Inhibition of Kv4.3/KChIP2.2 channels by bupivacaine and its modulation by the pore mutation Kv4.3V401I

BACKGROUND: The transient outward current Ito is an important repolarizing K current in human ventricular myocardium mediated by Kv4.3 and KChIP2.2 subunits. Inhibition of Ito by amino-amide local anesthetics may be involved in severe cardiotoxic side effects. This study elucidates the molecular mechanisms of bupivacaine interaction with complexes formed by Kv4.3 and KChIP2.2 as well as the modulatory effect of KChIP2.2. For this purpose, the pharmacologic effects of bupivacaine on Kv4.3wt/KChIP2.2 channels and on the pore mutant Kv4.3V401I were investigated. METHODS: Kv4.3/KChIP2.2 cDNA was transiently expressed in Chinese hamster ovary cells. Site-directed mutagenesis and patch clamp experiments were performed to analyze the effects of bupivacaine on wild-type and mutant channels. RESULTS: Inhibition of Kv4.3wt/KChIP2.2 channels by bupivacaine was concentration-dependent and reversible. The IC50s for inhibition of the charge conducted by Kv4.3wt/KChIP2.2 channels by bupivacaine and levobupivacaine were 55 +/- 8 and 50 +/- 5 microm, respectively. The local anesthetic accelerated macroscopic current decline of Kv4.3wt/KChIP2.2 and slowed recovery from inactivation without altering steady state inactivation. KChIP2.2 altered the response of Kv4.3wt channels to bupivacaine and bupivacaine modulated KChIP2.2 effects on Kv4.3wt channels. The pore mutation V401I slowed macroscopic current decline of Kv4.3 channels and recovery from inactivation, and it diminished modulation of gating by KChIP2.2. Bupivacaine inhibition of Kv4.3V401I resembled Kv4.3wt and was not changed by coexpression of KChIP2.2. CONCLUSIONS: These results indicate that bupivacaine blocks Kv4.3/KChIP2.2 channels from the open state. They furthermore give structural evidence that amino-amide local anesthetics interfere with the effects of KChIP2.2 on Kv4.3 by an indirect mechanism.     

The γ Subunit Determines Whether Anesthetic-induced Leftward Shift Is Altered by a Mutation at α1S270 in α1β2γ2L GABAA Receptors

Background: A major action of volatile anesthetics is enhancement of [gamma]-aminobutyric acid receptor type A (GABAAR) currents. In recombinant GABAARs consisting of several subunit mixtures, mutating the [alpha]1 subunit serine at position 270 to isoleucine [[alpha]1(S270I)] was reported to eliminate anesthetic-induced enhancement at low GABA concentrations. In the absence of studies at high GABA concentrations, it remains unclear whether [alpha]1(S270I) affects enhancement versus inhibition by volatile anesthetics. Furthermore, the majority of GABAARs in mammalian brain are thought to consist of [alpha]1, [beta]2, and [gamma]2 subunits, and the [alpha]1(S270I) mutation has not been studied in the context of this combination.

Methods: Recombinant GABAARs composed of [alpha]1[beta]2 or [alpha]1[beta]2[gamma]2L subunit mixtures were studied electrophysiologically in whole Xenopus oocytes in the voltage clamp configuration. Currents elicited by GABA (0.03 [mu]m to 1 mm) were measured in the absence and presence of isoflurane or halothane. Anesthetic effects on GABA concentration responses were evaluated for individual oocytes.

Results: In wild-type [alpha]1[beta]2[gamma]2L GABAA, anesthetics at approximately 2 minimum alveolar concentration (MAC) shifted GABA concentration response curves to the left approximately threefold, decreased the Hill coefficient, and enhanced currents at all GABA concentrations. The [alpha]1(S270I) mutation itself rendered the GABAAR more sensitive to GABA and reduced the Hill coefficient. At low GABA concentrations (EC5), anesthetic enhancement of peak current was much smaller in [alpha]1(S270I)[beta]2[gamma]2Lversus wild-type channels. Paradoxically, the leftward shift of the whole GABA concentration-response relation by anesthetics was the same in both mutant and wild-type channels. At high GABA concentrations, volatile anesthetics reduced currents in [alpha]1(S270I)[beta]2[gamma]2L GABAARs. In parallel studies on [alpha]1[beta]2 ([gamma]-less) GABAARs, anesthetic-induced leftward shifts in wild-type receptors were more than eightfold at 2 MAC, and the [alpha]1(S270I) mutation nearly eliminated anesthetic-induced leftward shift.     

Influence of GABAA Receptor γ2 Splice Variants on Receptor Kinetics and Isoflurane Modulation

Background: [gamma]-Aminobutyric acid type A (GABAA) receptors, the major inhibitory receptors in the brain, are important targets of many drugs, including general anesthetics. These compounds exert multiple effects on GABAA receptors, including direct activation, prolongation of deactivation kinetics, and reduction of inhibitory postsynaptic current amplitudes. However, the degree to which these actions occur differs for different agents and synapses, possibly because of subunit-specific effects on postsynaptic receptors. In contrast to benzodiazepines and intravenous anesthetics, there is little information available about the subunit dependency of actions of volatile anesthetics. Therefore, the authors studied in detail the effects of isoflurane on recombinant GABAA receptors composed of several different subunit combinations.

Methods: Human embryonic kidney 293 cells were transiently transfected with rat complementary DNAs of [alpha]1[beta]2, [alpha]1[beta]2[gamma]2L, [alpha]1[beta]2[gamma]2S, [alpha]5[beta]3, or [alpha]5[beta]3[gamma]2S subunits. Using rapid application and whole cell patch clamp techniques, cells were exposed to 10- and 2,000-ms pulses of [gamma]-aminobutyric acid (1 mm) in the presence or absence of isoflurane (0.25, 0.5, 1.0 mm). Anesthetic effects on decay kinetics, peak amplitude, net charge transfer and rise time were measured. Statistical significance was assessed using the Student t test or one-way analysis of variance followed by the Tukey post hoc test.

Results: Under control conditions, incorporation of a [gamma]2 subunit conferred faster deactivation kinetics and reduced desensitization. Isoflurane slowed deactivation, enhanced desensitization, and reduced peak current amplitude in [alpha][beta] receptors. Coexpression with a [gamma]2 subunit caused these effects of isoflurane to be substantially reduced or abolished. Although the two [gamma]2 splice variants imparted qualitatively similar macroscopic kinetic properties, there were significant quantitative differences between effects of isoflurane on deactivation and peak current amplitude in [gamma]2S- versus [gamma]2L-containing receptors. The net charge transfer resulting from brief pulses of [gamma]-aminobutyric acid was decreased by isoflurane in [alpha][beta] but increased in [alpha][beta][gamma] receptors.     

Effect of Halothane on Gαi-3 and Its Coupling to the M2 Muscarinic Receptor

Background: Halothane is an effective bronchodilator and inhibits airway smooth muscle contraction in part by inhibiting intracellular signaling pathways activated by the M2 muscarinic receptor and its cognate inhibitory heterotrimeric guanosine-5'-triphosphate (GTP)-binding protein (G protein), Gi. This study hypothesized that halothane inhibits nucleotide exchange at the [alpha] isoform-3 subunit of Gi (G[alpha]i-3), but only when regulated by the M2 muscarinic receptor.

Methods: GTP hydrolysis by G[alpha]i-3 and the G[alpha]i-3[beta]1[gamma]2HF heterotrimer expressed in Spodoptera frugiperda cells was measured using a phosphohydrolase assay with [[gamma]32Pi]-labeled GTP. Anesthetic binding to G[alpha]i-3 was measured by saturation transfer difference nuclear magnetic resonance spectroscopy. G[alpha]i-3 nucleotide exchange was measured in crude membranes prepared from COS-7 cells transiently coexpressing the M2 muscarinic receptor and G[alpha]i-3. A radioactive analog of GTP, [35S]GTP[gamma]S, was used as a reporter for G[alpha]i-3 nucleotide exchange.

Results: Although spectroscopy demonstrated halothane binding to G[alpha]i-3, this binding had no effect on [[gamma]32Pi]-labeled GTP hydrolysis by the G[alpha]i-3[beta]1[gamma]2HF heterotrimer expressed in Spodoptera frugiperda cells, nor basal G[alpha]i-3 nucleotide exchange measured in crude membranes when the muscarinic receptor agonist acetylcholine was omitted from the assay. Conversely, halothane caused a concentration-dependent inhibition of G[alpha]i-3 nucleotide exchange with acetylcholine included in the assay.     

Lidocaine Enhances Gαi Protein Function

Background: Local anesthetics inhibit several G protein-coupled receptors by interaction with the G[alpha]q protein subunit. It is not known whether this effect on G protein function can be extrapolated to other classes of G proteins. The authors investigated interactions of lidocaine with the human adenosine 1 receptor (hA1R)-coupled signaling pathway. Activated A1Rs couple to adenylate cyclase via the pertussis toxin sensitive G[alpha]i protein, thereby decreasing cyclic adenosine monophosphate formation. A1Rs are widely expressed and abundant in the spinal cord, brain, and heart. Interactions of LAs with the hA1R-coupled transduction cascade therefore might produce a broad range of clinically relevant effects.

Methods: The function of hA1Rs stably expressed in Chinese hamster ovary cells was determined with assays of cyclic adenosine monophosphate, receptor binding, and guanosine diphosphate/guanosine triphosphate [gamma]35S exchange by using reconstituted defined G protein subunits. Involvement of phosphodiesterase and G[alpha]i was characterized by using the phosphodiesterase inhibitor rolipram and pertussis toxin, respectively.

Results: Lidocaine (10-9-10-1 M) had no significant effects on agonist or antagonist binding to the hA1R or on receptor-G protein interactions. However, cyclic adenosine monophosphate levels were reduced significantly to 50% by the LAs, even in the absence of an A1R agonist or presence of an A1R antagonist. This effect was unaffected by rolipram (10 [mu]m), but abolished completely by pretreatment with pertussis toxin, which inactivates the G[alpha]i protein. Therefore, the main target site for LAs in this pathway is located upstream from adenylate cyclase.     

ED50 and ED95 of Intrathecal Hyperbaric Bupivacaine Coadministered with Opioids for Cesarean Delivery

Background: Successful cesarean delivery anesthesia has been reported with use of small doses (5-9 mg) of intrathecal bupivacaine coadministered with opioids. This double-blind, randomized, dose-ranging study determined the ED50 and ED95 of intrathecal bupivacaine (with adjuvant opioids) for cesarean delivery anesthesia.

Methods: Forty-two parturients undergoing elective cesarean delivery with use of combined spinal-epidural anesthesia received intrathecal hyperbaric bupivacaine in doses of 6, 7, 8, 9, 10, 11, or 12 mg in equal volumes with an added 10 [mu]g intrathecal fentanyl and 200 [mu]g intrathecal morphine. Sensory levels (pinprick) were evaluated every 2 min until a T6 level was achieved. The dose was a success(induction) if a bilateral T6 block occurred in 10 min; otherwise, it was a failure(induction). In addition to being a success(induction), the dose was a success(operation) if no intraoperative epidural supplement was required; otherwise, it was a failure(operation). ED50 and ED95 for both success(induction) and success(operation) were determined with use of a logistic regression model.

Results: ED50 for success(induction) and success(operation) were 6.7 and 7.6 mg, respectively, whereas the ED95 for success(induction) and success(operation) were 11.0 and 11.2 mg. Speed of onset correlated inversely with dose. Although no clear advantage for low doses could be demonstrated (hypotension, nausea, vomiting, pruritus, or maternal satisfaction), this study was underpowered to detect significance in these variables.     

Rapid and Direct Modulation of GABAA Receptors by Halothane

Background: Hypotheses regarding the nature of channel modulation by volatile anesthetics have focused primarily on "membrane actions" of anesthetics and more recently on direct actions of volatile agents on receptor proteins themselves. With the recognition that many channels are subject to modulation by intracellular enzymes, such as protein kinases and phosphatases, and recent demonstrations that the activity of these modulators themselves may be altered by anesthetic agents, a third possibility has been suggested:-anesthetic actions on channels may be indirect, produced, for example, via direct effects on intracellular enzyme systems.

Methods: To determine the contribution of indirect versus direct modulation, the authors compared effects of the volatile anesthetic halothane on [gamma]-aminobutyric acid A receptors under two conditions: in the whole cell configuration with intact intracellular regulatory systems, and in the excised patch configuration, in which intracellular signaling systems have been disrupted. They also evaluated the effects of rapid application and withdrawal of anesthetic to determine the time course of onset and offset of the anesthetic actions on these channels.

Results: Characteristic changes in [gamma]-aminobutyric acid A receptor function occurred in excised patches as in whole cells, did not require alteration of receptor phosphorylation, and were rapid (onset and offset of anesthetic action occured within milliseconds).     

The ED50 and ED95 of Intrathecal Isobaric Bupivacaine with Opioids for Cesarean Delivery

Background: The ideal intrathecal isobaric bupivacaine dose for cesarean delivery anesthesia is uncertain. While small doses (5-9 mg) of bupivacaine may reduce side effects such as hypotension, they potentially increase spinal anesthetic failures. This study determined the ED50 and ED95 of intrathecal isobaric bupivacaine (with adjuvant opioids) for cesarean delivery.

Methods: After institutional review board approval and written informed consent were obtained, 48 parturients undergoing elective cesarean delivery under combined spinal-epidural anesthesia were enrolled in this double-blind, randomized, dose-ranging study. Patients received a 5-, 6-, 7-, 8-, 9-, 10-, 11-, or 12-mg intrathecal isobaric bupivacaine dose with 10 [mu]g fentanyl and 200 [mu]g morphine. Overall anesthetic success was recorded when no intraoperative epidural supplement was required during the cesarean delivery. ED50 and ED95 values for overall anesthetic success were determined using a logistic regression model.

Results: ED50 and ED95 values for overall anesthetic success were 7.25 and 13.0 mg, respectively. No advantages for low doses could be demonstrated with regard to hypotension, nausea, vomiting, pruritus, or maternal satisfaction, although this study was underpowered to detect significant differences in secondary outcome variables.     

α2A Adrenoceptors Contribute to Feedback Inhibition of Capsaicin-induced Hyperalgesia

Background: Studies on receptor knockout mice have so far shown that of the three [alpha]2-adrenoceptor subtypes, the [alpha]2A adrenoceptor has a major role in mediating the powerful central analgesia induced by synthetic [alpha]2-adrenoceptor agonists. However, because a knockout of the gene for the [alpha]2A adrenoceptor has produced only little if any change in the pain sensitivity of control, nerve-injured, or inflamed animals, it has not been clear whether activation of [alpha]2A-adrenoceptors by endogenous ligands has a significant pain regulatory role.

Methods: The authors assessed spontaneous pain behavior and mechanical hypersensitivity induced by administration of capsaicin in the colon or paw of [alpha]2A-adrenoceptor knockout mice versus their wild-type controls.

Results: Enhanced pain hypersensitivity was observed in [alpha]2A-adrenoceptor knockout mice 20 min or more after administration of capsaicin, but before, hypersensitivity and spontaneous pain were of equal magnitude in [alpha]2A-adrenoceptor knockout and wild-type mice. When wild-type mice were pretreated with an [alpha]2-adrenoceptor antagonist, capsaicin-induced pain hypersensitivity increased to a level equal to that in [alpha]2A-adrenoceptor knockout mice. Capsaicin-induced hypersensitivity was suppressed in wild-type but not [alpha]2A-adrenoceptor knockout mice by a centrally acting [alpha]2-adrenoceptor agonist, whereas a peripherally acting [alpha]2-adrenoceptor agonist was without effect on hypersensitivity, although it attenuated capsaicin-induced spontaneous pain behavior in wild-type mice.     

Isoflurane and Nociception: Spinal α2A Adrenoceptors Mediate Antinociception while Supraspinal α1 Adrenoceptors Mediate Pronociception

Background: The authors recently established that the analgesic actions of the inhalation anesthetic nitrous oxide were mediated by noradrenergic bulbospinal neurons and spinal [alpha]2B adrenoceptors. They now determined whether noradrenergic brainstem nuclei and descending spinal pathways are responsible for the antinociceptive actions of the inhalation anesthetic isoflurane, and which [alpha] adrenoceptors mediate this effect.

Methods: After selective lesioning of noradrenergic nuclei by intracerebroventricular application of the mitochondrial toxin saporin coupled to the antibody directed against dopamine [beta] hydroxylase (D[beta]H-saporin), the antinociceptive action of isoflurane was determined. Antagonists for the [alpha]1 and [alpha]2 adrenoceptors were injected at spinal and supraspinal sites in intact and spinally transected rats to identify the noradrenergic pathways mediating isoflurane antinociception. Null mice for each of the three [alpha]2-adrenoceptor subtypes ([alpha]2A, [alpha]2B, and [alpha]2C) and their wild-type cohorts were tested for their antinociceptive response to isoflurane.

Results: Both D[beta]H-saporin treatment and chronic spinal transection enhanced the antinociceptive effects of isoflurane. The [alpha]1-adrenoceptor antagonist prazosin also enhanced isoflurane antinociception at a supraspinal site of action. The [alpha]2-adrenoceptor antagonist yohimbine inhibited isoflurane antinociception, and this effect was mediated by spinal [alpha]2 adrenoceptors. Null mice for the [alpha]2A-adrenoceptor subtype showed a reduced antinociceptive response to isoflurane.     

Halothane Does Not Inhibit the Functional Coupling between the β2-Adrenergic Receptor and the Gαs Heterotrimeric G Protein

Background: This study investigated whether halothane affects the functional coupling between the [beta]2 adrenergic receptor and the [alpha] subunit of its cognate stimulatory heterotrimeric guanosine-5'-triphosphate (GTP)-binding protein (G[alpha]s). The authors hypothesized that halothane does not affect isoproterenol-promoted guanosine nucleotide exchange at G[alpha]s and hence would not affect isoproterenol-induced relaxation of airway smooth muscle.

Methods: Halothane effects on isoproterenol-induced inhibition of calcium sensitivity were measured in permeabilized porcine airway smooth muscle. G[alpha]s nucleotide exchange was measured in crude membranes prepared from COS-7 cells transfected to transiently coexpress the human [beta]1 or [beta]2 receptor each with human short G[alpha]s. A radioactive, nonhydrolyzable analog of GTP, [35S]GTP[gamma]S, was used as the reporter for nucleotide exchange at G[alpha]s.

Results: Halothane (0.75 mm, approximately 2.8 minimum alveolar concentration [MAC] in pigs) did not affect isoproterenol-induced inhibition of calcium sensitivity. Isoproterenol caused a time- and concentration-dependent increase in G[alpha]s nucleotide exchange. Halothane, even at concentrations of 1.5 mm (approximately 5.6 MAC), had no effect on basal G[alpha]s nucleotide exchange in the absence of isoproterenol, whereas halothane inhibited isoproterenol-promoted G[alpha]s nucleotide exchange in both the [beta]1-G[alpha]s and [beta]2-G[alpha]s expressing membranes. However, the effect was significantly greater on [beta]1-G[alpha]s coupling compared with [beta]2-G[alpha]s coupling, with no effect on [beta]2-G[alpha]s coupling at 2.8 MAC halothane.     

Nitrous Oxide Produces Antinociceptive Response via [small alpha, Greek]2B and/or [small alpha, Greek]2C Adrenoceptor Subtypes in Mice

Background: Opiate receptors in the periaqueductal gray region and [small alpha, Greek]2 adrenoceptors in the spinal cord of the rat mediate the antinociceptive properties of nitrous oxide (N2 O). The availability of genetically altered mice facilitates the detection of the precise protein species involved in the transduction pathway. In this study, the authors establish the similarity between rats and mice in the antinociceptive action of N2 O and investigate which [small alpha, Greek]2 adrenoceptor subtypes mediate this response.

Methods: After obtaining institutional approval, antinociceptive dose-response and time-course to N2 O was measured in wild-type and transgenic mice (D79N), with a nonfunctional [small alpha, Greek]2A adrenoceptor using tail-flick latency. The antinociceptive effect of N2 O was tested after pretreatment systemically with yohimbine (nonselective [small alpha, Greek]2 antagonist), naloxone (opiate antagonist), L659,066 (peripheral [small alpha, Greek]2-antagonist) and prazosin ([small alpha, Greek]2B- and [small alpha, Greek] (2C-selective) antagonist). The tail-flick latency to dexmedetomidine (D-med), a nonselective [small alpha, Greek]2 agonist, was tested in wild-type and transgenic mice.

Results: N2 O produced antinociception in both D79N transgenic and wild-type litter mates, although the response was less pronounced in the transgenic mice. Antinociception from N2 O decreased over time with continuing exposure, and the decrement was more pronounced in the transgenic mice. The antinociceptive response could be dose dependently antagonized by opiate receptor and selective [small alpha, Greek]2B-/[small alpha, Greek]2C-receptor antagonists but not by a central nervous system-impermeant [small alpha, Greek]2 antagonist (L659,066). Whereas dexmedetomidine exhibited no antinociceptive response in the D79N mice, the robust antinociceptive response in the wild-type litter mates could not be blocked by a selective [small alpha, Greek]2B-/[small alpha, Greek]2C-receptor antagonist.     

Enflurane Actions on Spinal Cords from Mice That Lack the β3 Subunit of the GABAA Receptor

Background: [gamma]-Aminobutyric acid type A (GABAA) receptors are considered important in mediating anesthetic actions. Mice lacking the [beta]3 subunit of this receptor ([beta]3-/-) have a higher enflurane minimum alveolar concentration (MAC) than wild types (+/+). MAC is predominantly determined in spinal cord.

Methods: The authors measured three population-evoked responses in whole spinal cords, namely, the excitatory postsynaptic potential (pEPSP), the slow ventral root potential (sVRP), and the dorsal root potential. Synaptic and glutamate-evoked currents from motor neurons in spinal cord slices were also measured.

Results: Sensitivity of evoked responses to enflurane did not differ between +/+ and -/- cords. The GABAA receptor antagonist bicuculline significantly (P < 0.05) attenuated the depressant effects of enflurane on pEPSP, sVRP and glutamate-evoked currents in +/+ but not -/- cords. The glycine antagonist strychnine elevated the pEPSP to a significantly greater extent in -/- than in +/+ cords, but the interactions between strychnine and enflurane did not differ between -/- and +/+ cords.     

Direct Effects of [Greek small letter alpha]1- and [Greek small letter alpha]2-Adrenergic Agonists on Spinal and Cerebral Pial Vessels in Dogs

Background: The effects of adrenergic agonists, often used as local anesthetic additives or spinal analgesics, on spinal vessels have not been firmly established. The authors investigated the effects of [Greek small letter alpha]2- and [Greek small letter alpha]1-adrenergic agonists on spinal and cerebral pial vessels in vivo.

Methods: Pentobarbital-anesthetized dogs (n = 28) were prepared for measurement of spinal pial-vessel diameter in a spinal-window preparation. The authors applied dexmedetomidine, clonidine, phenylephrine, or epinephrine in three different concentrations (0.5, 5.0, and 50 [micro sign]g/ml; [2.1, 1.9, 2.5, and 2.3] x [10-6, 10-5, and 10-4] M, respectively) under the window (one drug in each dog) and measured spinal pial arteriolar and venular diameters in a sequential manner. To enable the comparison of their effects on cerebral vessels, the authors also administered these drugs under a cranial window.

Results: On topical administration, each drug constricted spinal pial arterioles in a concentration-dependent manner. Phenylephrine and epinephrine induced a significantly larger arteriolar constriction than dexmedetomidine or clonidine at 5 [micro sign]g/ml (8%, 11%, 0%, and 1%, respectively). Spinal pial venules tended to be less constricted than arterioles. In cerebral arterioles, greater constrictions were induced by dexmedetomidine and clonidine than those induced by phenylephrine and epinephrine (14%, 8%, 0%, and 1%, respectively). Cerebral pial venules tended to exhibit larger constrictions than cerebral arterioles (unlike in spinal vessels).     

Classic Benzodiazepines Modulate the Open-Close Equilibrium in α1β2γ2L γ-Aminobutyric Acid Type A Receptors

Background: Classic benzodiazepine agonists induce their clinical effects by binding to a site on [gamma]-aminobutyric acid type A (GABAA) receptors and enhancing receptor activity. There are conflicting data regarding whether the benzodiazepine site is allosterically coupled to [gamma]-aminobutyric acid binding versus the channel open-close (gating) equilibrium. The authors tested the hypothesis that benzodiazepine site ligands modulate [alpha]1[beta]2[gamma]2L GABAA receptor gating both in the absence of orthosteric agonists and when the orthosteric sites are occupied.

Methods: GABAA receptors were recombinantly expressed in Xenopus oocytes and studied using two-microelectrode voltage clamp electrophysiology. To test gating effects in the absence of orthosteric agonist, the authors used spontaneously active GABAA receptors containing a leucine-to-threonine mutation at residue 264 on the [alpha]1 subunit. To examine effects on gating when orthosteric sites were fully occupied, they activated wild-type receptors with high concentrations of a partial agonist, piperidine-4-sulfonic acid.

Results: In the absence of orthosteric agonists, the channel activity of [alpha]1L264T[beta]2[gamma]2L receptors was increased by diazepam and midazolam and reduced by the inverse benzodiazepine agonist FG7142. Flumazenil displayed very weak agonism and blocked midazolam from further activating mutant channels. In wild-type receptors activated with saturating concentrations of piperidine-4-sulfonic acid, midazolam increased maximal efficacy.     

Differential Effects of Volatile Anesthetics on M3 Muscarinic Receptor Coupling to the Gαq Heterotrimeric G Protein

Background: Halothane inhibits airway smooth muscle contraction in part by inhibiting the functional coupling between muscarinic receptors and one of its cognate heterotrimeric G proteins, G[alpha]q. Based on previous studies indicating a more potent effect of halothane and sevoflurane on airway smooth muscle contraction compared with isoflurane, the current study hypothesized that at anesthetic concentrations of 2 minimum alveolar concentration (MAC) or less, halothane and sevoflurane but not isoflurane inhibit acetylcholine-promoted G[alpha]q guanosine nucleotide exchange.

Methods: G[alpha]q guanosine nucleotide exchange was measured in crude membranes prepared from COS-7 cells transiently coexpressing the human M3 muscarinic receptor and human G[alpha]q. A radioactive, nonhydrolyzable analog of guanosine-5'-triphosphate, [35S]GTP[gamma]S, was used as a reporter for nucleotide exchange at G[alpha]q.

Results: Acetylcholine caused a concentration-dependent increase in G[alpha]q [35S]GTP[gamma]S-GDP exchange. Neither anesthetic affected constitutive G[alpha]q [35S]GTP[gamma]S-GDP exchange in the absence of acetylcholine. Conversely, each anesthetic caused a concentration-dependent and reversible inhibition of G[alpha]q [35S]GTP[gamma]S-GDP exchange when promoted by acetylcholine. At concentrations of 3 MAC or less, the effect of halothane and sevoflurane were significantly greater than that of isoflurane, with only a minimal inhibition by isoflurane observed at 2 MAC.     

Preparation of Barbiturate Optical Isomers and Their Effects on GABA (A) Receptors

Background: Barbiturate anesthetics are optically active and usually exist in two mirror-image enantiomeric forms. Their stereoselective effects in mammals are well known, but remarkably few data are available concerning their effects on anesthetic targets in vitro. This is in part because of the lack of availability of pure barbiturate enantiomers. Such in vitro data could be used to test the relevance of putative molecular targets.

Methods: A high-performance liquid chromatography technique using a permethylated [Greek small letter beta]-cyclodextrin column was used to separate the optical isomers of three barbiturates in preparative quantities. The effects of the isomers on GABA-induced currents in stably transfected mouse fibroblast cells were investigated using the whole-cell patch-clamp technique.

Results: Highly purified optical isomers of hexobarbital, pentobarbital, and thiopental were prepared, and their effects were studied on a [Greek small letter gamma]-aminobutyric acid type A receptor of defined subunit composition. For each of the three barbiturates, both enantiomers potentiated [Greek small letter gamma]-aminobutyric acid-induced currents at pharmacologically relevant concentrations, with the S-enantiomer being more potent than the R-enantiomer by a factor of between 1.7 and 3.5. The degree of stereoselectivity did not vary greatly with anesthetic concentration.     

Long QT 1 Mutation KCNQ1A344V Increases Local Anesthetic Sensitivity of the Slowly Activating Delayed Rectifier Potassium Current

Background: Anesthesia in patients with long QT syndrome (LQTS) is a matter of concern. Congenital LQTS is most frequently caused by mutations in KCNQ1 (Kv7.1), whereas drug-induced LQTS is a consequence of HERG (human ether-a-go-go-related gene) channel inhibition. The aim of this study was to investigate whether the LQT1 mutation A344V in the S6 region of KCNQ1, at a position corresponding to the local anesthetic binding site in HERG, may render drug insensitive KCNQ1 channels into a toxicologically relevant target of these pharmacologic agents. This may suggest that LQTS constitutes not only a nonspecific but also a specific pharmacogenetic risk factor for anesthesia.

Methods: The authors examined electrophysiologic and pharmacologic properties of wild-type and mutant KCNQ1 channels. The effects of bupivacaine, ropivacaine, and mepivacaine were investigated using two-electrode voltage clamp and whole cell patch clamp recordings.

Results: The mutation A344V induced voltage-dependent inactivation in homomeric KCNQ1 channels and shifted the voltage dependence of KCNQ1/KCNE1 channel activation by +30 mV. The mutation furthermore increased the sensitivity of KCNQ1/KCNE1 channels for bupivacaine 22-fold (KCNQ1wt/KCNE1: IC50 = 2,431 +/- 582 [mu]m, n = 20; KCNQ1A344V/KCNE1: IC50 = 110 +/- 9 [mu]m, n = 24). Pharmacologic effects of the mutant channels were dominant when mutant and wild-type channels were coexpressed. Simulation of cardiac action potentials with the Luo-Rudy model yielded a prolongation of the cardiac action potential duration and induction of early afterdepolarizations by the mutation A344V that were aggravated by local anesthetic intoxication.