Fast and slow interactions of n-alkanols with human 5-HT3A receptors: Implications for anesthetic mechanisms.

This is part of a continuing patch-clamp study exploring molecular actions of anesthetics and systematically varied related substances on 5-HT3A receptors as prototypes of ligand-gated ion channels. Specifically, n-alkanols, related to but simpler in structure than propofol, were studied to explore the complex actions of this leading intravenous anesthetic. Outside-out patches excised from HEK 293 cells heterologously expressing human 5-HT3A receptors were superfused with even-numbered n-alkanols (ethanol through n-tetradecanol) of different concentrations. Fast solution exchange for varying durations allowed separation of drug actions by their kinetics. Compared with propofol the electrophysiological responses to n-alkanols were not much simpler. n-Alkanols produced fast and slow inhibition or potentiation of current amplitudes, and acceleration of current rise and decay time constants, depending on exposure time, concentration, and chain-length of the drug. Inhibition dominated, characterized by fast and slow processes with time constants separated by two orders of magnitude which were similar for different n-alkanols and for propofol. Absolute interaction energies for ethanol to n-dodecanol (relative to xenon) ranged from -10.8 to -37.3kJmol(-1). No two n-alkanols act completely alike. Potency increases with chain length (until cutoff) mainly because of methylene groups interacting with protein sites rather than because of their tendency to escape from the aqueous phase. Similar wash-in time constants for n-alkanols and propofol suggest similar mechanisms, dominated by the kinetics of conformational state changes rather than by binding reactions.

Direct effects of morphine but not of fentanyl-type opioids on human 5-HT3A receptors in outside-out patch-clamp studies.

BACKGROUND: The alkaloid morphine is historically the oldest opiate, yet still today it has clinically important uses in analgesic therapies. The main analgesic effect of opioids, including synthetic opioids belonging to the family of 4-anilidopiperidines, is mediated via activation of opioid receptors spread throughout the peripheral and central nervous system. However, morphine acting as a 'dirty' drug also exhibits effects on other receptor systems, e.g., the serotonergic system and its 5-HT3 receptor. Therefore, this study focuses on the interaction of morphine and fentanyl-type opioids (alfentanil, remifentanil and sufentanil) with 5-HT3A receptors. METHODS: Excised outside-out patches from human embryonic kidney-293 cells, stably transfected with the human 5-HT3A receptor cDNA, were used to determine the opioid effects using the patch-clamp technique. RESULTS: Within clinical concentrations, the effects of morphine are concentration-dependent. Morphine reduced current amplitudes, as well as activation and decay time constants. These effects were not competitive. Contrary to these results, all fentanyl-type opioids only exerted effects far above their clinical concentration ranges. These effects were not homogenous but varying. CONCLUSIONS: Morphine is an opioid compound exhibiting special antagonistic interaction with 5-HT3A receptors. This interaction is not shared by the newer synthetic derivatives of the fentanyl-type opioids in the clinical relevant concentration range.

Which agonist properties are important for the activation of 5-HT3A receptors?

PURPOSE: Why do anesthetics not activate excitatory ligand-gated ion channels such as 5-HT3 receptors in contrast to inhibitory ligand-gated ion channels? This study examines the actions of structural closely-related 5-HT derivatives and 5-HT constituent parts on 5-HT3A receptors with the aim of finding simpler if not minimal agonists and thus determining requirements for successful agonist action. EXPERIMENTAL APPROACH: Responses to 5-HT derivatives of human 5-HT3A receptors stably expressed in HEK 293 cells have been examined with the patch-clamp technique in the outside-out configuration combined with a fast solution exchange system. RESULTS: Phenol, pyrrole and alkyl amines, constituents of 5-HT, even at high concentrations, cannot activate 5-HT3A receptors but they can inhibit them. To date, tyramines are the smallest known agonists. However, an aromatic ring is not required for activation as acetylcholine is also an agonist of similar strength. CONCLUSION: Simultaneous interactions of adequate strength at two separate subsites within the 5-HT binding domain appear to be essential for successful agonist function. Anesthetics either fail to achieve this or the activation they produce is so weak that it is masked by a comparatively very strong inhibition.

The site of anesthetic action.

The mechanisms of general anesthesia constitute one of the great unsolved problems of classical neuropharmacology. Since the discovery of general anesthesia, hundreds of substances have been tested and found to possess anesthetic activity. Anesthetics differ tremendously in their chemical, physical, and pharmacological properties, greatly varying in size, in chemically active groups, and in the combinations of interactions and chemical reactions that they can undergo. The large spectrum of targets makes it obvious that dealing with anesthetics pharmacologically is different from dealing with most other drugs used in pharmacology. Anesthetic potency often correlates with the lipophilicity of anesthetic compounds, i.e., their preference for dissolving in lipophilic phases. This suggests as a main characteristic of anesthetic interactions that they are weak and that for many of them there is overall an approximate balance of nonspecific hydrophobic interactions and weak specific polar interactions. These include various electrostatic (ions, permanent and induced dipoles, quadrupoles), hydrogen bonding, and hydrophobic interactions. There are many molecular targets of anesthetic action within the central nervous system, but there are many more still to be discovered. Molecular interaction sites postulated from functional studies include protein binding sites, protein cavities, lipid/protein interfaces, and protein/protein interfaces.

Correlation of the A-Line ARX index with acoustically evoked potential amplitude.

BACKGROUND: Automated indices derived from mid-latency auditory evoked potentials (MLAEP) have been proposed for monitoring the state of anaesthesia. The A-Line ARX index (AAI) has been implemented in the A-Line monitor (Danmeter, V1.4). Several studies have reported variable and, in awake patients, sometimes surprisingly low AAI values. The purpose of this study was to reproduce these findings under steady-state conditions and to investigate their causes. METHODS: Ten awake unmedicated volunteers were studied under steady-state conditions. For each subject, the raw EEG and the AAI were recorded with an A-Line monitor (V1.4) during three separate sessions of 45.0 (1.6) min duration each. MATLAB (Mathworks) routines were used to derive MLAEP responses from EEG data and to calculate maximal MLAEP amplitudes. RESULTS: The AAI values ranged from 15 to 99, while 11.4% fell below levels which, according to the manufacturer, indicate an anaesthetic depth suitable for surgery. Inter-individual and intra-individual variation was observed despite stable recording conditions. The amplitudes of the MLAEP varied from 0.8 to 42.0 microV. The MLAEP amplitude exceeded 2 microV in 75.3% of readings. The Spearman's rank correlation coefficient between the MLAEP amplitude and the AAI value was r=0.89 (P<0.0001). CONCLUSIONS: The version of the A-Line monitor used in this study does not exclude contaminated MLAEP signals. Previous publications involving this version of the A-Line monitor (as opposed to the newer A-Line/2 monitor series) should be reassessed in the light of these findings. Before exclusively MLAEP-based monitors can be evaluated as suitable monitors of depth of anaesthesia, it is essential to ensure that inbuilt validity tests eliminate contaminated MLAEP signals.

The effects of morphine on human 5-HT3A receptors.

5-HT3 receptors are ligand-gated ion channels that are involved in the modulation of emesis and pain. In this study, we investigated whether the opioid analgesic, morphine, exerts specific effects on human 5-HT3 receptors. Whole-cell patches from HEK-293 cells stably transfected with the human 5-HT3A receptor cDNA were used to determine the effects of morphine on the 5-HT-induced currents using the patch clamp technique. At negative membrane potentials, 5-HT induced inward currents in a concentration-dependent manner. The 5-HT3 receptor antagonist, ondansetron, (0.3 nM) reversibly inhibited the 5-HT-induced signals. Morphine reversibly suppressed 5-HT-induced peak currents as a function of concentration (IC50 = 1.1 microM, Hill coefficient = 1.2). The block by morphine decreased with increasing 5-HT concentrations, suggesting a competitive effect. In addition, the activation, as well as the inactivation, kinetics of the currents were significantly slowed in the presence of morphine. The morphine antagonist, naloxone, also inhibited 5-HT-induced currents (e.g., at 3 microM by 17%). The effects of morphine and naloxone were not additive. The potency of morphine and the competitivity of the blocking effect points to a specific mechanism at a receptor site rather than an unspecific membrane effect.

Influence of sodium substitutes on 5-HT-mediated effects at mouse 5-HT3 receptors.

1 The influence of sodium ion substitutes on the 5-hydroxytryptamine (5-HT)-induced flux of the organic cation [14C]guanidinium through the ion channel of the mouse 5-HT3 receptor and on the competition of 5-HT with the selective 5-HT3 receptor antagonist [3H]GR 65630 was studied, unless stated otherwise, in mouse neuroblastoma N1E-115 cells. 2 Under physiological conditions (135 mm sodium), 5-HT induced a concentration-dependent [14C]guanidinium influx with an EC50 (1.3 microm) similar to that in electrophysiological studies. 3 The stepwise replacement of sodium by increasing concentrations of the organic cation hydroxyethyl trimethylammonium (choline) concentration dependently caused both a rightward shift of the 5-HT concentration-response curve and an increase in the maximum effect of 5-HT. Complete replacement of sodium resulted in a 34-fold lower potency of 5-HT and an almost two times higher maximal response. A low potency of 5-HT in choline buffer was also observed in other 5-HT3 receptor-expressing rodent cell lines (NG 108-15 or NCB 20). 4 Replacement of Na+ by Li+ left the potency and maximal effects of 5-HT almost unchanged. Replacement by tris (hydroxymethyl) methylamine (Tris), tetramethylammonium (TMA) or N-methyl-d-glucamine (NMDG) caused an increase in maximal response to 5-HT similar to that caused by choline. The potency of 5-HT was only slightly reduced by Tris, to a high degree decreased by TMA (comparable to the decrease by choline), but not influenced by NMDG. 5 The potency of 5-HT in inhibiting [3H]GR65630 binding to intact cells was 35-fold lower when sodium was completely replaced by choline, but remained unchanged after replacement by NMDG. 6 The results are compatible with the suggestion that choline competes with 5-HT for the 5-HT3 receptor; the increase in maximal response may be partly due to a choline-mediated delay of the 5-HT-induced desensitization. For studies of 5-HT-evoked [14C]guanidinium flux through 5-HT3 receptor channels, NMDG appears to be an 'ideal' sodium substituent since it increases the signal-to-noise ratio without interfering with 5-HT binding.

Comparison of the concentration-dependent effect of sevoflurane on the spinal H-reflex and the EEG in humans.

BACKGROUND: It has been shown that spinal reflexes such as the H-reflex predict motor responses to painful stimuli better than cortical parameters derived from the EEG. The precise concentration-dependence of H-reflex suppression by anaesthetics, however, is not known. Here we investigated this concentration-response relationship and the equilibration between the alveolar and the effect compartment for sevoflurane. METHODS: In 26 patients, the H-reflex was recorded at a frequency of 0.1 Hz while anaesthesia was induced and maintained with sevoflurane at increasing and decreasing concentrations. Population pharmacodynamic modelling was performed using the NONMEM software package, yielding population mean parameters as well as indicators of interindividual variability. RESULTS: Suppression of H-reflex amplitude occurred at lower concentrations (mean EC(50) 1.04 +/- 0.10 vol%, SE of NONMEM estimate) than the effect on either BIS or SEF(95) of the EEG (mean EC(50) 1.55 +/- 0.08 and 1.72 +/- 0.18 vol%, respectively), and exhibited a higher interindividual variability. The concentration-response function for the H-reflex was also steeper (mean ë 2.83 +/- 0.25). In addition, the equilibration between alveolar and effect compartment was slower for the H-reflex (mean k(e0) 0.15 +/- 0.01 min(-1)) than for BIS or SEF(95) (mean k(e0) 0.22 +/- 0.02 and 0.41 +/- 0.05 min(-1)). CONCLUSION: The differences in EC(50) and slope of the concentration-response relationships for H-reflex suppression and the EEG parameters point to different underlying mechanisms. In addition, the differences in time constant for equilibration between alveolar and effect compartment confirm the notion that immobility is caused at a different anatomic site than suppression of the EEG.

Biophysical properties of Kv3.1 channels in SH-SY5Y human neuroblastoma cells.

Biophysical properties of delayed rectifier K channels in the human neuroblastoma SH-SY5Y were established using patch clamp recordings. The whole cell K+ conductance activated at membrane potentials positive to -20 mV. The midpoint of current activation was 9.6 +/- 5.1 mV, the equivalent charge was 3.7 +/-.6. Whole-cell currents inactivated slightly with time constants of 700 ms and 5 s. The K+ currents were sensitive to micromolar concentrations of TEA and 4-aminopyridine. RT-PCR experiments amplified a cDNA fragment specific for human Kv3.1 channels. Activation gating parameters in outside-out patches were shifted by approximately 14 mV in the hyperpolarizing direction.

Pharmacological modification of sodium channels from the human heart atrium in planar lipid bilayers: electrophysiological characterization of responses to batrachotoxin and pentobarbital.

BACKGROUND AND OBJECTIVE: To investigate the effects of barbiturates on batrachotoxin-modified sodium channels from different regions of the human heart. Single sodium channels from human atria were studied and compared with existing data from the human ventricle and from the central nervous system. METHODS: Sodium channels from preparations of human atrial muscle were incorporated into planar lipid bilayers in the presence of batrachotoxin, a sodium channel activator. The steady-state behaviour of single sodium channels was recorded in symmetrical 500 mmol NaCl before and after the addition of pentobarbital 0.34-1.34 mmol. RESULTS: The batrachotoxin-treated human atrial sodium channel had an average single-channel conductance of 23.8 +/- 1.6 pS in symmetrical 500 mmol NaCl and a channel fractional open time of 0.83 +/- 0.06. The activation mid-point potential was -98.0 +/- 2.3 mV. Extracellular tetrodotoxin (a specific sodium channel blocking agent) blocked these channels with a k(1/2) = 0.53 micromol at 0 mV. Pentobarbital reduced the time average conductance of single atrial sodium channels in a concentration-dependent manner (ID50 = 0.71 mmol). In the same way, the steady-state activation was shifted to more hyperpolarized potentials (-10.6 mV at 0.67 mmol pentobarbital). CONCLUSIONS: The properties of batrachotoxin-modified sodium channels from human atrial tissue did not differ greatly from those described for ventricular sodium channels in the literature. Our data yielded no explanation for the observed functional diversity. However, cardiac sodium channels differ from those found in the central nervous system.

Molecular and functional changes in voltage-dependent Na(+) channels following pilocarpine-induced status epilepticus in rat dentate granule cells.

Status epilepticus (S.E.) is known to lead to a large number of changes in the expression of voltage-dependent ion channels and neurotransmitter receptors. In the present study, we examined whether an episode of S.E. induced by pilocarpine in vivo alters functional properties and expression of voltage-gated Na(+) channels in dentate granule cells (DGCs) of the rat hippocampus. Using patch-clamp recordings in isolated DGCs, we show that the voltage-dependent inactivation curve is significantly shifted toward depolarizing potentials following S.E. (half-maximal inactivation at -43.2+/-0.6 mV) when compared with control rats (-48.2+/-0.8 mV, P<0.0001). The voltage-dependent activation curve is significantly shifted to more negative potentials following S.E., with half-maximal activation at -28.6+/-0.8 mV compared with -25.8+/-0.9 mV in control animals (P<0.05). The changes in voltage dependence resulted in an augmented window current due to increased overlap between the activation and inactivation curve. In contrast to Na(+) channel voltage-dependence, S.E. caused no changes in the kinetics of fast or slow recovery from inactivation. The functional changes were accompanied by altered expression of Na(+) channel subunits measured by real-time reverse transcription-polymerase chain reaction in dentate gyrus microslices. We investigated expression of the pore-forming alpha subunits Na(v)1.1-Na(v)1.3 and Na(v)1.5-Na(v)1.6, in addition to the accessory subunits beta(1) and beta(2). The Na(v)1.2 and Na(v)1.6 subunit as well as the beta(1) subunit were persistently down-regulated up to 30 days following S.E. The beta(2) subunit was transiently down-regulated on the first and third day following S.E. These results indicate that differential changes in Na(+) channel subunit expression occur in concert with functional changes. Because coexpression of beta subunits is known to robustly shift the voltage dependence of inactivation in a hyperpolarizing direction, we speculate that a down-regulation of beta-subunit expression may contribute to the depolarizing shift in the inactivation curve following S.E.

Direct inhibition by cannabinoids of human 5-HT3A receptors: probable involvement of an allosteric modulatory site.

Excised outside-out patches from HEK293 cells stably transfected with the human (h) 5-HT3A receptor cDNA were used to determine the effects of cannabinoid receptor ligands on the 5-HT-induced current using the patch clamp technique. In addition, binding studies with radioligands for 5-HT3 as well as for cannabinoid CB1 and CB2 receptors were carried out. The 5-HT-induced current was inhibited by the following cannabinoid receptor agonists (at decreasing order of potency): 9-THC, WIN55,212-2, anandamide, JWH-015 and CP55940. The WIN55,212-2-induced inhibition was not altered by SR141716A, a CB1 receptor antagonist. WIN55,212-3, an enantiomer of WIN55,212-2, did not affect the 5-HT-induced current. WIN55,212-2 did not change the EC50 value of 5-HT in stimulating current, but reduced the maximum effect. The CB1 receptor ligand [3H]-SR141716A and the CB1/CB2 receptor ligand [3H]-CP55940 did not specifically bind to parental HEK293 cells. In competition experiments on membranes of HEK293 cells transfected with the h5-HT3A receptor cDNA, WIN55,212-2, CP55940, anandamide and SR141716A did not affect [3H]-GR65630 binding, but 5-HT caused a concentration dependent-inhibition. In conclusion, cannabinoids stereoselectively inhibit currents through recombinant h5-HT3A receptors independently of cannabinoid receptors. Probably the cannabinoids act allosterically at a modulatory site of the h5-HT3A receptor. Thus the functional state of the receptor can be controlled by the endogenous ligand anandamide. This site is a potential target for new analgesic and antiemetic drugs.

Ketamine effects on human neuronal Na+ channels.

BACKGROUND AND OBJECTIVE: Similar doses of ketamine are employed in regional and general anaesthesia. In contrast to commonly used local anaesthetic agents, accidental systemic application of local anaesthetic doses of ketamine will not result in seizure, dysrhythmia or cardiovascular depression. However, there is some doubt about the quality of regional analgesia induced by ketamine. As human sodium channels constitute an important molecular target of local anaesthetics, the study was designed to establish concentration-dependent effects of ketamine on conductance, steady-state activation and steady-state inactivation of human neuronal sodium channels. This information--that has, so far, not been published--will help to characterize further local anaesthetic properties of ketamine. METHODS: Whole-cell patch-clamp recordings were made of sodium channels natively expressed in human neuroblastoma SH-SY5Y cells. RESULTS: The sodium channels activated at a threshold of -60 mV and exhibited a maximal peak current at -10 mV. The voltage of half-maximal activation was -20 mV. The Na+ currents depended on the prepulse potential. The voltage of half-maximal inactivation was -80 mV. Ketamine inhibited the sodium conductance in a concentration-dependent manner (IC50 = 1140 micromol). A concentration-dependent hyperpolarizing shift of both steady-state activation and steady-state inactivation accounted for at most 5 mV. CONCLUSIONS: The effects of ketamine on these human ion channels occur at clinical concentrations. They are consistent with the local anaesthetic action of ketamine. Whether ketamine helps to decrease the incidence of severe side-effects during regional anaesthesia needs to be addressed in further clinical studies.

Bupivacaine inhibits human neuronal Kv3 channels in SH-SY5Y human neuroblastoma cells.

BACKGROUND: Information on molecular targets that may be involved in the neurotoxicity of bupivacaine is limited. Suppression of Kv3 channels has been demonstrated to result in abnormal patterns in the electroencephalogram and in seizures. Inhibition of Kv3 channels by bupivacaine may consequently contribute to its neuroexcitatory side-effects. Data on the effects of bupivacaine on these potassium channels are lacking. We therefore characterized the effects of bupivacaine on human Kv3 channels natively expressed in SH-SY5Y cells. METHODS: Kv3 channels natively expressed in human SH-SY5Y cells were studied using a standard whole-cell patch-clamp protocol. RESULTS: Bupivacaine reversibly inhibited Kv3 channels in a concentration-dependent manner. The half-maximal inhibitory concentration (IC50) for conductance block was 57 microM and the Hill coefficient was close to unity. Bupivacaine accelerated macroscopic current decline by inducing inactivation-like behaviour. The midpoint of current activation was shifted to depolarized potentials in a concentration-dependent and reversible manner by a maximum of 26 mV. The IC50 was 47 microM and the Hill coefficient was 2.4. The free arterial plasma concentrations of bupivacaine that have been estimated to occur during convulsions in man would inhibit the Kv3 channels by at least 40% and would shift the midpoint of current activation by a minimum of 9 mV. CONCLUSIONS: Both inhibition of potassium channels and a depolarizing shift of their activation midpoint would increase neuronal excitability. The effects of bupivacaine on human Kv3 channels are thus compatible with a contributory role of Kv channel alteration in bupivacaine-induced neuronal excitation.

Interaction of volatile anesthetics with human Kv channels in relation to clinical concentrations.

BACKGROUND: Recent evidence shows that inhibition of human Kv3 channels by intravenous anesthetics occurs at clinical concentrations. The effects of volatile anesthetics on these human ion channels are unknown. This study was designed to establish whether minimum alveolar concentrations (MAC) of halothane, enflurane, isoflurane, and desflurane exhibit effects on Kv3 channeLs. To obtain an indication whether these findings may be specific to Kv3 channels, the effects of enflurane and isoflurane on human Kv1.1 channels were also investigated. METHODS: Kv3 channels natively expressed in SH-SY5Y cells and Kv1.1 channels expressed in HEK293 cells were measured with the whole cell patch clamp technique by standard protocols. Concentrations of volatile anesthetics were determined by gas chromatography. RESULTS: Halothane, enflurane, isoflurane, and desflurane reversibly inhibited Kv3 channels in a concentration-dependent manner. Concentrations at half-maximal effect (IC50 values) ranged between 1,800 and 4,600 microM. Hill coefficients were between 1.7 and 2.5. IC50 values for inhibition of Kv1.1 channels were 2,800 and 5,200 microM, and Hill coefficients were 3.9 and 5.6 for enflurane and isoflurane, respectively. CONCLUSION: Volatile anesthetics inhibit human Kv3 channels at clinical concentrations. At 1-3 MAC, inhibition would account on average for 2-12%. Inhibition would be highest with enflurane (between 3% and 22%) and lowest with isoflurane (between 0.2% and 3%). Kv1.1 channels would only be inhibited by enflurane at clinical concentrations (2% at 2 MAC and 8% at 3 MAC). Whether the degree of K channel inhibition by volatile anesthetics may contribute to their clinical action needs further study.

Human cardiac sodium channels are affected by pentobarbital.

BACKGROUND AND OBJECTIVE: To investigate the response to general anaesthetics of different sodium channel subtypes, we examined the effects of pentobarbital, a close thiopental analogue, on single sodium channels from human ventricular muscle and compared them with existing data from human brain channels. METHODS: Sodium channels from preparations of human ventricular muscle were incorporated into planar lipid bilayers in the presence of batrachotoxin, a sodium channel activator. Single channel currents were recorded in symmetrical 100 mmol L-1 and 500 mmol L-1 NaCl before and after the addition of the anaesthetic pentobarbital (0.34-1.34 mmol L-1). RESULTS: The blocking effect of pentobarbital on the fractional open time had an IC50 of 690 micromol L-1 in 500 mmol L-1 NaCl, whereas it had a significantly lower IC50 of 400 micromol L-1 in 100 mmol L-1 NaCl. Pentobarbital caused a significant shift of steady-state activation to hyperpolarized potentials (fmax = -42 mV, IC50 = 2 mmol L-1). This effect was independent of NaCl concentration. CONCLUSION: Despite pharmacological and electrophysiological differences between human cardiac and human brain sodium channels their responses to pentobarbital are similar. The finding of channel block being dependent on the electrolyte concentration is novel for sodium channels.

Slow recovery from inactivation regulates the availability of voltage-dependent Na(+) channels in hippocampal granule cells, hilar neurons and basket cells.

1. Fundamental to the understanding of CNS function is the question of how individual neurons integrate multiple synaptic inputs into an output consisting of a sequence of action potentials carrying information coded as spike frequency. The availability for activation of neuronal Na(+) channels is critical for this process and is regulated both by fast and slow inactivation processes. Here, we have investigated slow inactivation processes in detail in hippocampal neurons. 2. Slow inactivation was induced by prolonged (10-300 s) step depolarisations to -10 mV at room temperature. In isolated hippocampal dentate granule cells (DGCs), recovery from this inactivation was biexponential, with time constants for the two phases of slow inactivation tau(slow,1) and tau(slow,2) ranging from 1 to 10 s and 20 to 50 s, respectively. Both (slow,1) and tau(slow,2) were related to the duration of prior depolarisation by a power law function of the form tau(t) = a (t/a)b, where t is the duration of the depolarisation, a is a constant kinetic setpoint and b is a scaling power. This analysis yielded values of a = 0.034 s and b = 0.62 for tau(slow,1) and a = 24 s and b = 0.30 for tau(slow,2) in the rat. 3. When a train of action potential-like depolarisations of different frequencies (50, 100, 200 Hz) was used to induce inactivation, a similar relationship was found between the frequency of depolarisation and both tau(slow,1) and tau(slow,2) (a = 0.58 s, b = 0.39 for tau(slow,1) and a = 3.77 s and b = 0.42 for tau(slow,2)). 4. Using nucleated patches from rat hippocampal slices, we have addressed possible cell specific differences in slow inactivation. In fast-spiking basket cells a similar scaling relationship can be found (a = 3.54 s and b = 0.39) as in nucleated patches from DGCs (a = 2.3 s and b = 0.48) and non-fast-spiking hilar neurons (a = 2.57 s and b = 0.49). 5. Likewise, comparison of human and rat granule cells showed that properties of ultra-slow recovery from inactivation are conserved across species. In both species ultra-slow recovery was biexponential with both tau(slow,1) and tau(slow,2) being related to the duration of depolarisation t, with a = 0.63 s and b = 0.44 for tau(slow,1) and a = 25 s and b = 0.37 for tau(slow,2) for the human subject. 6. In summary, we describe in detail how the biophysical properties of Na(+) channels result in a complex interrelationship between availability of sodium channels and membrane potential or action potential frequency that may contribute to temporal integration on a time scale of seconds to minutes in different types of hippocampal neurons.

Ketamine and propofol differentially inhibit human neuronal K(+) channels.

BACKGROUND AND OBJECTIVE: Interaction of intravenous anaesthetic agents with voltage-dependent potassium channels significantly correlates with clinical concentrations. If potassium channels were to play an important part in anaesthesia, one might expect different effects at the molecular level of those anaesthetics that show different clinical effects. Our aim was to analyse the interaction of general anaesthetics with voltage-dependent K channels. METHODS: Whole cell patch-clamp experiments were analysed in detail in order to compare the effects of two clinically diverse intravenous hypnotics, ketamine and propofol, on voltage-dependent potassium channels in human neuroblastoma SH-SY5Y cells. RESULTS: Both anaesthetics inhibited the potassium conductance in a concentration-dependent and reversible manner with IC50-values of 300 microM and 45 microM for ketamine and propofol respectively. Whereas ketamine shifted the midpoint of current activation by maximally 14 mV to more hyperpolarized potentials, propofol had the opposite effect on the activation midpoint. Current inhibition by ketamine increased with voltage but decreased with propofol at higher membrane potentials. Propofol but not ketamine induced concentration-dependent but voltage-independent decline, akin to inactivation, of the voltage-dependent potassium channels. CONCLUSIONS: The anaesthetics differed not only in their clinical profiles but they also showed differential actions on voltage-dependent potassium channels in several ways. This provides additional evidence for the hypothesis that voltage-dependent potassium channels play an important role in anaesthesia.