Specific blockade of slowly activating I(sK) channels by chromanols -- impact on the role of I(sK) channels in epithelia

Chromanols, which were recently shown to inhibit cAMP-mediated Cl- secretion in colon crypts via a blockade of a cAMP-activated K+ conductance, were analyzed for their effects on distinct cloned K+ channels expressed in Xenopus oocytes. The lead chromanol 293B specifically inhibited I(sK) channels with an IC50 of 7 micromol/l without affecting the delayed rectifier Kv1.1 or the inward rectifier Kir2.1. Moreover, several other chromanols displayed the same rank order of potency for I(sK) inhibition as demonstrated in colon crypts. Finally, we tested the effects of the previously described I(sK) blocker azimilide on cAMP mediated Cl- secretion in rat colon crypts. Similar to 293B azimilide inhibited the forskolin induced Cl- secretion. These data suggest that I(sK) protein induced K+ conductances are the targets for the chromanol 293B and its analogues, and azimilide.     

cAMP-activation of amiloride-sensitive Na+ channels from guinea-pig colon expressed in Xenopus oocytes

Guinea-pig distal colonic mRNA injection into Xenopus laevis oocytes resulted in expression of functional active epithelial Na+ channels in the oocyte plasma membrane. Poly(A)+ RNA was extracted from distal colonic mucosa of animals fed either a high-salt (HS) or a low-salt (LS) diet. The electrophysiological properties of the expressed amiloride-sensitive Na+ conductances were investigated by conventional two-electrode voltage-clamp and patch-clamp measurements. Injection of poly(A)+ RNA from HS-fed animals [from hereon referred to as HS-poly(A)+ RNA] into oocytes induced the expression of amiloride-sensitive Na+ conductances. On the other hand, oocytes injected with poly(A)+ RNA from LS-fed animals [LS-poly(A)+ RNA] expressed a markedly larger amount of amiloride-blockable Na+ conductances. LS-poly(A)+ RNA-induced conductances were completely inhibitable by amiloride with a Ki of 77 nM, and were also blocked by benzamil with a Ki of 1.8 nM. 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA), even in high doses (25 "mu"M), had no detectable effect on the Na+ conductances. Expressed amiloride-sensitive Na+ channels could be further activated by cAMP leading to nearly doubled clamp currents. When Na+ was replaced by K+, amiloride (1 "mu"M) showed no effect on the clamp current. Single-channel analysis revealed slow gating behaviour, open probabilities (Po) between 0.4 and 0.9, and slope conductances of 3. 8 pS for Na+ and 5.6 pS for Li+. The expressed channels showed to be highly selective for Na+ over K+ with a permeability ratio PNa/PK > 20. Amiloride (500 nM) reduced channel Po to values < 0.05. All these features make the guinea-pig distal colon of LS-fed animals an interesting mRNA source for the expression of highly amiloride-sensitive Na+ channels in Xenopus oocytes, which could provide new insights in the regulatory mechanism of these channels.     

Block by propofol and thiopentone of the min K current (IsK) expressed in Xenopus oocytes

The slowly activating component of the delayed rectifier potassium current (I(Ks)) in the heart is important during the repolarization of the cardiac action potential. Injection into Xenopus oocytes of mRNA coding for the min K protein induces a similar current (IsK) and recent observations support the hypothesis that functional channels result from the association of the min K protein with an endogenous K+ channel similar to the recently cloned KvLQT1. The general anaesthetics propofol and thiopentone have been shown to suppress cardiac I(Ks) with no effect on the rapidly activating component of I(K) (Takahashi and Terrar 1995). It was therefore of interest to test whether IsK was also inhibited by propofol and thiopentone. IsK was induced following injection into oocytes of min K mRNA which was transcribed in vitro from a synthetic gene (Hausdorff et al. 1991). IsK was activated by step depolarizations to a series of potentials from a holding potential of -40 mV and measured as the deactivating tail current on repolarization to the holding potential. Following a 2 s depolarization to +45 mV, propofol and thiopentone caused concentration-dependent reductions in IsK. The estimated IC50 value for the block of IsK by propofol was 250 microM and by thiopentone was 56 microM. Block of IsK by both propofol and thiopentone was not dependent on voltage or time. The reductions in IsK caused by propofol and thiopentone are consistent with the previously reported effects of these anaesthetics on I(Ks) in the heart and support the hypothesis that the min K protein contributes to the molecular basis of the cardiac I(Ks) channel.     

Actions of the pyrethroid insecticides cismethrin and cypermethrin on house fly Vssc1 sodium channels expressed in Xenopus oocytes

Voltage-sensitive sodium channels encoded by the Vssc1 gene of the house fly (Musca domestica) were expressed in Xenopus laevis oocytes in combination with the tipE gene product of Drosophila melanogaster and were characterized by two-electrode voltage clamp. Vssc1/tipE sodium channels expressed in oocytes were highly sensitive to tetrodotoxin; half-maximal inhibition of sodium currents by tetrodotoxin was obtained at a concentration of 2.4 nM. Cismethrin, a pyrethroid that produces Type I effects on intact nerve, slowed the inactivation of sodium currents carried by Vssc1/tipE channels during a depolarizing pulse and induced a tail current after repolarization that decayed with a first-order time constant of approximately 650 ms. The voltage dependence of activation and steady-state inactivation of cismethrin-modified channels were shifted to more negative potentials. Cypermethrin, a pyrethroid with Type II effects on intact nerve, also prolonged the inactivation of Vssc1/tipE sodium channels and induced a tail current. However, the cypermethrin-induced tail current was extremely persistent, decaying with a first-order time constant of approximately 42 s. Unlike cismethrin, the effect of cypermethrin was use dependent, requiring repeated depolarizing pulses for the full development of modified sodium currents. The divergent effects of cismethrin and cypermethrin on Vssc1/tipE sodium channels expressed in oocytes are consistent with the actions of these and related compounds on sodium channels in invertebrate and vertebrate nerve preparations and provide insight into the mechanisms underlying the production of Type I and II effects on neuronal excitability.     

Chicken GABA(A) receptor beta4 subunits form robust homomeric GABA-gated channels in Xenopus oocytes

We investigated the effect of estrogen on the accumulation of ascorbic acid by human intestinal Caco-2 cells. 17beta-estradiol, synthetic estrogen diethylstilbestrol, and partial agonist tamoxifen were found to inhibit ascorbic acid accumulation in a dose-dependent fashion. The inhibitory effect of estrogens can be observed at as short as 5 min of incubation. An additive effect was observed when they were used in combination. Similar to dietary flavonoids, inhibition was also observed in two other intestinal cell lines, HT-29 and IEC-6. These chemicals affected both Na+ -dependent and Na+ -independent(K+ substituting Na+) accumulation of ascorbic acid and did not affect the efflux of accumulated ascorbic acid. Kinetic analysis of diethylstilbestrol showed a non-competitive inhibition with an apparent Ki of 23 microM. The hormone-ascorbic acid interaction in the intestinal cell could help to explain the known reduction in blood ascorbic acid level among oral contraceptive users and female guinea pigs given contraceptive hormones.     

Toxin and subunit specificity of blocking affinity of three peptide toxins for heteromultimeric, voltage-gated potassium channels expressed in Xenopus oocytes

The ability of voltage-gated potassium channel alpha-subunits to form heteromultimers has complicated efforts to use toxins to characterize potassium channels in native cells. Here I investigate the effects of subunit composition on toxin blocking affinity, using three members of the Shaker subfamily of potassium channel alpha-subunits (Kv1.1, Kv1.2 and Kv1.4), which are known to form heteromultimers in vivo, in the Xenopus oocyte expression system. These subunits were coexpressed as pairs in which one member was toxin-sensitive and the other relatively insensitive. The blocking affinity of two dendrotoxins (DTX-I and delta-DTX) and a scorpion toxin (tityustoxin-Kalpha) on the resulting mixed population of channels was measured to evaluate three models of toxin block. The single subunit model, in which a single toxin-sensitive subunit renders the channel toxin sensitive, best described all of the data for the two dendrotoxins and the block of tityustoxin-Kalpha for a mixed population of channels composed of Kv1.1 and Kv1.2 subunits. However, with tityustoxin-Kalpha, the data for a mixed population of Kv1.2 and Kv1.4 subunits was fit best by a model in which the toxin interacts with all four subunits for high-affinity block. The data suggest that subunit composition of potassium channels can have a large effect on toxin block and that different toxins yield strikingly diverse results with the same pair of subunits, even when they are nearly identical in blocking affinity for the toxin-sensitive subunit.     

Effect of extracellular pH on the myo-inositol transporter SMIT expressed in Xenopus oocytes

The myo-inositol transporter SMIT is expressed particularly at high extracellular osmolarity and serves to accumulate the osmolyte myo-inositol. Transport of myo-inositol is coupled to the cotransport of Na+ and is electrogenic. In Xenopus oocytes injected with mRNA encoding SMIT but not in water-injected oocytes, myo-inositol creates an inward current that is dependent on the ambient Na+ concentration. The present study has been performed to elucidate the pH dependence of myo-inositol-induced currents. Therefore, Xenopus oocytes were injected with mRNA encoding SMIT and two-electrode voltage-clamp studies were performed. The myo-inositol-induced currents in oocytes expressing SMIT were found to have a sigmoidal dependence on the ambient pH between pH 5.5 and 8.5 with an apparent Ki of 0.21+/-001 microM H+ and a Hill coefficient of 1.80+/-0.16. Kinetic analysis of the myo-inositol-induced currents at pH 8.0 and -90 mV holding potential revealed a Hill coefficient of 0.93+/-0.07 and an apparent Km for myo-inositol of 0.031+/-0.003 mM as well as a Hill coefficient of 1. 64+/-0.24 and an apparent Km of 38.8+/-4.1 mM for Na+. A decrease of the Na+ concentra-tion from 150 mM to 50 mM significantly altered the maximal observed current and increased the apparent Km for myo-inositol. Acidification to pH 6.5 significantly increased the apparent Km for myo-inositol and for Na+ to 0.057+/-0.005 mM and 73. 9+/-4.8 mM, respectively. The Hill coefficients for myo-inositol and Na+ were not affected and remained close to 1 for myo-inositol and 2 for Na+. In summary, acidification impedes SMIT-mediated myo-inositol transport at least partially by decreasing the affinity of the carrier for Na+. The impaired Na+ binding subsequently decreases binding and transport of myo-inositol.     

Blocking effects of benzyltetrahydropalmatine on delayed rectified K+ currents expressed in Xenopus oocytes and in toad oocytes

The effects of benzyltetrahydropalmatine (BTHP) on delayed rectified K+ currents (Ik) expressed in Xenopus oocytes and Ik of toad (Bufo bufo gargarizans) oocytes were studied. The Ik expressed in Xenopus oocytes was measured after microinjection of mRNA isolated from carp fish (C anratus L.) brains with double -microelectrode voltage clamp technique. The maximum and mean value of Ik expressed in Xenopus oocytes were 600 nA and 360 +/- 104 nA, respectively. BTHP reduced the current amplitude of Ik expressed in Xenopus oocytes in 10-1000 mumol.L-1 dose-dependently, EC50 was 29 mumol.L-1. Also, the reduction of Ik of toad oocytes was 9.1%, 29.1%, 54.7% and 68.6% by BTHP 10, 30, 100 and 1000 mumol.L-1, respectively, EC50 was 33 mumol.L-1. The results showed that BTHP possesses an inhibitory effect on Ik, the main ion mechanism of antiarrhythmic action of BTHP.     

Rapid chemical kinetic techniques for investigations of neurotransmitter receptors expressed in Xenopus oocytes

Xenopus laevis oocytes have been used extensively during the past decade to express and study neurotransmitter receptors of various origins and subunit composition and also to express and study receptors altered by site-specific mutations. Interpretations of the effects of structural differences on receptor mechanisms were, however, hampered by a lack of rapid chemical reaction techniques suitable for use with oocytes. Here we describe flow and photolysis techniques, with 2-ms and 100-microseconds time resolution, respectively, for studying neurotransmitter receptors in giant (approximately 20-microns diameter) patches of oocyte membranes, using muscle and neuronal acetylcholine receptors as examples. With these techniques, we find that the muscle receptor in BC3H1 cells and the same receptor expressed in oocytes have comparable kinetic properties. This finding is in contrast to previous studies and raises questions regarding the interpretations of the many studies of receptors expressed in oocytes in which an insufficient time resolution was available. The results obtained indicate that the rapid reaction techniques described here, in conjunction with the oocyte expression system, will be useful in answering many outstanding questions regarding the structure and function of diverse neurotransmitter receptors.     

Interaction of Ba2+ with the pores of the cloned inward rectifier K+ channels Kir2.1 expressed in Xenopus oocytes

Interactions of Ba2+ with K+ and molecules contributing to inward rectification were studied in the cloned inward rectifier K+ channels, Kir2.1. Extracellular Ba2+ blocked Kir2.1 channels with first-order kinetics in a Vm-dependent manner. At Vm more negative than -120 mV, the Kd-Vm relationship became less steep and the dissociation rate constants were larger, suggesting Ba2+ dissociation into the extracellular space. Both depolarization and increasing [K+]i accelerated the recovery from extracellular Ba2+ blockade. Intracellular K+ appears to relieve Ba2+ blockade by competitively slowing the Ba2+ entrance rate, instead of increasing its exit rate by knocking off action. Intracellular spermine (100 microM) reduced, whereas 1 mM [Mg2+]i only slightly reduced, the ability of intracellular K+ to repulse Ba2+ from the channel pore. Intracellular Ba2+ also blocked outward IKir2.1 in a voltage-dependent fashion. At Vm >/= +40 mV, where intrinsic inactivation is prominent, intracellular Ba2+ accelerated the inactivation rate of the outward IKir2.1 in a Vm-independent manner, suggesting interaction of Ba2+ with the intrinsic gate of Kir2.1 channels.     

Follicular tissues reduce drug effects on ion channels in oocytes of Xenopus laevis

The influence of follicular tissues on drug effects on ion channels in Xenopus oocytes was tested by investigating the pharmacological properties of a cloned potassium channel in oocytes with and without follicular tissues. The data show that the efficacy of blocking agents (ranging from metal ions to peptides) is drastically reduced by the follicular tissues (reductions by as much as 90% and increases of the IC50 values up to 30-fold). Furthermore, the time course of the blocking effect was slowed down by the tissues (increases of the t50 values up to 40-fold). The described impairment could be mitigated, but not abolished by partial removal of the follicular tissues (so-called defolliculation, leaving only the vitelline envelope and part of the follicle cells on the oocyte surface). The results indicate that the follicular tissues can induce significant errors in pharmacological measurements on membrane proteins in Xenopus oocytes.     

Pharmacology of the nicotinic acetylcholine receptor from fetal rat muscle expressed in Xenopus oocytes

BACKGROUND: Firefly luciferase is a 62 kDa protein that catalyzes the production of light. In the presence of MgATP and molecular oxygen, the enzyme oxidizes its substrate, firefly luciferin, emitting yellow-green light. The reaction proceeds through activation of the substrate to form an adenylate intermediate. Firefly luciferase shows extensive sequence homology with a number of enzymes that utilize ATP in adenylation reactions. RESULTS: We have determined the crystal structure of firefly luciferase at 2.0 A resolution. The protein is folded into two compact domains. The large N-terminal domain consists of a beta-barrel and two beta-sheets. The sheets are flanked by alpha-helices to form an alphabetaalphabetaalpha five-layered structure. The C-terminal portion of the molecule forms a distinct domain, which is separated from the N-terminal domain by a wide cleft. CONCLUSIONS: Firefly luciferase is the first member of a superfamily of homologous enzymes, which includes acyl-coenzyme A ligases and peptide synthetases, to have its structure characterized. The residues conserved within the superfamily are located on the surfaces of the two domains on either side of the cleft, but are too far apart to interact simultaneously with the substrates. This suggests that the two domains will close in the course of the reaction. Firefly luciferase has a novel structural framework for catalyzing adenylate-forming reactions.     

Progesterone-induced DNA polymerase activity in full-grown oocytes of Xenopus laevis (1)

DNA polymerase activity increases in full-grown oocytes of Xenopus laevis during in vitro progesterone-induced maturation. This increase is inhibited by cycloheximide. The presence of the oocyte's nucleus (germinal vesicle) seems essential for the induction of this increase: in previously enucleated oocytes, the level of DNA polymerase activity does not change during progesterone treatment. Furthermore, a new form of DNA polymerase is detectable by DEAE chromatography in in vitro matured oocytes.     

Differential interactions of gentamicin with mouse junctional and extrajunctional ACh receptors expressed in Xenopus oocytes

The nicotinic acetylcholine receptors (AChRs) from Torpedo electric organ and mouse muscles when expressed in Xenopus oocytes desensitize with different time courses. Initially, the role of cAMP-dependent phosphorylation on the gamma subunits in the different desensitization rates was investigated by expressing normal and mutant AChRs in the oocytes cultured in the presence of gentamicin. Mutant Torpedo AChRs lacking the potential cAMP-dependent phosphorylation sites in the gamma subunit appear to desensitize like normal Torpedo AChRs. Similarly, mutant mouse extrajunctional AChRs containing a newly created phosphorylation site in the gamma subunit appeared to desensitize like normal mouse AChRs, which lack the potential cAMP-dependent phosphorylation site in the gamma subunit. These results suggest that different rates of desensitization between the Torpedo and muscle extrajunctional AChRs are not attributable to differential cAMP-dependent phosphorylation of these AChRs. Subsequently, to determine whether gentamicin used in culturing oocytes differentially interacts with muscle junctional and extrajunctional AChRs, we analyzed rates of current decay following different gentamicin treatments. Both chronic and acute treatment with gentamicin profoundly accelerated the decay of whole-cell currents mediated by both types of AChR. The effect of prolonged gentamicin treatment on junctional AChRs was long lasting when compared to treatment on extrajunctional AChRs. Although the two types of AChR still desensitize differently in the absence of gentamicin, these results suggest that the characteristic desensitization of junctional and extrajunctional AChRs observed previously is largely due to differential interactions of gentamicin with the two types of AChR.     

Kappa-opioid receptors couple to inwardly rectifying potassium channels when coexpressed by Xenopus oocytes

Xenopus oocytes expressed kappa-opioid specific binding sites after injection of cRNA prepared from a clone of the rat kappa-opioid receptor. Coinjection of kappa receptor cRNA with cRNA coding for a G protein-linked, inwardly rectifying, K+ channel (GIRK1, or KGA) resulted in oocytes that responded to the kappa agonist U-69593 by activating a large (1.0-1.5-microA) K+ current. U-69593 exhibited an EC50 of 260 +/- 50 nM and was blocked by the opioid antagonists norbinaltorphimine and naloxone. The kappa agonist bremazocine was 200-fold more potent than U-69593 in eliciting K+ current but exhibited a partial agonist profile in this expression system. The present results indicate that stimulation of inwardly rectifying K+ channels may be a potential effector mechanism for kappa-opioid receptors.     

Nicotinic agonists competitively antagonize serotonin at mouse 5-HT3 receptors expressed in Xenopus oocytes

Leptin is an adipocyte-derived blood-borne satiety factor that decreases food intake and increases energy expenditure, thereby leading to a substantial decrease in body weight. To explore the possible roles of the hypothalamic melanocortin system in leptin action, we examined the effects of intracerebroventricular (i.c.v.) injection of leptin with or without SHU9119, a potent antagonist of alpha-melanocyte stimulating hormone, on food intake, body weight, and mitochondrial uncoupling protein-1 (UCP-1) mRNA expression in the brown adipose tissue (BAT) in rats. A single i.c.v. injection of leptin decreased cumulative food intake and body weight gain, and increased UCP-1 mRNA expression during 3 h at the onset of the dark phase. Inhibition of food intake and body weight change with leptin was reversed by co-injection of SHU9119 in a dose-dependent manner. Co-injection of SHU9119 also inhibited completely the leptin-induced increase in UCP-1 mRNA expression in the BAT. Treatment with SHU9119 alone did not affect food intake, body weight, and UCP-1 mRNA expression in rats. The present study provides evidence that the hypothalamic melanocortin system plays a central role in both satiety effect and sympathetic activation of leptin.     

Effects of the abused solvent toluene on recombinant N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors expressed in Xenopus oocytes

Previous studies have shown that toluene, which is commonly abused, depresses neuronal activity and causes behavioral effects in both animals and man similar to those observed for ethanol. In this study, the oocyte expression system was used to test the hypothesis that toluene, like ethanol, inhibits the function of ionotropic glutamate receptors. Oocytes were injected with mRNA for specific N-methyl-D-aspartate (NMDA) or non-NMDA subunits and currents were recorded using conventional two-electrode voltage clamp. To enhance the low water solubility of toluene, drug solutions were prepared by mixing toluene with alkamuls (ethoxylated castor oil) at a 1:1 ratio (v:v) and diluting this mixture to the appropriate concentration with barium-containing normal frog Ringer solution. Alkamuls, up to 0.1%, had no significant effects on membrane leak currents or on NMDA-induced currents. Toluene, up to approximately 9 mM, had only minor effects on membrane leak currents but dose-dependently inhibited NMDA-mediated currents in oocytes. The inhibition of NMDA receptor currents by toluene was rapid, reversible and the potency for toluene's effects was subunit dependent. The NR1/2B subunit combination was the most sensitive with an IC50 value for toluene-induced inhibition of 0.17 mM. The NR1/2A and NR1/2C receptors were 6- and 12-fold less sensitive with IC50 values of 1.4 and 2.1 mM, respectively. In contrast, toluene up to approximately 9 mM did not inhibit kainate-induced currents in oocytes expressing GluR1, GluR1(+)R2 or GluR6 subunits. These results suggest that some of the effects of toluene on neuronal activity and behavior may be mediated by inhibition of NMDA receptors.     

Interaction of positive allosteric modulators with human and Drosophila recombinant GABA receptors expressed in Xenopus laevis oocytes

1. A comparative study of the actions of structurally diverse allosteric modulators on mammalian (human alpha 3 beta 2 gamma 2L) or invertebrate (Drosophila melanogaster Rdl or a splice variant of Rdl) recombinant GABA receptors has been made using the Xenopus laevis oocyte expression system and the two electrode voltage-clamp technique. 2. Oocytes preinjected with the appropriate cRNAs responded to bath applied GABA with a concentration-dependent inward current. EC50 values of 102 +/- 18 microM; 152 +/- 10 microM and 9.8 +/- 1.7 microM were determined for human alpha 3, beta 1 gamma 2L, Rdl splice variant and the Rdl receptors respectively. 3. Pentobarbitone enhanced GABA-evoked currents mediated by either the mammalian or invertebrate receptors. Utilizing the appropriate GABA EC10, the EC50 for potentiation was estimated to be 45 +/- 1 microM, 312 +/- 8 microM and 837 +/- 25 microM for human alpha 3, beta 1 gamma 2L, Rdl splice variant and Rdl receptors respectively. Maximal enhancement (expressed relative to the current induced by the EC10 concentration of GABA where this latter response = 1) at the mammalian receptor (10.2 +/- 1 fold) was greater that at either the Rdl splice variant (5.5 +/- 1.3 fold) or Rdl (7.9 +/- 0.8 fold) receptors. 4. Pentobarbitone directly activated the human alpha 3 beta 1 gamma 2L receptor with an EC50 of 1.2 +/- 0.03 mM and had a maximal effect amounting to 3.3 +/- 0.4 fold of the response evoked by the EC10 concentration of GABA. Currents evoked by pentobarbitone were blocked by 10-30 microM picrotoxin and potentiated by 0.3 microM flunitrazepam. Pentobarbitone did not directly activate the invertebrate GABA receptors. 5. 5 alpha-Pregnan-3 alpha-ol-20-one potentiated GABA-evoked currents mediated by the human alpha 3 beta 1 gamma 2L receptor with an EC50 of 87 +/- 3 nM and a maximal enhancement of 6.7 +/- 0.8 fold of that produced by the GABA EC10 concentration. By contrast, relatively high concentrations (3-10 microM) of this steroid had only a modest effect on the Rdl receptor and its splice variant. 6. A small direct effect of 5 alpha-pregnan-3 alpha-ol-20-one (0.3-10 microM) was detected for the human alpha 3 beta 1 gamma 2L receptor (maximal effect only 0.08 +/- 0.01 times that of the GABA EC10). This response was antagonized by 30 microM picrotoxin and enhanced by flunitrazepam (0.3 microM). 5 alpha-Pregnan-3 alpha-ol-20-one did not directly activate the invertebrate GABA receptors. 7. Propofol enhanced GABA-evoked currents mediated by human alpha 3 beta 1 gamma 2L and Rdl splice variant receptors with EC50 values of 3.5 +/- 0.1 microM and 8 +/- 0.3 microM respectively. The maximal enhancement was similar at the two receptor types (human 11 +/- 1.8 fold; invertebrate 8.8 +/- 1.4 fold that of the GABA EC10). 8. Propofol directly activated the human alpha 3 beta 1 gamma 2L receptor with an EC50 of 129 +/- 10 microM, and at a maximally effective concentration, evoked a current amounting to 3.5 +/- 0.5 times that elicited by a concentration of GABA producing 10% of the maximal response. The response to propofol was blocked by 10-30 microM picrotoxin and enhanced by flunitrazepam (0.3 microM). Propofol did not directly activate the invertebrate Rdl splice variant receptor. 9. GABA-evoked currents mediated by the human alpha 3 beta 1 gamma 2L receptor were potentiated by etomidate (EC50 = 7.7 +/- 0.2 microM) and maximally enhanced to 8 +/- 0.8 fold of the response to an EC10 concentration of GABA. By contrast, the Rdl, or Rdl splice variant forms of the invertebrate GABA receptor were insensitive to the positive allosteric modulating actions of etomidate. Neither the mammalian nor the invertebrate receptors, were directly activated by etomidate. 10. delta-Hexachlorocyclohexane enhanced GABA-evoked currents with EC50 values of 3.4 +/- 0.1 microM and 3.0 +/- 0.1 microM for the human alpha 3 beta 1 gamma 2L receptor and the Rdl splice variant receptor respectively. The maximal enhancement was 4.5     

Four pharmacologically distinct subtypes of alpha4beta2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes

Nicotinic receptors generally are presumed to consist of two alpha and three non-alpha subunits. We varied the relative levels of expression of the neuronal nicotinic alpha4 and beta2 receptor subunits in Xenopus laevis oocytes by nuclear injection of cDNAs coding for these subunits in alpha:beta ratios of 9:1, 1:1, and 1:9. The sensitivities of the receptors to acetylcholine and d-tubocurarine were investigated in voltage-clamp experiments. For receptors expressed at the 9:1 and 1:1 alpha:beta ratios, the EC50 value of acetylcholine is approximately 60 microM. For the majority of the receptors expressed at the 1:9 alpha:beta ratio, the sensitivity to acetylcholine is enhanced 30-fold. No evidence for more than one type of acetylcholine binding site in a single receptor is obtained. The sensitivity to d-tubocurarine decreases with decreasing alpha:beta ratio. IC50 values of d-tubocurarine are 0.2, 0.5, and 2 microM for the 9:1, 1:1, and 1:9 alpha:beta ratios, respectively. At the 1:9 alpha:beta ratio, additional receptors with an IC50 value of 163 microM d-tubocurarine are expressed. At least two components with distinct sensitivities to d-tubocurarine are required to account for the shift in IC50. The combined agonist and antagonist effects reveal four distinct subtypes of alpha4beta2 nicotinic receptors. The results imply that the subunit stoichiometry of heteromeric alpha4beta2 acetylcholine receptors is not restricted to 2alpha:3beta.     

Ethanol, flunitrazepam, and pentobarbital modulation of GABAA receptors expressed in mammalian cells and Xenopus oocytes

GABAA receptors composed of human alpha 1 beta 2 gamma 2L, alpha 1 beta 2 gamma 2S, alpha 1 beta 3 gamma 2S, alpha 6 beta 3 gamma 2S, and alpha 5 beta 3 gamma 3 subunits as well as bovine alpha 1 beta 1 gamma 2L and alpha 1 beta 1 subunits were stably expressed in mammalian L(tk-) cells and transiently expressed in Xenopus oocytes. Effects of muscimol, ethanol, flunitrazepam, and pentobarbital on receptor function were compared for the two expression systems using a 36Cl- flux assay for cells and an electrophysiological assay for oocytes. Muscimol activated all receptors in both expression systems but was more potent for L(tk-) cells than oocytes; this difference ranged from 2.6-to 26-fold, depending upon subunit composition. The most pronounced differences between receptors and expression systems were found for ethanol. In L(tk-) cells, low (5-50 mM) concentrations of ethanol potentiated muscimol responses only with receptors containing the gamma 2L subunit. In oocytes, concentrations of 30-100 mM produced small enhancements for most subunit combinations. Flunitrazepam enhanced muscimol responses for all receptors except alpha 6 beta 3 gamma 2S and alpha 1 beta 1, and this enhancement was similar for both expression systems. Pentobarbital also enhanced muscimol responses for all receptors, and this enhancement was similar for L(tk-) cells and oocytes, except for alpha 6 beta 3 gamma 2S where the pentobarbital enhancement was much greater in oocytes than cells. The alpha 6 beta 3 gamma 2S receptors were also distinct in that pentobarbital produced direct activation of chloride channels in both expression systems. Thus, the type of expression/assay system markedly affects the actions of ethanol on GABAA receptors and also influences the actions of muscimol and pentobarbital on this receptor. Differences between these expression systems may reflect posttranslational modifications of receptor subunits.