the mouse 5-hydroxytryptamine(3A) receptor.

The nicotinic receptor drug candidate, 3-(2,4-dimethoxybenzylidene)-anabaseine (also known as GTS-21; DMXBA), its hydroxy metabolites, and some related analogs were evaluated with the two-electrode voltage-clamp technique in mouse 5-hydroxytryptamine (5-HT)(3A) receptors expressed in Xenopus oocytes. Although DMXBA lacked partial agonist activity, its hydroxy-benzylidene metabolites and related analogs were partial agonists, displaying the following rank order of potency (EC(50)) and apparent efficacy: 5-HT, 0.9 +/- 0.06 microM (100% efficacy) > 3-(2-hydroxy,4-methoxybenzylidene)-anabaseine (2-OH-MBA), 2.0 +/- 0.3 microM (63% efficacy) > 3-(2,4-dihydroxybenzylidene)-anabaseine, 2.6 +/- 0.3 microM (63% efficacy) > 3-(2-methoxy,4-hydroxybenzylidene)-anabaseine, 17.2 +/- 1.0 microM (30% efficacy). To examine the influence of a benzylidene ring hydroxy substituent, the agonist actions of the three possible monohydroxy isomers were examined. The rank order of potency, based on EC(50) determinations, and apparent efficacy was: 3-(2-hydroxybenzylidene)-anabaseine, 20.3 +/- 2.6 microM (63% efficacy) > 3-(4-hydroxybenzylidene)-anabaseine, 32.3 +/- 5.9 microM (14% efficacy) > 3-(3-hydroxybenzylidene)-anabaseine (3-OH-BA) (no agonist activity). Both DMXBA and 3-OH-BA antagonized 5-HT-mediated currents, with IC(50) values of 15.7 +/- 0.9 and 27.5 +/- 4.7 microM, respectively. DMXBA demonstrated both competitive and noncompetitive forms of antagonism over the range of concentrations tested. These results suggest that a hydroxy substituent at the 2' position of the benzene ring is necessary and sufficient for partial agonist activity; substitution at the 4' position with a hydroxy or methoxy group further enhances agonist potency. Because 2-OH-MBA is a primary metabolite of DMXBA, it may contribute to the physiological, biochemical, and behavioral effects of the parent compound when administered in vivo.

Bicuculline antagonizes 5-HT(3A) and alpha2 glycine receptors expressed in Xenopus oocytes.

The present study examined the effects of bicuculline on the mouse 5-hydroxytryptamine(3A) receptor (5-HT(3A) receptor and the human alpha2 subunit of the glycine receptor. Bicuculline antagonized both the 5-HT(3A) receptor (IC(50)=20.12+/-0.39 microM) and the alpha2 glycine receptor (IC(50)=169.40+/-1.73 microM). A competitive form of antagonism by bicuculline was suggested by experiments in which the EC(50)s for 5-HT and glycine were increased in the 5-HT(3A) and alpha2 glycine receptors, respectively, as bicuculline concentrations were increased. A competitive nature of antagonism by bicuculline at the 5-HT(3A) receptor was also suggested by displacement of the competitive antagonist, [3H]GR65630 in SF21 insect cells expressing the 5-HT(3A) receptor (K(i)=19.01+/-0.71 microM). Our data and that of others reveal that bicuculline, a purported selective antagonist of the GABA(A) receptor, antagonizes at least one receptor subclass in every member of the superfamily of ligand-gated ion channels.

Competitive antagonism of the mouse 5-hydroxytryptamine3 receptor by bisindolylmaleimide I, a "selective" protein kinase C inhibitor.

We examined the effects of several protein kinase C (PKC) inhibitors on the murine 5-hydroxytryptamine3 (5-HT3) receptor to determine whether they acted directly on the receptor. The 5-HT-evoked currents in Xenopus laevis oocytes expressing the recombinant 5-HT3 receptor were measured with the two-electrode voltage-clamp technique. The PKC inhibitors bisindolylmaleimide I (BIM, GF109203x) and staurosporine, but not calphostin C or chelerythrine, decreased the 5-HT3 receptor-mediated currents when coapplied with 5-HT. BIM blocked 0.5 microM 5-HT-elicited currents with an IC50 value of 7 nM, whereas in the presence of 5 microM staurosporine, 42% inhibition of 0.5 microM 5-HT-mediated currents was observed. Increasing concentrations of BIM resulted in a rightward shift of the 5-HT concentration-response curve, without altering efficacy. A Schild plot was generated, which had a slope of -1.01, suggesting competitive antagonism. The Ki value of BIM was determined to be 29 nM. To confirm competitive antagonism, a competitive binding assay was performed on Sf21 insect cells infected with the mouse 5-HT3 receptor cDNA in a baculovirus expression vector. BIM completely displaced binding of the selective 5-HT3 receptor antagonist [3H]GR65630. BIM bound to the 5-HT3 receptor with a Ki value of 61 nM, which was slightly less potent than that of the selective 5-HT3 receptor antagonist MDL72222 (27 nM). The PKC inhibitor BIM is a potent competitive antagonist at the 5-HT3 receptor.

Characterization of interaction of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester with Torpedo californica nicotinic acetylcholine receptor and 5-hydroxytryptamine3 receptor.

The widely used calcium channel antagonist 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8) has been identified as a noncompetitive antagonist (NCA) and open-channel blocker of both muscle- and neuronal-type nicotinic acetylcholine receptors (AChRs). To further examine the interaction of TMB-8 with the AChR, the compound was tested as a competitor for the binding of two NCAs of the Torpedo californica AChR, phencyclidine and 3-trifluoromethyl-3-(m[125I]iodophenyl)diazirine, for which the binding to the AChR has been pharmacologically well characterized and a channel binding loci has been established. TMB-8 fully inhibited specific photoincorporation of 3-trifluoromethyl-3-(m[125I]iodophenyl)diazirine into the resting AChR channel (IC50 = 3.1 microM) and inhibited high-affinity [3H]phencyclidine binding to the desensitized AChR (IC50 = 2.4 microM). We conclude that TMB-8 is a potent NCA of the nicotinic AChR, interacting with the resting, open-channel, and desensitized channel conformations. TMB-8 was next tested as an inhibitor of the structurally homologous 5-hydroxytryptamine (5-HT)3 receptor (5-HT3R). Using 5-HT3R containing Sf21 cell membranes, TMB-8 completely inhibited specific binding of the radiolabeled 5-HT3R antagonist [3H]GR65630 (Ki = 2.5 microM). Furthermore, TMB-8 antagonized 5-HT-evoked currents of both mouse and human 5-HT3Rs expressed in Xenopus laevis oocytes, and additional analysis was consistent with a competitive antagonistic mechanism of action. These results, taken together, indicate that TMB-8 antagonizes the function of the AChR and 5-HT3R by different mechanisms. Given the sequence similarity and emerging evidence of structural homology in the channels of these two receptors, these results underscore the existence of subtle yet important structural differences in each channel.

Mutation of putative phosphorylation sites in the 5-hydroxytryptamine3 receptor does not eliminate its modulation by ethanol.

The function of the 5-hydroxytryptamine3 (5-HT3) receptor is enhanced by ethanol, but the amino acid residue(s) that confers sensitivity to ethanol remains to be identified. Phosphorylation of the related GABA(A) receptor has been implicated in conferring its sensitivity to ethanol. In common with the GABA(A) receptor, the 5-HT3 receptor contains multiple consensus sites for protein kinases. To evaluate the possibility that phosphorylation of the 5-HT3 receptor underlies its ethanol sensitivity, we examined the ability of ethanol to enhance 5-HT-mediated currents in a mutant 5-HT3 receptor containing no intracellular serines, threonines, or tyrosines. Mutation of these 13 residues in the intracellular loops produced a modest leftward shift in the 5-HT concentration response curve, with the EC50's for 5-HT decreasing from 0.839 +/- 0.03 microM in the wild-type receptor to 0.713 +/- 0.03 microM in the mutant receptor. Cooperativity of the 5-HT binding sites was enhanced by the mutations, with Hill coefficients of 2.92 for the wild-type receptor and 3.74 for the mutant receptor, respectively. In oocytes expressing mutant receptors, ethanol (50 to 200 mM) enhanced the currents produced by low concentrations of 5-HT by approximately 5 to 45%, which was not statistically different from the potentiation produced by ethanol in wild-type receptors. These results suggest that ethanol enhancement of the 5-HT3 receptor function does not require receptor phosphorylation.

Colchicine competitively antagonizes glycine receptors expressed in Xenopus oocytes.

Ligand-gated ion channel association with the cytoskeleton may play an important role in receptor distribution and function. However, the microtubule depolymerizing agent, colchicine, has inhibitory effects on glycine receptors that are independent of microtubule depolymerization. The actions of colchicine and other microtubule-modifying drugs were examined on glycine alpha1 and alpha2 receptors expressed in Xenopus oocytes. The potency of colchicine was much greater in alpha2 than alpha1 receptors, with IC50s of approximately 64 and 324 microM in alpha2 and alpha1 receptors, respectively. Colchicine inhibition of receptor function was instantaneous. Pre-incubation with colchicine failed to enhance its inhibition, and washout of colchicine's inhibition could be observed in 30 s. Incubation of oocytes on ice for 2 h to depolymerize microtubules failed to alter colchicine's antagonism of glycine receptors. Taxol, a microtubule polymerizing agent, and nocodazole, a depolymerizing drug, had no effect on receptor function when co-applied with glycine. The antagonism of glycine-mediated currents by colchicine (100-600 microM) was competitive. Thus, the action of colchicine at the agonist recognition site of the glycine receptor suggests that this drug should be used with care when studying microtubule-associated changes in ligand-gated ion channel function.

Direct channel-gating and modulatory effects of triiodothyronine on recombinant GABA(A) receptors.

We have previously shown that triiodothyronine (T3) inhibits gamma-aminobutyric acid type A (GABA(A)) receptors in synaptoneurosomes and transfected cells. To further characterize this phenomenon, the effect of T3 on recombinant GABA(A) receptors expressed in Xenopus oocytes was investigated using the two-electrode voltage-clamp method. T3 inhibited GABA-gated chloride currents in a non-competitive manner and yielded an IC50 of 7.3 +/- 0.8 microM in oocytes coexpressing alpha1beta2gamma2L receptor subunits. T3 had no inhibitory effect on alpha6beta2gamma2L or beta2gamma2L receptor constructs, indicating that the alpha1 subunit imparts T3 sensitivity to the receptor. In addition to the inhibitory effect of T3 on GABA responses, T3 alone induced a current in oocytes expressing alpha1beta2gamma2L, alpha6beta2gamma2L and beta2gamma2L constructs. This current displayed a reversal potential identical to that of GABA-gated chloride currents, and was blocked by picrotoxin (10 microM), but not by bicuculline (50 microM), indicating that T3 gates the chloride channel by binding to a site other than the GABA-binding site. The direct channel-gating action of T3 was concentration-dependent, with an EC50 of 23 +/- 5 microM. The actions of T3 are unique in that T3 acts as a noncompetitive antagonist in the presence of GABA but can directly gate the chloride channel in the absence of GABA.

Selective actions of a detergent on ligand-gated ion channels expressed in Xenopus oocytes.

Cytoclean a commercially available detergent, has selective actions on ligand-gated ion channels. Cytoclean (0.0005-0.01% v/v) potentiated 50 microM glycine responses in oocytes expressing alpha-2 glycine receptors by 23 +/- 7% to 342 +/- 43%. Cytoclean is composed of five components dissolved in water, but only one reagent, Bio-Soft D-62, modulated responses of oocytes expressing alpha-2 glycine receptors. Bio-Soft D-62 (0.00005-0.001% w/v), potentiated 50 microM glycine responses by 13 +/- 1% to 474 +/- 50%. Bio-Soft D-62 is composed of linear alkylbenzene sulfonate (> 95% C12 chain). The effects of Cytoclean or Bio-Soft D-62 were examined on alpha-1 beta-2 and alpha-1 beta-2 gamma-2L gamma-aminobutyric acidA, gamma-aminobutyric acid rho 1, DL-alpha-amino-3-hydroxy-5-methyl-4-isoxalonepropionic acid, kainate and 5-hydroxytryptamine3 receptors expressed in Xenopus laevis oocytes. Enhancement of gamma-aminobutyric acidA receptor function ranged from approximately 21% to 458% with Cytoclean (0.0001-0.01%), respectively. Bio-Soft D-62 (0.001%) inhibited GABA rho 1 receptor function by approximately 72%. Cytoclean had no effect on 5-hydroxytryptamine3 or GluR6 function, but Cytoclean (0.005% and 0.01%) inhibited GluR3-mediated currents by approximately 21% and approximately 41%, respectively. These results suggest that trace amounts of Cytoclean, such as amounts adhering to glassware, may modulate ion channel function and potentially confound experimental results.

Enhancement of homomeric glycine receptor function by long-chain alcohols and anaesthetics.

1. The effects of n-alcohols (ethanol to dodecanol) and anaesthetics on strychnine-sensitive glycine receptors were studied in Xenopus oocytes expressing homomeric alpha 1 or alpha 2 glycine receptor subunits, with the two electrode voltage-clamp recording technique. 2. The glycine-induced chloride conductance of homomeric alpha glycine receptors was potentiated by all the alcohols tested when an EC2 concentration of glycine was used. Homomeric alpha 1 and alpha 2 receptors were potentiated similarly by the n-alcohols, except that low concentrations of ethanol produced greater potentiation with alpha 1, as previously reported. 3. Increasing the n-alcohol carbon number has been shown to increase the potency of the alcohols up to decanol at concentrations corresponding to EC50s for producing loss of righting reflex in tadpoles. However, dodecanol was no more potent than decanol, and only modest potentiation (30-60%) was obtained with dodecanol, in contrast to marked (150-200%) potentiation with the other alcohols. Thus, a "cut-off' occurred at about dodecanol. 4. Propofol, alphaxalone, pentobarbitone, halothane and enflurane, reversibly potentiated the function of homomeric alpha 1 glycine receptors at concentrations which represent approximately twice the EC50 for production of anaesthesia in mammals, but ketamine and etomidate were ineffective. 5. Two novel cyclobutane compounds were tested; the anaesthetic compound (1-chloro-1,2,2-trifluorocyclobutane) from 0.5 to 5 mM potentiated the action of glycine in a concentration-dependent manner; however, the non-anaesthetic analogue (1,2-dichloro-hexfluorocyclobutane) had no effect on glycine receptor function at concentrations (25 to 80 microM) predicted to be anaesthetic, based on the lipid solubility of this compound. 6. These results suggest that the alpha subunits of strychnine-sensitive glycine receptors contain sites of action for n-alcohols, propofol, alphaxalone, pentobarbitone and volatile anaesthetics, but not for ketamine and etomidate. Potentiation of glycine receptor function may contribute to the anaesthetic action of n-alcohols and volatile agents.

Actions of anesthetics on ligand-gated ion channels: role of receptor subunit composition.

Molecular cloning of cDNAs coding for ligand-gated ion channel subunits makes it possible to study the pharmacology of recombinant receptors with defined subunit compositions. Many laboratories have used these techniques recently to study actions of agents that produce general anesthesia. We review the effects of volatile and intravenous anesthetics on recombinant GABAA, glycine, AMPA, kainate, NMDA, and 5HT3 receptors. Evidence for and against specific ligand-gated ion channel subunits as targets responsible for anesthesia or the side effects of anesthetic agents is discussed for each type of receptor. Subunit specific actions of some of the agents suggest that construction and testing of certain chimeric receptor subunits may be useful for defining the amino acid sequences responsible for anesthetic actions.

Tyrosine kinase phosphorylation of GABAA receptors.

Phosphorylation of purified bovine brain GABAA receptors by the tyrosine kinase, pp60v-src was examined. pp60v-src phosphorylated two bands of 54-62 kDa and 48-51 kDa that migrated to approximately the same position as bands recognized by antisera against the beta 2 and gamma 2 GABAA receptor subunits, respectively. Bacterially expressed proteins containing the putative large cytoplasmic loops of the beta 1 and gamma 2L subunits were phosphorylated by pp60v-src, indicating that the phosphorylation sites are located in these subunit domains. The tyrosine kinase inhibitors, genistein and the tyrphostins B-42 and B-44, inhibited muscimol-stimulated 36Cl- uptake in mouse brain membrane vesicles (microsacs). magnitude of the tyrphostin B-44-induced inhibition of muscimol-stimulated 36Cl- uptake was significantly reduced in microsacs that were lysed and resealed under conditions that inhibit phosphorylation. GABA-gated Cl- currents were also inhibited by genistein and tyrphostin B-44 in Xenopus oocytes expressing alpha 1 beta 1 and alpha 1 beta 1 gamma 2L subunits. Consequently, protein tyrosine kinase-dependent phosphorylation appears to be another mechanism of regulating the function of GABAA receptors.

Alcohols and anesthetics enhance the function of 5-hydroxytryptamine3 receptors expressed in Xenopus laevis oocytes.

One subunit of the 5-hydroxytryptamine3 (5-HT3) receptor has been cloned, and expression of cDNA coding for this protein in Xenopus oocytes results in the formation of homomeric ion channels. In the present study, this system was used to define the sensitivity of the 5-HT3 receptor to alcohols and anesthetics. Ethanol, in pharmacologically relevant concentrations, potentiated 5-HT-mediated currents, with the greatest potentiation observed at lower concentrations of 5-HT. Likewise, butanol stimulated the receptor but with greater efficacy and potency than ethanol. The volatile anesthetics isoflurane, halothane and 1,2,2-trifluorocyclobutane (F3) all enhanced 5-HT3 receptor function. Concentrations of these anesthetics below the minimal alveolar concentration for anesthesia (MAC) produced significant stimulation of 5-HT-mediated currents. Similar to the alcohols, the greatest enhancement of 5-HT3 receptor function by anesthetics was seen at lower concentrations of 5-HT. However, anesthetics were substantially more efficacious than ethanol in enhancing 5-HT3 receptor function. In the presence of 0.5 microM 5-HT, maximal stimulation by ethanol was approximately 50%, but anesthetic enhancement of 5-HT3 receptor-mediated currents did not reach a maximum. Over the concentrations tested, anesthetics potentiated 0.5 microM 5-HT-mediated currents by approximately 25% to 400%. The intravenous anesthetic propofol did not enhance 5-HT3 receptor function or change the potentiation of this receptor by halothane. These results suggest that alcohols and volatile anesthetics have similar actions on 5-HT3 receptor function, which is in agreement with results of studies with other members of the superfamily of ligandgated ion channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunochemical characterization of the beta 2 subunit of the GABAA receptor.

To date three beta subunits of the GABAA receptor have been identified in rat brain as a result of cDNA library screening. The beta 2 subunit has been reported to have a wide distribution in rat brain based on in situ hybridization studies quantifying beta 2 mRNA. To study the beta 2 subunit more directly, we have raised a polyclonal antibody to a synthetic peptide representing residues 315-334 of the intracellular loop of the beta 2 subunit. The antibody, which had been affinity-purified, recognized the beta 2 peptide but did not immunolabel homologous beta 1 and beta 3 subunit peptides, indicating that this antibody is specific for the beta 2 subunit of the receptor. In western blots of the purified receptor, the antibody recognized a major diffuse band of 54-58 kDa and exhibited minor labeling of lower-molecular-mass polypeptides. In western blots of cortex homogenate, the antibody exhibited nervous system-specific labeling of a 55-kDa band that comigrated with the 55-kDa band of the purified receptor. Quantitative immunolabeling of this 55-kDa polypeptide permitted direct determination of the relative amounts of the beta 2 subunit in different brain regions. The brainstem contained the highest relative specific activity of the beta 2 subunit, followed by the inferior colliculus, olfactory lobe, and cerebellum. Lower levels of immunolabeling were seen in hypothalamus, hippocampus, thalamus, and cortex.

Phosphorylation of cytochrome P4502E1 (CYP2E1) by calmodulin dependent protein kinase, protein kinase C and cAMP dependent protein kinase.

Phosphorylation of pure cytochrome P4502E1 (CYP2E1) was achieved in vitro using Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), protein kinase C (PKC) and cAMP-dependent protein kinase (PKA). The stoichiometry and time-course of phosphorylation were determined. CaM kinase II was the most efficient enzyme capable of catalyzing this phosphorylation reaction: the maximum incorporation of 32PO4 was 0.8 mol/mol CYP2E1 in 20 min. PKA phosphorylated a maximum of 0.7 mol of 32PO4/mol of cytochrome within 60 min. The phosphorylation by PKC reached a maximum of 0.19 mol of 32PO4/mol of cytochrome and this occurred within a few minutes of incubation. Limited digestion by S. aureus V8 protease (SAP) of CYP2E1, which had been phosphorylated by either PKA and PKC, yielded a single major phosphopeptide with an M(r) of approximately 18,000. Limited digestion of CYP2E1, that had been phosphorylated by CaM kinase II, yielded phosphorylated polypeptides with M(r) of approximately 18,000 and 15,000. These results raise the possibility that these three kinases may be involved in the regulation of CYP2E1.

Ca2+/calmodulin-dependent protein kinase II and protein kinase C phosphorylate a synthetic peptide corresponding to a sequence that is specific for the gamma 2L subunit of the GABAA receptor.

The gamma 2 subunit of the GABA receptor (GABAA-R) is alternatively spliced. The long variant (gamma 2L) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylation. Previous studies have demonstrated that a peptide or fusion protein containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP-dependent protein kinase A (PKA)-stimulated phosphorylation. We have examined the ability of PKA, PKC, and Ca2+/calmodulin-dependent protein kinase (CAM kinase II) to phosphorylate a synthetic peptide corresponding to residues 336-351 of the intracellular loop of the gamma 2L subunit and inclusive of the alternatively spliced phosphorylation consensus sequence site. PKC and CAM kinase II produced significant phosphorylation of this peptide, but PKA was ineffective. The Km values for PKC- and CAM kinase II-stimulated phosphorylation of this peptide were 102 and 35 microM, respectively. Maximal velocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC and CAM kinase II, respectively. The phosphorylation site in the eight-amino-acid insert of the gamma 2L subunit has been shown to be necessary for ethanol potentiation of the GABAA-R. Thus, our results suggest that PKC, CAM kinase II, or both may play a role in the effects of ethanol on GABAergic function.

Ethanol and voltage- or receptor-mediated increases in cytosolic Ca2+ in brain cells.

Dissociated brain cells were isolated from newborn rat pups and loaded with fura-2. Different mechanisms for stimulating increased free intracellular Ca2+ concentrations [( Ca2+]i) were examined in the absence and presence of ethanol. KCl, carbachol, and kainate concentration-dependently increased [Ca2+]i. Quisqualate also elevated [Ca2+]i but did not produce clear concentration-dependent increases. KCl, carbachol, and quisqualate responses reached peak levels within 10-30 s and then desensitized within 90 s. However, kainate-stimulated increases in [Ca2+]i plateaued and did not decline after 90 s. Of these different [Ca2+]i-mediated processes, only 60 mM KCl stimulation was significantly inhibited by 100 mM ethanol, while lower KCl concentrations were not affected. Carbachol-induced release of intracellular Ca2+ and activation of non-NMDA (i.e., kainate, quisqualate) excitatory amino acid receptor-operated cation channels were also not significantly inhibited by 100 mM ethanol. Thus, in acutely dissociated brain cells from newborn rats, only Ca2+ influx via voltage- and, as reported previously, NMDA-operated Ca2+ channels were sensitive to ethanol inhibition.

Ethanol has no effect on cAMP-dependent protein kinase-, protein kinase C-, or Ca(2+)-calmodulin-dependent protein kinase II-stimulated phosphorylation of highly purified substrates in vitro.

The actions of ethanol on kinase stimulated phosphorylation were examined using highly purified protein kinases and a variety of purified substrates. Ethanol (25-200 mM) failed to alter the phosphorylation of histone IIa and histone IIIs by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), respectively. Moreover, ethanol (25-200 mM) did not affect the phosphorylation of synapsin I by Ca(2+)-calmodulin-dependent protein kinase II (CAM kinase II). Finally, neither PKA nor PKC stimulated phosphorylation of the GABAA receptor (GABAA-R) was modulated by ethanol at any concentration of ethanol tested. These results suggest that ethanol, in pharmacological concentrations, has no direct actions on the ability of these kinases to catalyze the phosphorylation of specific substrate proteins. In particular, ethanol does not appear to directly influence GABAA-R phosphorylation by either PKA or PKC.

Cyclic AMP-dependent protein kinase decreases gamma-aminobutyric acidA receptor-mediated 36Cl- uptake by brain microsacs.

The effect of cyclic AMP (cAMP)-dependent protein phosphorylation on gamma-aminobutyric acidA (GABAA) receptor function was examined using isolated brain membrane vesicles (microsacs). Muscimol-stimulated 36Cl- uptake was studied in mouse brain microsacs permeabilized to introduce the catalytic subunit of cAMP-dependent protein kinase (PKA). At both submaximal and maximally effective concentrations of muscimol, PKA inhibited muscimol-stimulated 36Cl- uptake by approximately 25%. In parallel experiments, PKA and [gamma-32P]ATP were introduced into the microsacs, and we attempted to immunoprecipitate the entire GABAA receptor complex, under nondenaturing conditions, using an anti-alpha 1-subunit antibody. Data from such experiments show that PKA increases the phosphorylation of several microsac proteins, including a 66-kDa polypeptide specifically immunoprecipitated with the GABAA receptor anti-alpha 1 subunit antibody. Phosphopeptide mapping of the 66-kDa polypeptide demonstrated a 14-kDa fragment similar to that obtained with the purified, PKA-phosphorylated GABAA receptor. These results provide evidence that the catalytic subunit of PKA inhibits the function of brain GABAA receptors and demonstrate that this functional change is concomitant with an increase in protein phosphorylation.