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.     

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.     

Native serotonin 5-HT3 receptors expressed in Xenopus oocytes differ from homopentameric 5-HT3 receptors

Efficacies of the 5-hydroxytryptamine (serotonin) 5-HT3 receptor (5-HT3R) agonists 2-methyl-5-HT, dopamine, and m-chlorophenylbiguanide on 5-HT3R native to N1E-115 cells and on homopentameric 5-HT3R expressed in Xenopus oocytes were determined relative to that of 5-HT. Efficacies of 2-methyl-5-HT and dopamine on 5-HT3R native to differentiated N1E-115 cells are high (54 and 36%) as compared with their efficacies on homopentameric 5-HT3R-A(L) and 5-HT3R-A(S) receptors expressed in oocytes (4-8%). m-Chlorophenylbiguanide does not distinguish between 5-HT3R in N1E-115 cells and in oocytes. The distinct pharmacological profile of 5-HT3R native to differentiated N1E-115 cells is conserved when poly(A)+ mRNA from these cells is expressed in oocytes. The results indicate that, apart from the known 5-HT3R subunits, N1E-115 cells express additional proteins involved in 5-HT3R function.     

The agonistic action of pentobarbital on GABAA beta-subunit homomeric receptors

Murine gamma-aminobutyric acid type A (GABAA) receptor beta 1, beta 2, and beta 3 subunits were expressed in Xenopus oocytes and studied using the two electrode voltage clamp technique. Although all three beta-subunits were unresponsive to GABA when expressed as homomers, the intravenous general anaesthetics pentobarbital, etomidate and propofol induced currents in beta 2 and beta 3 homomers. The pentobarbital-induced currents in beta 3 homomers showed a dose dependence with an ED50 of 89 +/- 8.9 microM and a Hill coefficient of 0.94 +/- 0.08. Zinc (50 microM) blocked (61.1 +/- 5.6% of control) and 200 microM lanthanum potentiated (139 +/- 8.6% of control) the pentobarbital-induced current. This current was also blocked by picrotoxin but was insensitive to the GABAA receptor antagonist bicuculline. These observations indicate that the full expression of the agonistic action of GABA requires the presence of an alpha-subunit, in contrast to the agonistic action of intravenous general anesthetics, where the presence of a beta2 or beta 3-subunit is sufficient. The difference in the agonistic action of intravenous anaesthetics among these highly homologous beta-subunits suggests that the beta-subunit homomeric receptors may be useful to further define the molecular sites of action of intravenous general anaesthetics and other functional domains on GABAA receptors.     

Selective modulation of GABAA receptors by aluminum

Aluminum has been implicated in several neurodegenerative conditions including Alzheimer's disease. Because the mammalian olfactory system has an unusual capacity for the uptake and transneuronal spread of inhaled substances such as aluminum, whole cell recording techniques were used to examine the actions of aluminum on basic membrane properties and amino acid receptors on rat olfactory bulb mitral/tufted (M/T) neurons in culture. Aluminum had little direct effects on M/T neurons. Aluminum (100 microM) did not evoke a membrane current or alter action-potential shape or duration. Aluminum also had no marked effects on the family of voltage-gated membrane currents evoked by a series of 10-mV, 50-ms depolarizing steps. However, aluminum dramatically potentiated the current evoked by 30 microM gamma-aminobutyric acid (GABA) at concentrations <100 microM. Conversely, higher concentrations of aluminum blocked the GABA-evoked current. The effects of aluminum on GABA-evoked currents were not voltage dependent. Aluminum (100 microM) equally potentiated both inward currents at -30 mV and outward currents at + 30 mV. At 300 microM, aluminum blocked both inward and outward currents to a similar extent. In some neurons, aluminum only blocked the current and potentiation was not observed. The biphasic action of aluminum on GABA-evoked currents suggests separate binding sites: a high-affinity potentiating site and a low-affinity inhibiting site. Despite its effects on GABA-evoked currents, aluminum did not alter membrane currents evoked by glutamate, N-methyl-D-aspartate, kainate, or glycine. Aluminum also did not reduce spontaneous excitatory synaptic activity, suggesting little, if any, effect on glutamate release. Although a causal role for aluminum in Alzheimer's disease and other neuropathological conditions remains controversial, it is clear that elevated aluminum concentrations in the brain are associated with a variety of cognitive impairments. The present results indicate that aluminum can alter the function of GABAA receptors and may suggest that aluminum can contribute to cognitive impairment through disruption of inhibitory circuits.     

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.     

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.     

Region-specific changes of GABAA receptors by tolerance to and dependence upon pentobarbital

Shigella sonnei is a major agent of diarrhoeal disease in developed as well as in developing countries. Several phenotypic methods to define strain differences have been applied to this species of Shigella including, more recently, analysis of extrachromosomal and chromosomal DNA. In this study, 432 endemic and epidemic strains isolated between 1975 and 1991 in Italy, France and Switzerland were submitted to rRNA gene restriction pattern analysis, after digestion of whole-cell DNA by Hinc II, and to concomitant biotyping. Thirteen ribotypes, H1 to H13, and five biotypes, a, d, e, f, g, were detected. Ninety-five percent of the sporadic strains were assigned to ribotypes H1 to H4, which could be subtyped, except for H4, in different biotypes. Strains from each of seven different outbreaks had indistinguishable ribotype-biotype patterns. In contrast, 65 strains, isolated in Sicily in 1980 over an extended period of apparently epidemic increase of isolations and which had previously been considered to be a single bacterial clone on the basis of resistance pattern and phage type, were found to belong to two different and scarcely related ribotypes. Ribotyping and biochemical subtyping appear to be a useful epidemiological tool in studies on the circulation and distribution of strains of S. sonnei.     

Gating of I(sK) channels expressed in Xenopus oocytes

The channel underlying the slow component of the voltage-dependent delayed outward rectifier K+ current, I(Ks), in heart is composed of the minK and KvLQT1 proteins. Expression of the minK protein in Xenopus oocytes results in I(Ks)-like currents, I(sK), due to coassembly with the endogenous XKvLQT1. The kinetics and voltage-dependent characteristics of I(sK) suggest a distinct mechanism for voltage-dependent gating. Currents recorded at 40 mV from holding potentials between -60 and -120 mV showed an unusual "cross-over," with the currents obtained from more depolarized holding potentials activating more slowly and deviating from the Cole-Moore prediction. Analysis of the current traces revealed two components with fast and slow kinetics that were not affected by the holding potential. Rather, the relative contribution of the fast component decreased with depolarized holding potentials. Deactivation and reactivation, after a short period of repolarization (100 ms), was markedly faster than the fast component of activation. These gating properties suggest a physiological mechanism by which cardiac I(Ks) may suppress premature action potentials.     

Coupling of calcium receptors to inositol phosphate and cyclic AMP generation in mammalian cells and Xenopus laevis oocytes and immunodetection of receptor protein by region-specific antipeptide antisera

We hypothesized that manganese superoxide dismutase (MnSOD), known to be induced in rat mesothelial cells by asbestos fibers, cytokines, and hyperoxia, may also be induced in asbestos-related pleural diseases such as mesothelioma. MnSOD was assessed in healthy human pleural mesothelium (n = 6), in biopsy samples of human pleural mesothelioma (n = 7), in transformed nonmalignant human mesothelial cells (Met5A), and in two human mesothelioma cell lines (M14K and M38K) established from the tumor tissue of mesothelioma patients. There was no MnSOD immunoreactivity in five of the six samples of healthy pleural mesothelium, whereas MnSOD immunoreactivity was high in the tumor cells in all the mesothelioma samples. Northern blotting, immunohistochemistry, Western blotting, and specific activity measurements showed lower MnSOD in the nonmalignant Met5A mesothelial cells than in the M14K and M38K mesothelioma cells. In additional experiments the mesothelial and mesothelioma cells were exposed to menadione, which generates superoxide intracellularly, and to epirubicin, a cytotoxic drug commonly used to treat mesothelioma. The M38K mesothelioma cells were most resistant to menadione and epirubicin when assessed by LDH release or by adenine nucleotide (ATP, ADP, and AMP) depletion. These same cells showed not only the highest MnSOD levels, but also the highest mRNA levels and activities of catalase, whereas glutathione peroxidase and glutathione reductase levels did not differ significantly. We conclude that MnSOD expression is low in healthy human pleural mesothelium and high in human malignant mesothelioma. The most resistant mesothelioma cells contained coordinated induction of MnSOD and catalase.     

AMPA and NMDA receptors expressed by differentiating Xenopus spinal neurons

N-methyl--aspartate (NMDA) receptors are often the first ionotropic glutamate receptors expressed at early stages of development and appear to influence neuronal differentiation by mediating Ca2+ influx. Although less well studied, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors also can generate Ca2+ elevations and may have developmental roles. We document the presence of AMPA and NMDA class receptors and the absence of kainate class receptors with whole cell voltage-clamp recordings from Xenopus embryonic spinal neurons differentiated in vitro. Reversal potential measurements indicate that AMPA receptors are permeable to Ca2+ both in differentiated neurons and at the time they first are expressed. The PCa/Pmonocation of 1.9 is close to that of cloned Ca2+-permeable AMPA receptors expressed in heterologous systems. Ca2+ imaging reveals that Ca2+ elevations are elicited by AMPA or NMDA in the absence of Mg2+. The amplitudes and durations of these agonist-induced Ca2+ elevations are similar to those of spontaneous Ca2+ transients known to act as differentiation signals in these cells. Two sources of Ca2+ amplify AMPA- and NMDA-induced Ca2+ elevations. Activation of voltage-gated Ca2+ channels by AMPA- or NMDA-mediated depolarization contributes approximately 15 or 30% of cytosolic Ca2+ elevations, respectively. Activation of either class of receptor produces elevations of Ca2+ that elicit further release of Ca2+ from thapsigargin-sensitive but ryanodine-insensitive stores, contributing an additional approximately 30% of Ca2+ elevations. Voltage-clamp recordings and Ca2+ imaging both show that these spinal neurons express functional AMPA receptors soon after neurite initiation and before expression of NMDA receptors. The Ca2+ permeability of AMPA receptors, their ability to generate significant elevations of [Ca2+]i, and their appearance before synapse formation position them to play roles in neural development. Spontaneous release of agonists from growth cones is detected with glutamate receptors in outside-out patches, suggesting that spinal neurons are early, nonsynaptic sources of glutamate that can influence neuronal differentiation in vivo.     

Ethanol increases GABAA responses in cells stably transfected with receptor subunits

Ethanol enhancement of GABAA receptor function has been found in some, but not all, studies. These results suggest the existence of ethanol-sensitive and -resistant receptors that may differ in subunit composition, although methodological differences (e.g., 36Cl- flux versus membrane currents) could also contribute to the different results. To examine these possibilities, we used mouse L(tk-) cells stably transfected with alpha 1 + beta 1 or alpha 1 + beta 1 + gamma 2L GABAA receptor subunit DNAs and compared 36Cl- flux with whole-cell, patch-clamp measurements of GABAA receptor function. Both techniques detected a similar modulation of the GABA receptor by ethanol, flunitrazepam, and pentobarbital. The potentiating action of ethanol required the gamma-subunit and was maximal at a concentration of 10 mM. Similar ethanol potentiation was obtained with brief (20 msec) or long (2 sec) applications of GABA. Analysis of data obtained from individual cells expressing alpha 1 beta 1-gamma 2L subunits showed considerable variability in sensitivity to ethanol, particularly with concentrations of 30 and 100 mM. Ethanol potentiated GABA action if the cells were grown on coverslips coated with polylysine, but had no effect on GABAA receptors of cells grown on uncoated coverslips. Thus, ethanol action was influenced by the growth matrix. Taken together, these data indicate that a gamma-subunit is necessary, but not sufficient, for ethanol sensitivity in this cell system. We suggest that posttranslational processing, particularly receptor phosphorylation, may also be important and that stably transfected cells will be useful in elucidating these events.     

Local anesthetics inhibit receptors coupled to phosphoinositide signaling in Xenopus oocytes

Effects and the mechanism of action of quaternary amine local anesthetics on ligand- and voltage-activated ion currents were studied using voltage-clamped ovarian follicles and oocytes from Xenopus laevis. The fast inward and slow outward currents in response to acetylcholine were unaltered by procaine, whereas the oscillatory and smooth inward chloride currents (ICl) were abolished. Potassium currents (IK) elicited by norepinephrine and oscillatory ICl elicited by lysophosphatidic acid were blocked. Procaine caused a noncompetitive inhibition of oscillatory ICl mediated by heterologously expressed neurotransmitter receptors from the rat brain. Threefold differences were found in the procaine sensitivity of the 5-HT2a and 5-HT2c receptors. The rank order of intrinsic inhibitory activity of local anesthetics was: procaine > lidocaine > dibucaine > tetracaine. Extra- or intracellular application of procaine did not alter the Ca2+-activated Cl- current, indicating that neither the endogenous voltage-gated Ca2+ nor the Ca2+-activated Cl- channels account for the inhibition. Procaine caused only a slight reduction in ICl elicited by photolysis of caged inositol 1,4,5-trisphosphate (InsP3) and did not abolish ICl triggered by GTP[gamma-S]-induced direct activation of G proteins. For receptors coupling to the phosphoinositide/Ca2+ signal transduction pathway, the primary and physiologically relevant site of procaine action appears to be on the extracellular surface, upstream from the G protein, presumably on the receptor.     

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.     

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.     

Endogenous trypsin receptors in Xenopus oocytes: linkage to internal calcium stores

The effects of the protease trypsin, externally applied to full-grown oocytes of Xenopus laevis, were studied using electrophysiology and fluorometry. The following results were obtained: trypsin in concentrations of 0.1 microgram/ml to 1 mg/ml liberated Ca2+ from internal stores and evoked large transient currents of up to 5 microA in bath solutions containing 1 mM or no Ca2+. The response desensitized for 50 minutes and recovered at longer times. Transient currents could also be elicited by tryptic impurities in commercially available collagenase used for defolliculation of oocytes. Application of chymotrypsin (0.01 or 1 mg/ml) or of thrombin (3.4 ng/ml or 0.34 mg/ml) neither evoked currents nor desensitized trypsin responses. Incubation with 1 microgram/ml Pertussis toxin for 20 to 25 hours prevented the Ca2+ release from internal stores and the activation of transient currents by trypsin. We propose that endogenous receptors in the oolemma, specific for trypsin, are linked to internal Ca2+ stores via Pertussis toxin-sensitive G proteins. Thus, receptor activation by external trypsin raises internal Ca2+ and thereby opens Ca(2+)-activated Cl channels in the oolemma.     

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.     

Differential subunit dependence of the actions of the general anesthetics alphaxalone and etomidate at gamma-aminobutyric acid type A receptors expressed in Xenopus laevis oocytes

Atrial electrical activities during hypothermic, K(+)-induced cardioplegic arrest correlate with an increased incidence of postoperative supraventricular dysrhythmias in coronary artery bypass graft patients. Surface electrocardiogram (ECG) (S-ECG) may be insufficiently sensitive to detect such activity intraoperatively, and invasive methods are impractical and traumatic. From induction of anesthesia until the end of surgery, esophageal ECG signals were detected with a new bipolar esophageal probe and a new high-resolution preamplifier (frequency range 0.01-2000 Hz). The S-ECG and the esophageal ECG (E-ECG) were evaluated independently in 18 patients. Eight of 18 patients presented during cardioplegic arrest a mean of 483 +/- 119 high-amplitude, biphasic P components (mean amplitude 0.7 +/- 0.1 mV, range 0.35-1.15 mV) per patient (mean 36 +/- 6 [5-59] potentials/min) similar to those coinciding with the surface ECG P-waves during sinus rhythm. Six of these eight patients presented a mean of 29 +/- 11 low atrial activities (mean amplitude 0.14 +/- 0.023 mV; range 0.1-0.25 mV) per patient (mean 8.4 +/- 5.6 [2.3-48] potentials/min) in the E-ECG. In the S-ECG, one patient of these eight presented 26 P waves during cardioplegic arrest simultaneously with activities in the E-ECG. During the first 5 days, seven of eight (88%) patients with atrial activities in the E-ECG versus 3 of 10 (30%) patients without atrial activities developed supraventricular tachyarrhythmias postoperatively (P < 0.05). This new high-resolution E-ECG device detected in a beat-to-beat technique more atrial activity during cardioplegic arrest than a S-ECG and offered the advantages of artifact exclusion and better prediction of postoperative supraventricular dysrhythmias.     

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.