Kevin J. Gingrich

Dependence of the GABAA receptor gating kinetics on the alpha‐subunit isoform: implications for structure‐function relations and synaptic transmission.

1. To examine the dependence of gamma‐aminobutyric acid (GABAA) receptor gating on the alpha‐subunit isoform, we studied the kinetics of GABA‐gated currents (IGABA) of receptors that differed in the alpha‐subunit subtype, alpha 1 beta 2 gamma 2S and alpha 3 beta 2 gamma 2S. cDNAs encoding rat brain subunits were co‐expressed heterologously in HEK‐293 cells and the resultant receptors studied with the whole‐cell patch clamp technique and rapidly applied GABA pulses (5‐10 s). 2. IGABA of both receptors showed a loosely similar dependence on GABA concentration over a wide range (1‐5000 microM). Generally, IGABA manifested activation reaching an early current peak, subsequent slower spontaneous desensitization, and deactivation of open channels at pulse termination. Lowering GABA concentrations reduced peak currents and slowed activation and desensitization kinetics. 3. The presence of alpha 3 altered the peak IGABA concentration‐response relationship by shifting the fitted Hill equation to tenfold greater GABA concentrations (GABA concentration at half amplitude: alpha 1, 7 microM; and alpha 3, 75 microM) without affecting Hill coefficients (alpha 1, 1.6; alpha 3, 1.5). These findings indicate a reduction in the apparent activating site affinity and are consistent with previous reports. 4. To investigate differences in gating, we normalized for apparent activating site affinities by analysing the time course of macroscopic gating at equi‐activating GABA concentrations. The presence of alpha 3 slowed activation fourfold (time to current peak (means +/‐ S.E.M.): alpha 1, 1.2 +/‐ 0.06 s (2 microM); alpha 3, 4.7 +/‐ 0.5 s (20 microM)), desensitization nearly twofold (reciprocal of time to 80% decay: alpha 1, 2.5 +/‐ 0.48 s‐1 (100 microM); alpha 3, 1.5 +/‐ 0.15 s‐1 (1000 microM)) and deactivation threefold (monoexponential decay time constant: alpha 1, 0.22 +/‐ 0.026 s (2 microM); alpha 3, 0.68 +/‐ 0.1 s (20 microM)). 5. To gain an insight into the gating mechanisms underlying macroscopic desensitization, we extended a previous gating model of GABAA receptor single‐channel activity to include a desensitization pathway. Such a mechanism reproduced empirical alpha 1 beta 2 gamma 2S activation, desensitization and deactivation kinetics. 6. To identify molecular transitions underlying the gating differences between alpha 1 beta 2 gamma 2S and alpha 3 beta 2 gamma 2S receptors, we explored parameter alterations of the alpha 1 beta 2 gamma 2S gating model that provided an accounting of alpha 3 beta 2 gamma 2S empirical responses. Remarkably, alteration of rates and rate constants involved in ligand binding alone allowed reproduction of alpha 3 beta 2 gamma 2S activation, desensitization and deactivation. 7. These results indicate that substitution of the alpha 3 subunit variant in an alpha 1 beta 2 gamma 2S receptor alters transition rates involved in ligand binding that underlie changes in apparent activating site affinity and macroscopic current gating. Furthermore, they argue strongly that the structural determinants of these functional features reside on the alpha‐subunit.

A randomized, double-blind comparison of the NK1 antagonist, aprepitant, versus ondansetron for the prevention of postoperative nausea and vomiting

BACKGROUND: Antiemetics currently in use are not totally effective. Neurokinin-1 receptor antagonists are a new class of antiemetic that have shown promise for chemotherapy-induced nausea and vomiting. This is the first study evaluating the efficacy and tolerability of the neurokinin-1 receptor antagonist, aprepitant, for the prevention of postoperative nausea and vomiting. METHODS: In this multicenter, double-blind trial, we randomly assigned 805 patients receiving general anesthesia for open abdominal surgery to a preoperative dose of aprepitant 40 mg orally, aprepitant 125 mg orally, or ondansetron 4 mg IV. Vomiting, nausea, and use of rescue therapy were assessed over 48 h after surgery. Treatments were compared using logistic regression. RESULTS: Incidence rates for the primary end point (complete response [no vomiting and no use of rescue] over 0–24 h after surgery, tested for superiority of aprepitant) were not different across groups (45% with aprepitant 40 mg, 43% with aprepitant 125 mg, and 42% with ondansetron). The incidence of no vomiting (0–24 h) was higher with aprepitant 40 mg (90%) and aprepitant 125 mg (95%) versus ondansetron (74%) (P < 0.001 for both comparisons), although between-treatment use of rescue and nausea control was not different. Both aprepitant doses also had higher incidences of no vomiting over 0–48 h (P < 0.001). No statistically significant differences were seen among the side effect profiles of the treatments. CONCLUSIONS: Aprepitant was superior to ondansetron for prevention of vomiting in the first 24 and 48 h, but no significant differences were observed between aprepitant and ondansetron for nausea control, use of rescue, or complete response.

meperidine and Lidocaine Block of Recombinant Voltage-dependent Na+ Channels : Evidence that Meperidine is a Local Anesthetic

BACKGROUND The opioid meperidine induces spinal anesthesia and blocks nerve action potentials, suggesting it is a local anesthetic. However, whether it produces effective clinical local anesthesia in peripheral nerves remains unclear. Classification as a local anesthetic requires clinical local anesthesia but also blockade of voltage-dependent Na+ channels with characteristic features (tonic and phasic blockade and a negative shift in the voltage-dependence of steady-state inactivation) involving an intrapore receptor. The authors tested for these molecular pharmacologic features to explore whether meperidine is a local anesthetic. METHODS The authors studied rat skeletal muscle mu1 (RSkM1) voltage-dependent Na+ channels or a mutant form heterologously coexpressed with rat brain Na+ channel accessory beta1, subunit in Xenopus oocytes. Polymerase chain reaction was used for mutagenesis, and mutations were confirmed by sequencing. Na+ currents were measured using a two-microelectrode voltage clamp. Meperidine and the commonly used local anesthetic lidocaine were applied to oocytes in saline solution at room temperature. RESULTS Meperidine and lidocaine produced tonic current inhibition with comparable concentration dependence. Meperidine caused phasic current inhibition in which the concentration-response relationship was shifted to fivefold greater concentration relative to lidocaine. Meperidine and lidocaine negatively shifted the voltage dependence of steady-state inactivation. Mutation of a putative local anesthetic receptor reduced phasic inhibition by meperidine and lidocaine and tonic inhibition by lidocaine, but not meperidine tonic inhibition. CONCLUSIONS Meperidine blocks Na+ channels with molecular pharmacologic features of a local anesthetic. The findings support classification of meperidine as a local anesthetic but with less overall potency than lidocaine.

Zn2+ inhibition of recombinant GABAA receptors: an allosteric, state-dependent mechanism determined by the γ-subunit

1 The γ‐subunit in recombinant γ‐aminobutyric acid (GABAA) receptors reduces the sensitivity of GABA‐triggered Cl− currents to inhibition by Zn2+ and transforms the apparent mechanism of antagonism from non‐competitive to competitive. To investigate underlying receptor function we studied Zn2+ effects on macroscopic and single‐channel currents of recombinant α1β2 and α1β2γ2 receptors expressed heterologously in HEK‐293 cells using the patch‐clamp technique and rapid solution changes. 2 Zn2+ present for > 60 s (constant) inhibited peak, GABA (5 μM)‐triggered currents of α1β2 receptors in a concentration‐dependent manner (inhibition equation parameters: concentration at half‐amplitude (IC50) = 0.94 μM; slope related to Hill coefficient, S= 0.7) that was unaffected by GABA concentration. The γ2 subunit (α1β2γ2 receptor) reduced Zn2+ sensitivity more than fiftyfold (IC50= 51 μM, S= 0.86); increased GABA concentration (100 μM) antagonized inhibition by reducing apparent affinity (IC50= 322 μM, S= 0.79). Zn2+ slowed macroscopic gating of α1β2 receptors by inducing a novel slow exponential component in the activation time course and suppressing a fast component of control desensitization. For α1β2γ2 receptors, Zn2+ accelerated a fast component of apparent desensitization. 3 Zn2+ preincubations lasting up to 10 s markedly increased current depression and activation slowing of α1β2 receptors, but had little effect on currents from α1β2γ2 receptors. 4 Steady‐state fluctuation analysis of macroscopic α1β2γ2 currents (n= 5) resulted in control (2 μM GABA) power density spectra that were fitted by a sum of two Lorentzian functions (relaxation times: 37 ± 5.6 and 1.41 ± 0.15 ms, means ± s.e.m.). Zn2+ (200 μM) reduced the total power almost sixfold and accelerated the slow (23 ± 2.8 ms, P < 0.05) without altering the fast (1.40 ± 0.16 ms) relaxation time. The ratio (fast/slow) of Lorentzian areas was increased by Zn2+ (control, 3.39 ± 0.55; Zn2+, 4.9 ± 0.37, P < 0.05). 5 Zn2+ (500 μM) depression of previously activated current amplitudes (% control) for α1β2γ2 receptors was independent of GABA concentration (5 μM, 13.2 ± 0.72 %; 100 μM, 12.2 ± 2.9 %, P < 0.8, n= 5). Both onset and offset inhibition time courses were biexponential. Onset rates were enhanced by Zn2+ concentration. Inhibition onset was also biexponential for preactivated α1β2 receptors with current depression more than fourfold less sensitive (5 μM GABA, IC50= 3.8 μM, S= 0.84) relative to that in constant Zn2+. 6 The results lead us to propose a general model of Zn2+ inhibition of GABAA receptors in which Zn2+ binds to a single extracellular site, induces allosteric receptor inhibition involving two non‐conducting states, site affinity is state‐dependent, and the features of state dependence are determined by the γ‐subunit.

Mathematical model of cellular mechanisms contributing to presynaptic facilitation

Presynaptic facilitation of transmitter release from sensory neurons is an important mechanism contributing to nonassociative and associative learning in Aplysia. In a previous modeling study (28,29), we concluded that enhancement of the postsynaptic potential (PSP) during presynaptic facilitation is mediated by at least two processes; spike broadening, which has been observed experimentally, and a process that we modeled as mobilization of transmitter. In an effort to gain insight into the relative contribution of these two mechanisms of presynaptic facilitation, we have extended our earlier model to include more detailed descriptions of: a) the kinetics of the Ca2+ channel, b) the diffusion of Ca2+ through the cytoplasm, c) the process of transmitter release, and d) the PSP. The present quantitative model provides an accurate description of the input-output relationship for synapses of sensory neurons, and predicts changes in the shape of postsynaptic potentials as a function of mobilization and spike broadening. The results confirm and extend previous experimental studies (33) and indicated that cellular analogs of sensitization (facilitation of nondecremented responses) is mediated primarily by spike broadening; whereas, analogs of dishabituation (facilitation of depressed responses) require mobilization.

Quaternary ammonium block of mutant Na+ channels lacking inactivation: features of a transition‐intermediate mechanism

1 The quaternary ammonium (QA) lidocaine derivative QX‐314 (2‐(triethylamino)‐N‐(2,6‐dimethylphenyl)‐acetamide) induces internal pore blockade of single cardiac Na+ channels enzymatically modified (papain) to eliminate fast inactivation. The mechanism involves dual, interacting blocking modes (rapid and discrete) with binding domains deep in the pore from the cytoplasmic mouth, and where the rapid blocked configuration serves as a transition‐intermediate for the development of discrete block. The primary goals of this study were to test for this mechanism in a recombinant Na+ channel genetically engineered to selectively lack fast inactivation, and if present, to explore the underlying structural features. 2 Fast inactivation was removed in rat skeletal muscle μ1 Na+ channels (RSkM1) with an IFM‐QQQ mutation in the cytoplasmic III‐IV interdomain (QQQ). QQQ was expressed in Xenopus oocytes and single‐channel activity was studied in cell‐free, inside‐out membrane patches. Application of QX‐314 (QX, 0‐4 mM) to the cytoplasmic membrane surface caused two distinct modalities of single‐channel blockade: reduction of unitary current and interruptions of current lasting tens of milliseconds. These are consistent with rapid and discrete pore block, respectively. The voltage and concentration dependence of block indicates that the modes interact and have binding sites that share a deep location in the pore, at ≈65 % of the membrane electric field in from the cytoplasmic mouth. 3 Mutation of phenylalanine (F1579) in domain IV‐S6, critical in local anaesthetic block, to alanine in QQQ (QQQ‐F1579A) disabled discrete block but notably failed to alter rapid block, single‐channel gating and slope conductance. 4 Amplitude distribution analysis was applied to long bursts (> 50 ms) of QQQ‐F1579A activity to investigate the kinetics of rapid block. Computed rapid blocking and unblocking rate constants are 42 000 ± 18 000 M−1 ms−1 and 82 ± 22 ms−1, respectively (n= 3, ‐20 mV). 5 The results support a general transition‐intermediate mechanism that governs internal QX and local anaesthetic pore block of voltage‐gated Na+ channels and provide insight into underlying structural features.

Ketamine blockade of voltage-gated sodium channels: evidence for a shared receptor site with local anesthetics.

Background The general anesthetic ketamine is known to be an N-methyl-d-aspartate receptor blocker. Although ketamine also blocks voltage-gated sodium channels in a local anesthetic–like fashion, little information exists on the molecular pharmacology of this interaction. We measured the effects of ketamine on sodium channels. Methods Wild-type and mutant (F1579A) recombinant rat skeletal muscle sodium channels were expressed in Xenopus oocytes. The F1579A amino acid substitution site is part of the intrapore local anesthetic receptor. The effect of ketamine was measured in oocytes expressing wild-type or mutant sodium channels using two-electrode voltage clamp. Results Ketamine blocked sodium channels in a local anesthetic–like fashion, exhibiting tonic blockade (concentration for half-maximal inhibition [IC50] = 0.8 mm), phasic blockade (IC50 = 2.3 mm), and leftward shift of the steady-state inactivation; the parameters of these actions were strongly modified by alteration of the intrapore local anesthetic binding site (IC50 = 2.1 mm and IC50 = 10.3 mm for tonic and phasic blockade, respectively). Compared with lidocaine, ketamine showed greater tonic inhibition but less phasic blockade. Conclusions Ketamine interacts with sodium channels in a local anesthetic–like fashion, including sharing a binding site with commonly used clinical local anesthetics.

Nimodipine prevents transient cognitive dysfunction after moderate hypoxia in adult mice.

Background Cognitive changes associated with moderate hypoxia may be related to the elevation of cytosolic calcium (Ca2+) levels which may, in turn, affect neurotransmitter synthesis and metabolism. We tested whether treatment with nimodipine (NIMO), an L-type Ca2+ channel blocker, would preserve working memory after hypoxic hypoxia. Methods We randomized 157 Swiss-Webster, 30 to 35 g mice (6 to 8 wk) to 6 groups, which were exposed to the following gas mixtures for 1 hour: (1) O2 21%; (2) O2 21% followed by 0.1 mg/kg of subcutaneous NIMO; (3) O2 21% followed by vehicle (60% polyethylene glycol/40% methanol); (4) O2 10%; (5) O2 10% then NIMO; (6) O2 10% then vehicle. The Object Recognition Test (ORT) was given once either on Day 1 or Day 7 to assess changes in short-term memory. ORT exploits the tendency of mice to prefer novel over familiar objects. Two identical objects were placed in an arena for 15 minutes of training. During the testing 1 hour later, one of the objects was replaced by a new object. Recognition Index (RI) was used to compare performance. It is defined as the time spent exploring the novel object divided by the time spent exploring both objects, the novel plus the familiar, and this ratio is converted to a percentage. RI was analyzed with analysis of variance. Tukey Honestly Significant Difference tests were used for post hoc comparisons when appropriate. P values <0.05 were considered significant. Results RI for the control group was 68.3% (SE±3.6%). RI was 53.7% (SE±3.8%) for the 10% O2 group on the first posttreatment day. O2 saturation (SpO2) for the hypoxic group was 71.7% (SE±0.5%). By Day 7, RI for the 10% O2 group increased to 64.2% (SE±4.7%), which was not significantly different from control. On Day 1, RI was 68.6% (SE±5.2%) for hypoxic rodents treated with NIMO. These results were statistically significant. Low RI indicates impaired working memory and high RI indicates intact working memory. These results suggest that NIMO prevented impairment of working memory after moderate hypoxia. Conclusions NIMO reverses the disturbance of short-term working memory caused by moderate hypoxia in mice. The results may have implications for cognitive changes linked to Ca2+ homeostasis in the postoperative period.

Anesthetic complications in pediatric patients undergoing cochlear implantation

Cochlear implantation (CI) is effective in the treatment of childhood sensorineural hearing loss and is associated with minimal surgical complications. We investigated the incidence of anesthetic complications in young patients undergoing general anesthesia for CI.

Pentobarbital Produces Activation and Block of α1β2γ2S GABAA Receptors in Rapidly Perfused Whole Cells and Membrane Patches: Divergent Results Can Be Explained by Pharmacokinetics

Millimolar concentrations of the barbiturate pentobarbital (PB) activate γ-aminobutyric acid (GABA) type A receptors (GABARs) and cause blockade reported by a paradoxical current increase or “tail” upon washout. To explore the mechanism of blockade, we investigated PB-triggered currents of recombinant α1β2γ2S GABARs in whole cells and outside-out membrane patches using rapid perfusion. Whole cell currents showed characteristic bell-shaped concentration dependence where high concentrations triggered tail currents with peak amplitudes similar to those during PB application. Tail current time courses could not be described by multi-exponential functions at high concentrations (≥3,000 μM). Deactivation time course decayed over seconds and was slowed by increasing PB concentration and application time. In contrast, macropatch tail currents manifested eightfold greater relative amplitude, were described by multi-exponential functions, and had millisecond rise times; deactivation occurred over fractions of seconds and was insensitive to PB concentration and application time. A parsimonious gating model was constructed that accounts for macropatch results (“patch” model). Lipophilic drug molecules migrate slowly through cells due to avid partitioning into lipophilic subcellular compartments. Inclusion of such a pharmacokinetic compartment into the patch model introduced a slow kinetic component in the extracellular exchange time course, thereby providing recapitulation of divergent whole cell results. GABA co-application potentiated PB blockade. Overall, the results indicate that block is produced by PB concentrations sixfold lower than for activation involving at least three inhibitory PB binding sites, suggest a role of blocked channels in GABA-triggered activity at therapeutic PB concentrations, and raise an important technical question regarding the effective rate of exchange during rapid perfusion of whole cells with PB.