Abstract SNACC-38

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Modulation of alpha9/10 Nicotinic Acetylcholine Receptors by General Anesthetics

Carlson J, Jenkins A
EMORY UNIVERSITY, ATLANTA, Georgia, United states

Background: Ligand-gated ion channels contain binding sites for many general anesthetic drugs. The best structurally understood member of this genetic superfamily is the nicotinic acetylcholine receptor (nAChR). Functional pentameric receptors are composed of admixtures of 10 alpha and 4 beta subunits, often, but not always, with several other auxillary subunits. With regards to sensitivity to volatile anesthetics, nAChRs range from the insensitive (alpha1beta1 containing receptors) to the supersensitive (alpha4beta2 containing receptors); the former being sensitive to only lethal concentrations of drug, while the latter are completely inhibited by subanesthetic concentrations of inhaled anesthetics.

Goal: The aim of this study was to characterize the anesthetic sensitivity of alpha9alpha10 receptors. These receptors have a unique physiologic function and pharmacology. They are unique among nicotinic receptors in that they hyperpolarize, rather than excite neurons. Also, nicotine acts as a competitive antagonist, rather than an agonist at alpha9alpha10 receptors.

Methods: Xenopus laevis oocytes were injected with RNAs encoding the human alpha9 and alpha10 nAChR subunits. 24-72 hours after injection, receptor function was assayed using 2-electrode voltage clamp using an 8-channel Opus Express. Oocytes were voltage clamped at -60 mV and superfused with extracellular buffer. Agonists and anesthetics were delivered using an automated robotized perfusion system. Membrane currents were recorded pClamp and stored for offline analysis with MatLab.

Results: Acetylcholine activated cationic currents in a concentration dependent manner, with an EC(50) of 15.5±2.7 µM and Hill coefficient of 1.6±0.1 (n=12). Nicotine blocked cholinergic currents with an IC(50) of 0.7±0.2 µM (n=4). Both sevoflurane and isoflurane exhibited similar biphasic effects on receptor activation. At low concentrations (0.2 – 1 MAC) both anesthetics potentiated receptor function in a concentration dependent manner. At 1 MAC, isoflurane and sevoflurane potentiated currents activated by 5 µM Ach by 148±31% and 140±35% respectively. In the clinically useful range (1-5 MAC) the potentiation persisted, but generally decreased with increasing anesthetic concentration.

Conclusions: The biphasic effects described here are reminiscent of the actions of volatile anesthetics on one of the more primitive members of the LGIC superfamily, the 5-HT3 receptor, where clinically useful concentrations of inhaled anesthetics all enhance receptor activation and higher concentrations results in greater desensitization and ultimately inhibition. Such observations may lead to a better understanding of how anesthetic binding sites have evolved. Finally, recent structural studies of anesthetic-LGIC complexes have demonstrated that anesthetics locate in intra- and inter- subunit transmembrane cavities. However, how the relative occupation of these two types of cavity alters channel open time is not well understood. A comparison of receptor models of 3 structurally similar AChRs receptors (alpha1:beta1, alpha4:beta2, alpha9alpha10) with very different anesthetic responses will likely shed light on the relative roles these cavities play in controlling ion channel opening.


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