Bernd W. Urban

A novel mechanism underlying drug resistance in chronic epilepsy.

The development of resistance to pharmacological treatment is common to many human diseases. In chronic epilepsy, many patients develop resistance to anticonvulsant drug treatment during the course of their disease, with the underlying mechanisms remaining unclear. We have studied cellular mechanisms underlying drug resistance in resected hippocampal tissue from patients with temporal lobe epilepsy by comparing two groups of patients, the first displaying a clinical response to the anticonvulsant carbamazepine and a second group with therapy‐resistant seizures. Using patch‐clamp recordings, we show that the mechanism of action of carbamazepine, use‐dependent block of voltage‐dependent Na+ channels, is completely lost in carbamazepine‐resistant patients. Likewise, seizure activity elicited in human hippocampal slices is insensitive to carbamazepine. In marked contrast, carbamazepine‐induced use‐dependent block of Na+ channels and blocked seizure activity in vitro in patients clinically responsive to this drug. Consistent with these results in human patients, we also show that use‐dependent block of Na+ channels by carbamazepine is absent in chronic experimental epilepsy. Taken together, these data suggest that a loss of Na+ channel drug sensitivity may constitute a novel mechanism underlying the development of drug‐resistant epilepsy. Ann Neurol 2003

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.

Anticonvulsant pharmacology of voltage‐gated Na+ channels in hippocampal neurons of control and chronically epileptic rats

Voltage‐gated Na+ channels are a main target of many first‐line anticonvulsant drugs and their mechanism of action has been extensively investigated in cell lines and native neurons. Nevertheless, it is unknown whether the efficacy of these drugs might be altered following chronic epileptogenesis. We have, therefore, analysed the effects of phenytoin (100u2003µm), lamotrigine (100u2003µm) and valproate (600u2003µm) on Na+ currents in dissociated rat hippocampal granule neurons in the pilocarpine model of chronic epilepsy. In control animals, all three substances exhibited modest tonic blocking effects on Na+ channels in their resting state. These effects of phenytoin and lamotrigine were reduced (by 77 and 64%) in epileptic compared with control animals. Phenytoin and valproate caused a shift in the voltage dependence of fast inactivation in a hyperpolarizing direction, while all three substances shifted the voltage dependence of activation in a depolarizing direction. The anticonvulsant effects on Na+ channel voltage dependence proved to be similar in control and epileptic animals. The time course of fast recovery from inactivation was potently slowed by lamotrigine and phenytoin in control animals, while valproate had no effect. Interestingly, the effects of phenytoin on fast recovery from inactivation were significantly reduced in chronic epilepsy. Taken together, these results reveal that different anticonvulsant drugs may exert a distinct pattern of effects on native Na+ channels. Furthermore, the reduction of phenytoin and, to a less pronounced extent, lamotrigine effects in chronic epilepsy raises the possibility that reduced pharmacosensitivity of Na+ channels may contribute to the development of drug resistance.

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 τslow,1 and τslow,2 ranging from 1 to 10 s and 20 to 50 s, respectively. Both τslow,1 and τslow,2 were related to the duration of prior depolarisation by a power law function of the form τ(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 τslow,1 and a= 24 s and b= 0.30 for τslow,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 τslow,1 and τslow,2 (a= 0.58 s, b= 0.39 for τslow,1 and a= 3.77 s and b= 0.42 for τslow,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 τslow,1 and τslow,2 being related to the duration of depolarisation t, with a= 0.63 s and b= 0.44 for τslow,1 and a= 25 s and b= 0.37 for τslow,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.

Carbamazepine Effects on Na+ Currents in Human Dentate Granule Cells from Epileptogenic Tissue

Summary: Purpose: Carbamazepine (CBZ) is a well‐established drug in the therapy of temporal lobe epilepsy (TLE). The anticonvulsant action of CBZ has been explained mainly by use‐dependent effects on voltage‐dependent Na+ channels in various nonhuman cell types. However, it is unclear whether Na+ currents in neurons within the focal epileptogenic area of patients with medically intractable TLE show similar characteristics.

Electrophysiological characterization of Na+ currents in acutely isolated human hippocampal dentate granule cells

1 Properties of voltage‐dependent Na+ currents were investigated in forty‐two dentate granule cells (DGCs) acutely isolated from the resected hippocampus of twenty patients with therapy‐refractory temporal lobe epilepsy (TLE) using the whole‐cell patch‐clamp technique. 2 Depolarizing voltage commands elicited large, rapidly activating and inactivating Na+ currents (140 pS μm−2; 163 mm extracellular Na+) that were reduced in amplitude by lowering the Na+ gradient (43 mm extracellular Na+). At low temperatures (8‐12 °C), the time course of Na+ currents slowed and could be well described by the model of Hodgkin & Huxley. 3 Na+ currents were reversibly blocked by tetrodotoxin (TTX) and saxitoxin (STX) with a half‐maximal block of 4.7 and 2.6 nm, respectively. In order to reduce series resistance errors, the Na+ current was partially blocked by low toxin concentrations (10‐15 nm) in the experiments described below. Under these conditions, Na+ currents showed a threshold of activation of about ‐50 mV, and the voltages of half‐maximal activation and inactivation were ‐29 and ‐55 mV, respectively. 4 The time course of recovery from inactivation could be described with a double‐exponential function (time constants, 3‐20 and 60‐200 ms). The rapid and slow time constants showed a distinct voltage dependence with maximal values around ‐55 and ‐80 mV, respectively. These properties contributed to a reduction of the Na+ currents during repetitive stimulation that was more pronounced with higher stimulation frequencies and also showed a dependence on the holding potential. 5 In summary, the most striking features of DGC Na+ currents were the large current density and the presence of a current component showing a slow recovery from inactivation. Our data provide a basis for comparison with properties of Na+ currents in animal models of epilepsy, and for the study of drug actions in therapy‐refractory epilepsy.

The voltage-dependent action of pentobarbital on batrachotoxin-modified human brain sodium channels

The voltage-dependent action of the intravenous anesthetic pentobarbital on human brain sodium channels activated by batrachotoxin was examined using planar lipid bilayer methods. Fractional open time-data were fitted by Boltzmann functions to yield simple parameters characterizing the voltage-dependence of the fractional open time. Pentobarbital caused a dose-dependent reduction of the maximum fractional open time of the sodium channel and a shift of the potential of half-maximal open time towards hyperpolarized potentials, whereas the slope parameter of the Boltzmann-fits was unaffected. A statistically significant increase of the variability of these parameters was found only in the case of the maximum fractional open time, indicating a random fluctuation of pentobarbital-induced suppression of the sodium channels over time. The voltage-dependent action of pentobarbital probably results from either a pentobarbital-modification of channel activation gating and/or a modification of the pentobarbital action by the gating process itself.

Single sodium channels from human ventricular muscle in planar lipid bilayers.

Abstract Sodium channels from human ventricular muscle membrane vesicles were incorporated into planar lipid bilayers and the steady-state behavior of single sodium channels were examined in the presence of batrachotoxin.In symmetrical 500 mM NaCl the averaged single channel conductance was 24.7 ± 1.3 pS and the channel fractional open time was 0.85 ± 0.04. The activation midpoint potential was −99.5 ± 3.1 mV. Extracellular tetrodotoxin blocked the channel with a κ1/2 of 414 nM at 0 mV. In 7 out of 13 experiments subconductance states were observed (9.2 ± 1.2 pS).When sodium chloride concentration was lowered to 100 mM, single channel conductance decreased to 19.0 ± 0.9 pS, steady-state activation shifted by −17.3 ± 5.1 mV, tetrodotoxin sensitivity increased to 324 nM, and sub-conductance states were invariably observed in single channel records (7.9 ± 0.7 pS).In the planar lipid bilayer system the properties of cardiac sodium channels from different species are not very different, but there are significant differences between sodium channels from human heart and from human CNS.

Etomidat unterdrückt einen neuronalen Kaliumstrom des Menschen

ZusammenfassungBislang liegen keine detaillierten Studien zum Einfluß von Allgemeinanästhetika auf Ionenkanäle in neuronalen Zellen des Menschen vor. Deshalb wurde der Effekt von Etomidat auf einen neuronalen Kaliumstrom des Menschen mittels der Ganzzell Patch-Clamp-Technik untersucht.Methode: Die Kaliumströme der menschlichen Neuroblastomzellinie SH-SY5Y wurden mit einem EPC-7 Patch-Clamp Verstärker (List medical electronics) und Pclamp Version 5.7.1 (Axon instruments) untersucht. Die Zellen wurden in RPMI-Medium (mit Pen-Strep und FKS) bei 37°u2005C kultiviert. Das Haltepotential betrug −80 mV. Die Kaliumströme wurden durch Depolarisationen des Membranpotentials auf Werte von −50 mV bis +90 mV ausgelöst.nErgebnisse: Etomidat unterdrückte den Spitzenauswärts- und Gleichgewichts-Kaliumstrom mit unterschiedlichen Konzentrations-Wirkungskurven. Etomidat rief intaktivierungsartiges Verhalten des Kaliumstroms hervor und veränderte seine Aktivierung.nSchlußfolgerungen: Ein Allgemeinanästhetikum kann mehrere Effekte auf eine neuronale Zielstruktur mit verschiedenen Konzentrations-Wirkungskurven haben. Die Unterdrückung neuronaler Kaliumströme durch Etomidat könnte zu den bei der Narkoseeinleitung beobachteten Myoklonien beitragen.AbstractIntroduction: There is no data available on the action of etomidate on ion currents in human neuronal cells. Therefore the effects of etomidate on a human neuronal delayed rectifer potassium current were investigated.Method: Outward rectifying potasium currents of human neuroblastoma SH-SY5Y cells were measured using the whole cell patch-clamp technique. Cells were grown in RPMI-medium (+Pen/Strep and FCS) at 37°u2005C and 5% CO2. The holding potential was −80 mV. Potassium currents were evoked by depolarizing the membrane potential to values from −50 mV to +90 mV in 10 mV steps using an EPC-7 patch-clamp amplifier (List medical electronics) and pclamp version 5.71 (Axon instruments).nResults: Etomidate differentially inhibited steady-state and peak potassium current with IC50-values of 170 µM for peak current suppression and 120 µM for steady-state current, respectively. Etomidate induced inactivation-like behaviour of the potassium current and changed the voltage dependence of potassium current activation.nConclusion: The results demonstrate that etomidate has more than one effect on the potassium current, indicating the complexity of general anaesthetic actions on neuronal targets. The actions of etomidate on human neuronal potassium currents may potentially contribute to the myocloni observed with this general anaesthetic agent during induction of anaeshtesia.

Single sodium channels from human skeletal muscle in planar lipid bilayers: characterization and response to pentobarbital.

PurposeTo investigate the response to general anesthetics of different sodium-channel subtypes, we examined the effects of pentobarbital, a close thiopental analogue, on single sodium channels from human skeletal muscle and compared them to existing data from human brain and human ventricular muscle channels.MethodsSodium channels from a preparation of human skeletal muscle were incorporated into planar lipid bilayers, and the steady-state behavior of single sodium channels and their response to pentobarbital was examined in the presence of batrachotoxin, a sodium-channel activator. Single-channel currents were recorded before and after the addition of pentobarbital (0.34–1.34u2009mM).ResultsIn symmetrical 500u2009mM NaCl, human skeletal muscle sodium channels had an averaged single-channel conductance of 21.0 ± 0.6u2009pS, and the channel fractional open time was 0.96 ± 0.04. The activation midpoint potential was −96.2 ± 1.6u2009mV. Extracellular tetrodotoxin blocked the channel with a half-maximal concentration (k1/2) of 60u2009nM at 0u2009mV. Pentobarbital reduced the time-averaged conductance of single skeletal muscle sodium channels in a concentration-dependent manner (inhibitory concentration 50% [IC50] = 0.66u2009mM). The steady-state activation was shifted to more hyperpolarized potentials (−16.7u2009mV at 0.67u2009mM pentobarbital).ConclusionIn the planar lipid bilayer system, skeletal muscle sodium channels have some electrophysiological properties that are significantly different compared with those of sodium channels from cardiac or from central nervous tissue. In contrast to the control data, these different human sodium channel subtypes showed the same qualitative and quantitative response to the general anesthetic pentobarbital. The implication of these effects for overall anesthesia will depend on the role the individual channels play within their neuronal networks, but suppression of both central nervous system and peripheral sodium channels may add to general anesthetic effects.