Activity-dependent depression of neuronal sodium channels by the general anaesthetic isoflurane.

BACKGROUND: The mechanisms by which volatile anaesthetics such as isoflurane alter neuronal function are poorly understood, in particular their presynaptic mechanisms. Presynaptic voltage-gated sodium channels (Na(v)) have been implicated as a target for anaesthetic inhibition of neurotransmitter release. We hypothesize that state-dependent interactions of isoflurane with Na(v) lead to increased inhibition of Na(+) current (I(Na)) during periods of high-frequency neuronal activity. METHODS: The electrophysiological effects of isoflurane, at concentrations equivalent to those used clinically, were measured on recombinant brain-type Na(v)1.2 expressed in ND7/23 neuroblastoma cells and on endogenous Na(v) in isolated rat neurohypophysial nerve terminals. Rate constants determined from experiments on the recombinant channel were used in a simple model of Na(v) gating. RESULTS: At resting membrane potentials, isoflurane depressed peak I(Na) and shifted steady-state inactivation in a hyperpolarizing direction. After membrane depolarization, isoflurane accelerated entry (τ(control)=0.36 [0.03] ms compared with τ(isoflurane)=0.33 [0.05] ms, P<0.05) and slowed recovery (τ(control)=6.9 [1.1] ms compared with τ(isoflurane)=9.0 [1.9] ms, P<0.005) from apparent fast inactivation, resulting in enhanced depression of I(Na), during high-frequency stimulation of both recombinant and endogenous nerve terminal Na(v). A simple model of Na(v) gating involving stabilisation of fast inactivation, accounts for this novel form of activity-dependent block. CONCLUSIONS: Isoflurane stabilises the fast-inactivated state of neuronal Na(v) leading to greater depression of I(Na) during high-frequency stimulation, consistent with enhanced inhibition of fast firing neurones.

Pentobarbital inhibition of human recombinant alpha1A P/Q-type voltage-gated calcium channels involves slow, open channel block.

BACKGROUND AND PURPOSE: Pre-synaptic neurotransmitter release is largely dependent on Ca(2+) entry through P/Q-type (Ca(V)2.1) voltage-gated Ca(2+) channels (PQCCs) at most mammalian, central, fast synapses. Barbiturates are clinical depressants and inhibit pre-synaptic Ca(2+) entry. PQCC barbiturate pharmacology is generally unclear, specifically in man. The pharmacology of the barbiturate pentobarbital (PB) in human recombinant alpha(1A) PQCCs has been characterized. EXPERIMENTAL APPROACH: PB effects on macroscopic Ca(2+)(I(Ca)) and Ba(2+)(I(Ba)) currents were studied using whole-cell patch clamp recording in HEK-293 cells heterologously expressing (alpha(1A))(human)(beta(2a)alpha(2)delta-1)(rabbit) PQCCs. KEY RESULTS: PB reversibly depressed peak current (I(peak)) and enhanced apparent inactivation (fractional current at 800 ms, r(800)) in a concentration-dependent fashion irrespective of charge carrier (50% inhibitory concentration: I(peak), 656 microM; r(800), 104 microM). Rate of mono-exponential I(Ba) decay was linearly dependent on PB concentration. PB reduced channel availability by deepening non-steady-state inactivation curves without altering voltage dependence, slowed recovery from activity-induced unavailable states and produced use-dependent block. PB (100 microM) induced use-dependent block during physiological, high frequency pulse trains and overall depressed PQCC activity by two-fold. CONCLUSION AND IMPLICATIONS: The results support a PB pharmacological mechanism involving a modulated receptor with preferential slow, bimolecular, open channel block (K(d)= 15 microM). Clinical PB concentrations (<200 microM) inhibit PQCC during high frequency activation that reduces computed neurotransmitter release by 16-fold and is comparable to the magnitude of Ca(2+)-dependent facilitation, G-protein modulation and intrinsic inactivation that play critical roles in PQCC modulation underlying synaptic plasticity. The results are consistent with the hypothesis that PB inhibition of PQCCs contributes to central nervous system depression underlying anticonvulsant therapy and general anaesthesia.

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.

Dominant gating governing transient GABA(A) receptor activity: a first latency and Po/o analysis.

Steady-state, single-channel gating of GABA(A) receptors (GABARs ) is complex. Simpler gating may dominate when triggered by rapid GABA transients present during fast inhibitory synaptic transmission and is critical to understanding the time course of fast IPSCs. We studied the single-channel activity of expressed alpha1beta1gamma2 GABARs in outside-out patches from human embryonic kidney 293 cells triggered by rapidly applied GABA (10-2000 microm) pulses (2-300 msec). Activation was analyzed with the time to first channel opening after GABA presentation, or first latency (FL). FL distributions are monoexponential at low GABA concentrations and biexponential above 30 microm GABA. The fast rate increases supralinearly to a plateau of approximately 1100 sec(-1), the apparent activation rate. The slow rate and amplitude are insensitive to GABA concentration. The results argue that doubly liganded receptors can rapidly desensitize before opening. Gating after the first opening was quantified with analysis of open probability conditioned on the first opening (P(o/o)). P(o/o) functions are biexponential, dominated by a fast component, and insensitive to GABA concentration. This suggests that open channels convert primarily to fast but also to slow desensitized states. Furthermore, dual modes of fast desensitization may influence IPSC amplitude and thereby synaptic efficacy. The findings provided for the construction of a mathematical gating model that accounts for FL and P(o/o) functions. In addition, the model predicts the time course of macroscopic current responses thought to mimic IPSCs. The results provide new insights into dominant gating that is likely operational during fast GABAergic synaptic transmission.

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 mu1 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 approximately 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.

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 gamma-subunit.

1. The gamma-subunit in recombinant gamma-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 alpha 1 beta 2 and alpha 1 beta 2 gamma 2 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 microM)-triggered currents of alpha 1 beta 2 receptors in a concentration-dependent manner (inhibition equation parameters: concentration at half-amplitude (IC50) = 0.94 microM; slope related to Hill coefficient, S = 0.7) that was unaffected by GABA concentration. The gamma 2 subunit (alpha 1 beta 2 gamma 2 receptor) reduced Zn2+ sensitivity more than fiftyfold (IC50 = 51 microM, S = 0.86); increased GABA concentration (100 microM) antagonized inhibition by reducing apparent affinity (IC50 = 322 microM, S = 0.79). Zn2+ slowed macroscopic gating of alpha 1 beta 2 receptors by inducing a novel slow exponential component in the activation time course and suppressing a fast component of control desensitization. For alpha 1 beta 2 gamma 2 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 alpha 1 beta 2 receptors, but had little effect on currents from alpha 1 beta 2 gamma 2 receptors. 4. Steady-state fluctuation analysis of macroscopic alpha 1 beta 2 gamma 2 currents (n = 5) resulted in control (2 microM 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 microM) 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 microM) depression of previously activated current amplitudes (% control) for alpha 1 beta 2 gamma 2 receptors was independent of GABA concentration (5 microM, 13.2 +/- 0.72%; 100 microM, 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 alpha 1 beta 2 receptors with current depression more than fourfold less sensitive (5 microM GABA, IC50 = 3.8 microM, 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 gamma-subunit.

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.

Ultra-deep blockade of Na+ channels by a quaternary ammonium ion: catalysis by a transition-intermediate state?

1. Individual Na+ channels from isolated guinea-pig ventricular heart cells were studied using the patch-clamp technique. To localize the selectivity region of the channels we investigated their blockade by a permanently charged quaternary ammonium ion (QX-314, 2-(triethylamino)-N-(2,6-dimethylphenyl)acetamide, 0-5 mM) that was applied to the cytoplasmic side of the channel. 2. Resolution of individual blocking events was enhanced by covalent removal of fast inactivation following brief internal exposure to the enzyme papain. The improved resolution reveals the existence of two distinct modalities of blockade: reduction of unitary current, and millisecond interruptions of current. 3. Both modes of internal block could be potentiated by lowering external Na+ concentration. This finding argues that the two corresponding sites of interaction are both located within the channel pore. 4. Analysis of the voltage dependence of block placed both binding sites deep within the pore, at 70% of the electric field from the cytoplasmic entrance. Combined with recent studies localizing block by external Cd2+, the present results argue that the selectivity region of Na+ channels is quite narrow (spanning about 10% of the electric field), and located near the external side of the channel. 5. The manner in which the two blocking processes interact, along with the physical proximity of their binding sites, leads us to propose that the block configuration responsible for the reduction in unitary current serves as a transition intermediate that catalyses formation of the discrete-block complex.

Modeling the hemodynamic response to dopamine in acute heart failure.

A descriptive incremental nonlinear single-input-multiple-output (SIMO) model of the hemodynamic response [cardiac output (CO) and mean aortic pressure (MAP)] to the inotropic drug dopamine in acute ischemic heart failure was constructed to facilitate the design of closed-loop control systems. The structure of the CO component of the model is a first-order system with a sigmoidal relationship. The MAP component is a first-order system with a threshold. Parameter identification was performed on data collected during positive step (drug on) and negative step (drug off) testing using multiple levels (2-6 mcg/kg/min) of infusion of dopamine in a canine model of acute ischemic heart failure. Parameter estimation utilized a least squares objective function and a linearized form of the step response of the model in the time domain. The model provides good approximations to the mean empirical responses.

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.

Single-cell neuronal model for associative learning.

Recently, a novel cellular mechanism, activity-dependent neuromodulation, was identified in sensory neurons mediating the gill and tail withdrawal reflexes in Aplysia. This mechanism may explain associative learning on a behavioral level. The present study was designed to mathematically model subcellular events that may underlie this mechanism and to examine the ability of the model to fit available empirical data. In this associative model, the reinforcing or unconditioned stimulus (US) leads to non-specific enhancement of transmitter release from sensory neurons by activating a cAMP cascade. Spike activity in sensory neurons, the conditioned stimulus (CS), transiently elevates intracellular Ca2+. The CS-triggered increases of intracellular Ca2+ "primes" the cyclase and amplifies the US-mediated cAMP synthesis. As a result of pairing specific amplification of cAMP levels, transmitter release is enhanced beyond that produced by unpaired stimuli or by application of the US alone. The model is capable of fitting empirical data on activity-dependent neuromodulation and predicts a characteristic interstimulus interval (ISI) curve. At the subcellular level, the model's ISI function is related to the time course of the buffering of intracellular Ca2+. The magnitude and duration of the pairing specific enhancement of transmitter release is related to the levels and time course of intracellular cAMP stimulation.

Simulation of synaptic depression, posttetanic potentiation, and presynaptic facilitation of synaptic potentials from sensory neurons mediating gill-withdrawal reflex in Aplysia.

The defensive gill-withdrawal reflex in Aplysia has proven to be an attractive system for analyzing the neural mechanisms underlying simple forms of learning such as habituation, sensitization, and classic conditioning. Previous studies have shown that habituation is associated with synaptic depression and sensitization with presynaptic facilitation of transmitter release from sensory neurons mediating the reflex. The synaptic depression, in turn, is associated with a decrease in Ca2+ currents in the sensory neurons, whereas presynaptic facilitation with increased Ca2+ currents produced indirectly by a decrease in a novel serotonergic sensitive K+ current. The present work represents an initial quantitative examination of the extent to which these mechanisms account for each of these types of synaptic plasticity. To address these issues a lumped parameter mathematical model of the sensory neuron release process was constructed. Major components of this model include Ca2+-channel inactivation, Ca2+-mediated neurotransmitter release and mobilization, and readily releasable and upstream feeding pools of neurotransmitter. In the model, release of neurotransmitter has a linear function of Ca2+ concentration and is not affected directly by residual Ca2+. The model not only simulates the data of synaptic depression and recovery from depression, but also qualitatively predicts other features of neurotransmitter release that it was not designed to fit. These include features of synaptic depression with high and low levels of transmitter release, posttetanic potentiation, a steep relationship between action potential duration and transmitter release, enhanced release produced by broadening the sensory neuron action potential (presynaptic facilitation), and dramatic synaptic depression with two closely spaced tetraethylammonium (TEA) spikes. The model cannot account fully for synaptic depression with empirically observed somatic Ca2+-current kinetics. Rather a large component of synaptic depression is due to reduction to the pools of releasable neurotransmitter (depletion). In the model when spike durations are greater than 15-20 ms, spike broadening produces little facilitation. However, when spike durations are more physiological, spike broadening leads to enhanced transmitter release.