Jeffrey F Pregenzer

Chloride channel expression with the tandem construct of alpha 6-beta 2 GABAA receptor subunit requires a monomeric subunit of alpha 6 or gamma 2.

Despite the presence of the multiple subunits (α, β, γ, and δ) and their isoforms for γ-aminobutyric acid, type A (GABAA) receptors in mammalian brains, the αxβ2γ2 subtypes appear to be the prototype GABAA receptors sharing many properties with native neuronal receptors. In order to gain insight into their subunit stoichiometry and orientation, we prepared a tandem construct of the α6 and β2 subunit cDNAs where the carboxyl-terminal of α6 is linked to the amino-terminal of β2 via a linker encoding 10 glutamine residues. Transfection of human embryonic kidney 293 cells with the tandem construct alone failed to induce GABA-dependent Cl− currents, but its cotransfection with the cDNA for α6 or γ2, but not β2, led to the appearance of GABA currents which were picrotoxin-sensitive and, in the case of γ2 containing receptors, responded to a benzodiazepine agonist, U-92330. The high affinity GABA site, however, was detected with [3H]muscimol binding in all combinations of the receptor subunits, including the tandem construct alone or with the β2. No appreciable differences were found in their Kd (2.5 nM) and Bmax values (1.4 pmol/mg of protein). These data are consistent with the view that the polypeptides arising from the tandem construct were expressed with the high affinity GABA site, but unable to form GABA channels. The requirement of a specific monomeric subunit (α6 or γ2) for the tandem construct to express Cl− currents supports a pentameric structure of GABAA receptors consisting of two α6, two β2, and one γ2 for the α6β2γ2 and three α6 and two β2 for the α6β2 subtype.

Efficient functional coupling of the human D3 dopamine receptor to Go subtype of G proteins in SH-SY5Y cells

The D3 dopamine receptor presumably activates Gi/Go subtypes of G‐proteins, like the structurally analogous D2 receptor, but its signalling targets have not been clearly established due to weak functional signals from cloned receptors as heterologously expressed in mostly non‐neuronal cell lines. In this study, recombinant human D3 receptors expressed in a human neuroblastoma cell line, SH‐SY5Y, produced much greater signals than those expressed in a human embryonic kidney cell line, HEK293. Quinpirole, a prototypic agonist, markedly inhibited forskolin‐stimulated cyclic AMP production and Ca2+‐channel (N‐type) currents in SH‐SY5Y cells, and enhanced GTPγ35S binding in isolated membranes, nearly ten times greater than that observed in HEK293 cell membranes. GTPγ35S‐bound Gα subunits from quinpirole‐activated and solubilized membranes were monitored upon immobilization with various Gα‐specific antibodies. Gαo subunits (not Gαi) were highly labelled with GTPγ35S in SH‐SY5Y, but not in HEK293 cell membranes, despite their abundance in the both cell types, as shown with reverse transcription‐polymerase chain reaction and Western blots. N‐type Ca2+ channels and adenylyl cyclase V (D3‐specific effector), on the other hand, exist only in SH‐SY5Y cells. More efficient coupling of the D3 receptor to Go subtypes in SH‐SY5Y than HEK293 cells may be attributed, at least in part, to the two D3 neuronal effectors only present in SH‐SY5Y cells (N‐type Ca2+‐channels and adenylyl cyclase V). The abundance of Go subtypes in the both cell lines seems to indicate their availability not a limiting factor.

Differential pharmacology between the guinea-pig and the gorilla 5-HT1D receptor as probed with isochromans (5-HT1D-selective ligands).

Both the 5‐HT1D and 5‐HT1B receptors are implicated in migraine pathophysiology. Recently isochromans have been discovered to bind primate 5‐HT1D receptors with much higher affinity than 5‐HT1B receptors. In the guinea‐pig, a primary animal model for anti‐migraine drug testing, however, isochromans bound the 5‐HT1D receptor with lower affinity than the gorilla receptor. This species‐specific pharmacology was investigated, using site‐directed mutagenesis on cloned guinea‐pig receptors heterologously expressed in human embryonic kidney 293 cells. Mutations of threonine 100 and arginine 102 at the extracellular side of transmembrane II of the guinea‐pig 5‐HT1D receptor to the corresponding primate residues, isoleucine and histidine, respectively, enhanced its affinity for isochromans to that of the gorilla receptor, with little effects on its affinities for serotonin, sumatriptan and metergoline. Free energy change from the R102H mutation was about twice as much as that from the T100I mutation. For G protein‐coupling, serotonin marginally enhanced GTPγ35S binding in membranes expressing the guinea‐pig 5‐HT1D receptor and its mutants, but robustly in membranes expressing the gorilla receptor. Sumatriptan enhanced GTPγ35S binding in the latter nearly as much as serotonin, and several isochromans by 30–60% of serotonin. We discovered key differences in the function and binding properties of guinea‐pig and gorilla 5‐HT1D receptors, and identified contributions of I100 and H102 of primate 5‐HT1D receptors to isochroman binding. Among common experimental animals, only the rabbit shares I100 and H102 with primates, and could be useful for studying isochroman actions in vivo.

Stimulation and inhibition of iron-dependent lipid peroxidation by desferrioxamine.

Peroxidation of rat brain synaptosomes was assessed by the formation of thiobarbituric acid reactive products in either 50 mM potassium phosphate buffer (pH 7.4) or pH adjusted saline. In phosphate, addition of Fe2+ resulted in a dose-related increase in lipid peroxidation. In saline, stimulation of lipid peroxidation by Fe2+ was maximal at 30 uM, and was less at concentrations of 100 uM and above. Whereas desferrioxamine caused a dose-related inhibition of iron-dependent lipid peroxidation in phosphate, it stimulated lipid peroxidation with Fe2+ by as much as 7-fold in saline. The effects of desferrioxamine depended upon the oxidation state of iron, and the concentration of desferrioxamine and lipid. The results suggest that lipid and desferrioxamine compete for available iron. The data are consistent with the hypothesis that either phosphate or desferrioxamine may stimulate iron-dependent lipid peroxidation under certain circumstances by favoring formation of Fe2+/Fe3+ ratios.

Effects of GABA and various allosteric ligands on TBPS binding to cloned rat GABAA receptor subtypes

1 [35S]t‐butylbicyclophosphorothionate (TBPS) is a high affinity ligand for the picrotoxin site of GABAA receptors. Here we examined TBPS binding to the cloned receptors made of α1, α3 or α6 in combination with β2 or β2 and γ2 subunits, in the presence of GABA and several allosteric ligands (diazepam, methyl 6,7‐dimethoxy‐4‐methyl‐β‐carboline‐3‐carboxylate (DMCM), 3α,21‐dihydroxy‐5α‐pregnan‐20‐one (5α‐THDOC), pentobarbitone and Zn). The cloned receptors were transiently expressed in SF‐9 insect cells by infecting with recombinant baculoviruses. 2 In αβ subtypes, GABA at nanomolar concentrations enhanced TBPS binding but inhibited binding at micromolar concentrations. Half maximal GABA concentrations for enhancement or inhibition of TBPS binding were correlated with high and low affinity GABA binding sites, respectively, in individual subtypes. The maximal enhancement of binding also varied according to the α isoform (α3β2>>α1β2>α6β2). In αβγ subtypes, TBPS binding was unaffected by GABA at nanomolar concentrations, but was inhibited by GABA at micromolar concentrations. Addition of γ2 thus appeared to abolish conformational coupling between high affinity GABA sites and TBPS sites, and also altered low affinity GABA sites; in particular, the half maximal GABA concentration for inhibition of TBPS binding changed from > 100 (α6β2) to 1 μm (α6β2γ2). 3 Allosteric ligands also altered TBPS binding to sensitive GABAA receptor subtypes. For instance, diazepam only in the α1β2γ2 and α3β2γ2 subtypes, and 5α‐THDOC in all the subtypes enhanced TBPS binding in the absence of GABA, and intensified the inhibitory effect of GABA. Pentobarbitone exhibited only the latter effect in all the subtypes we examined. 4 DMCM and Zn, inhibitors of GABA‐induced Cl currents in αβγ and αβ subtypes, respectively, produced opposite effects to agonists, decreasing TBPS binding in the absence of GABA and attenuating (or eliminating in the case of Zn) the inhibitory effect of GABA on TBPS binding. 5 These results show that GABA binding sites and their conformational coupling with TBPS sites are differentially affected by the α isoform (particularly α6 as compared to α1 or α3) and by quaternary interactions involving the γ2 subunit. Moreover, changes in TBPS binding by allosteric ligands include not only direct (allosteric) effects on TBPS sites but also indirect effects via GABA sites, and are consistent with their known subtype selectivity and functionality from previous studies.

Agonist-induced GTPγ35S binding mediated by human 5-HT2C receptors expressed in human embryonic kidney 293 cells

Abstract The 5-HT2C receptor as heterologously expressed in various mammalian cells mediates inositol 1,4,5-triphosphate (IP3) signal by activating Gq/11 subtypes of G proteins, but minimally promotes agonist-induced GTPγ35S binding in membranes due to slow GTP turnover rates of the G proteins. Here we discovered robust (over 200%) agonist-induced GTPγ35S binding mediated by the human receptor expressed in human embryonic kidney (HEK) 293 cells, and investigated its pharmacology. Agonists concentration-dependently increased GTPγ35S binding in isolated membranes, which was competitively blocked by antagonists. Intrinsic efficacies of agonists from GTPγ35S binding were comparable to those from IP3 measurement. Pertussis toxin treatment largely blocked serotonin-induced GTPγ35S binding, serotonin high affinity sites by 70% without altering the total binding sites, and reduced IP3 release by 40%. GTPγ35S-bound Gα subunits from serotonin-activated membranes were mainly Gαi, judging from immobilization studies with various Gα-specific antibodies. Inhibition of forskolin-stimulated cAMP formation, however, was not observed. Apparently, the 5-HT2C receptor-mediated GTPγ35S binding is a unique phenotype observed in HEK293 cells, reflecting its coupling to pertussis toxin-sensitive Gi subtypes, which contribute to the IP3 signal, along with pertussis toxin-insensitive Gq/11 subtypes.

Agonist-induced [35S]GTPγS binding in the membranes of Spodoptera frugiperda insect cells expressing the human D3 dopamine receptor

In the membranes of Spodoptera frugiperda (Sf-9) insect cells heterologously expressing the human D3 dopamine receptor, agonists selective for the receptor, but not antagonists, robustly enhanced [35S]GTPgammaS binding. Quinpirole, for instance, dose-dependently enhanced [35S]GTPgammaS binding with a half-maximal concentration of 2.3 +/- 0.2 nM. Its action was absent in the cells infected with wild type viruses, and competitively blocked by an antagonist, YM-09151-2. A number of known agonists enhanced [35S]GTPgammaS binding to variable degrees, probably reflecting their differential efficacy to activate target G-proteins via the receptor. This agonist-induced [35S]GTPgammaS binding was abolished by N-ethylmaleimide, a selective blocking agent for Gi/Go proteins, with no appreciable effect on ligand binding. We propose coupling of the cloned D3 receptor to endogenous G-proteins in Sf-9 cells, probably homologs of mammalian Gi/Go proteins. Despite the apparent coupling of the D3 receptor to G-proteins, GTPgammaS (10 microM) failed to decrease agonist binding ([3H]dopamine) to the D3 receptor, probably due to small affinity differences between low and high affinity states for agonists in the D3 receptor, as well as due to high receptor density in Sf-9 cells. We conclude that agonist-induced [35S]GTPgammaS binding for the D3 receptor is suitable for estimating ligand intrinsic efficacy and pharmacological characterizations of ligand-receptor interactions.

Contributions of cysteine 114 of the human D3 dopamine receptor to ligand binding and sensitivity to external oxidizing agents

1 Cysteine 114 (C114) of the human dopamine D3 receptor is located at the helical face of transmembrane segment III (TMIII) near aspartate 110, a counterion for the amine group of catecholamines. The contributions of C114 to receptor function were investigated here using site‐directed mutagenetis of C114 to serine. 2 The C114S mutant, as expressed in Sf‐9 cells, bound aminotetralin antagonists (UH‐232 and AJ‐76) and several agonists ((−)3‐PPP, apomorphine, pramipexole and quinpirole) with markedly lower affinities as compared to the wild type D3 receptor, but bound other structurally diverse dopaminergic ligands with only minor changes in affinity. Because an N‐propyl substituent is the only common structural feature among most affected ligands, we propose that the mutation alters ‘a propyl cleft’ on the receptor. The mutation hardly affected quinpirole‐dependent [35S]‐GTPγS binding, suggesting C114 plays a minimal role in receptor‐G‐protein coupling. 3 N‐Ethylmaleimide(NEM), a sulfhydryl modifying agent, blocked ligand binding to the D3 receptor, but not to the C114S mutant. We infer that C114 is the primary residue on the D3 receptor vulnerable to external oxidizing agents. Dopamine D2long and D42 receptors contain highly homologous TMIII sequences including an equivalent cysteine residue. However, only the D2long receptor, not the D42 receptor, displayed NEM sensitivity similar to that of the D3 receptor. 4 We conclude that C114 is critical for high affinity interactions between the D3 receptor and ligands containing an N‐propyl substituent, and unlike its counterpart in the D42 receptor, is highly susceptible to external oxidizing agents.

Acceleration of GABA-dependent desensitization by mutating threonine 266 to alanine of the α6 subunit of rat GABAA receptors

Various GABAA receptor subunits share four highly homologous putative transmembrane domains (M1 to M4) and have been proposed to form an ion channel of pentameric structure with M2 lining the pore. The carboxyl terminal side of M2 contains three amino acid residues containing a hydroxyl group, which are Thr 265, 266 and serine 268 in the alpha 6 subunit. In order to study their functional role, we generated mutants of the alpha 6 subunit carrying a single point mutation of threonine 265 or 266 to alanine, or serine 268 to glycine. Co-expression of the mutants with beta 2 and gamma 2 subunits in human embryonic kidney cells produced functional receptors which are similar to the wild type in their sensitivity to a benzodiazepine agonist (U-92330), insensitivity to Zn, anion permeability, and GABA dose-response profiles as monitored with the whole cell patch clamp technique. Only in the alpha 6T266A beta 2 gamma 2 subtype, however, GABA-induced Cl- currents decayed much more rapidly than the wild type (about 10 times faster). Analysis of the GABA dependency of desensitization indicates that the T266A mutation enhanced the desensitization rate with little effect on the recovery rate from desensitization or on the half-maximal GABA concentration. We conclude that threonine 266 in the alpha 6 subunit plays a pivotal role in desensitization processes of GABAA receptors.

Advantages of heterologous expression of human D2long dopamine receptors in human neuroblastoma SH-SY5Y over human embryonic kidney 293 cells

The human D2long dopamine receptor when expressed heterologously in a human neuronal cell line, SH‐SY5Y, produced more robust functional signals than when expressed in a human embryonic kidney cell line, HEK293. Quinpirole (agonist)‐induced GTPγ35S binding and high affinity sites were 3–4 fold greater in SH‐SY5Y than in HEK293 cells. N‐type Ca2+ channel currents present in SH‐SY5Y cells, but not HEK293 cells, were inhibited potently by quinpirole with a half‐maximal inhibitory concentration of 0.15±0.03 nM. Inhibition of adenylyl cyclases by agonists, on the other hand, was of similar potency and efficacy in the two cell lines. GTPγ35S‐Bound Gα subunits from quinpirole‐activated and solubilized membranes were monitored upon immobilization with various Gα‐specific antibodies. Gαi and Gαo subunits were highly labelled with GTPγ35S in SH‐SY5Y cells, but only Gαi subunits were labelled in HEK293 cells. The additional Go coupling in SH‐SY5Y cells could arise, at least in part, from the presence of Go coupled‐effectors, such as the N‐type Ca2+ channel, and may contribute to robust agonist‐induced GTPγ35S binding, which is a reliable means for measuring ligand intrinsic efficacy. It appears that expression of neuronal G protein‐coupled receptors in neuronal environments could reveal additional functional characteristics that are absent in non‐neuronal cell lines. This appears to be due to, at least in part, to the presence of neuron‐specific effectors. These findings underscore the importance of the cellular environment in which drug actions are examined, particularly in the face of intensive efforts to develop drugs for G protein‐coupled receptors of various origins.