[Interactions of intravenous anesthetics with human CNS ion channels. Electrophysiologic studies with a new type of voltage clamp technique].
Despite a widespread approach to explain the molecular mechanisms of anaesthetic agents, the complex state of "general anaesthesia" is still not completely understood. Voltage-activated sodium channels from human brain cortex served as a model membrane protein to investigate anaesthetic drug-protein interactions using a novel electrophysiological voltage-clamp technique. Sodium channels are already well-characterized important integral membrane proteins responsible for the generation of the fast-propagated action potential and thus are vital components for neuronal signal integration and cell communication. In order to elucidate the molecular interactions of intravenous anaesthetics with single human brain sodium channels, representative compounds of four different clinical intravenous anaesthetic groups were used to correlate different types of clinical anaesthesia with differential anaesthetic effects on the molecular level. METHODS. Single sodium channels from human brain cortex were incorporated into artificial phospholipid bilayers and studied under our standard experimental conditions (Electrolyte solution: 500 mM NaCl, 10 mM HEPES, pH 7.4, Temp. 22-25 degrees C) with an electrophysiological voltage-clamp technique. In the presence of a channel activator (1 microM batrachotoxin) single-channel characteristics (fractional open time, single-channel conductance and amplitude, steady-state activation behaviour) were characterized for control conditions and in the presence of various doses of four different anaesthetic agents (pentobarbital, propofol, ketamine, midazolam). RESULTS. During control measurements the investigated human brain sodium channels showed stable and reproducible characteristics on the range expected for batrachotoxin-modified sodium channels in bilayers. After completion of the control measurement the effects of the four different general anaesthetics pentobarbital, propofol, ketamine and midazolam were investigated on the same control sodium channels. All four substances demonstrated a blocking effect of sodium channel conductance (pentobarbital: K50: concentration for 50% block of the maximal conductance block: 0.69 mM; blockmax: maximal conductance block (%): 100%; propofol: K50: 0.02 mM, blockmax: 28%; ketamine: K50: 1.1 mM, blockmax: 71%; midazolam: K50: 0.52 mM, blockmax: 100%). Furthermore, a destabilization of the steady-state activation process could be demonstrated. These effects were dose dependent, but only pentobarbital and propofol demonstrated these effects at or near clinically relevant serum concentrations.


CONCLUSIONS
At the clinical level, "general anaesthesia" is a highly complex phenomenon. Similarly, anaesthetics may demonstrate a multimechanistic mode of action also at the molecular level. In this study all four investigated anaesthetic compounds interacted with at least two primary sodium channel functions, leading to a voltage-independent reduction of the fractional channel open time and an interaction with the steady-state activation behaviour, respectively. The effects of pentobarbital and propofol were detectable at concentrations within the range of serum concentrations achieved during clinical anaesthesia, whereas ketamine and midazolam demonstrated qualitatively similar effects exceeding this range 10- to 50-fold. Thus, the human brain sodium channel might serve as a molecular target only for pentobarbital and propofol. This suggests that different types of clinical anaesthesia may correlate with differential actions of anaesthetics on the molecular level.
Frenkel
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Klinik und Poliklinik für Anästhesiologie und spezielle Intensivmedizin, Rheinischen Friedrich-Wilhelms-Universität Bonn.
Urban
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BW
D001921:Brain / Q000187:drug effects* / Q000502:physiology; D004594:Electrophysiology / Q000379:methods*; D006801:Humans; D007473:Ion Channels / Q000187:drug effects* / Q000502:physiology; D007649:Ketamine / Q000494:pharmacology*; D008874:Midazolam / Q000494:pharmacology*; D010424:Pentobarbital / Q000494:pharmacology*; D015742:Propofol / Q000494:pharmacology*
D007473:Ion Channels; D007649:Ketamine; D010424:Pentobarbital; D008874:Midazolam; D015742:Propofol