MOLECULAR STUDIES OF ANESTHETIC-MUSCARINIC INTERACTIONS

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Muscarinic acetylcholine receptors play important roles in both the peripheral and central nervous systems. They play a prominent role in the brain, and muscarinic antagonists are known to interfere with wakefulness, memory and learning. In addition, they are part of sympathetic and parasympathetic functioning, exercising profound influences on heart rate and sweating as well as on bronchial and gastrointestinal smooth muscle tone and glandular activity. Volatile anesthetics have been shown to inhibit muscarinic signaling, and some non-volatile anesthetic agents, such as ketamine and althesin, similarly appear to affect the system. These actions on muscarinic receptors could explain some of the effects as well as side-effects of the drugs. However, the studies are difficult to interpret as most times mixtures of receptor subtypes were used; single, high doses of anesthetic were studied; many studies address effects on binding rather than functional assays; and no molecular localization of the site of action was attempted. The molecular cloning of the five subtypes of muscarinic receptor now allows us to study these interactions in isolation and more detail. In this proposal, m1, m2 and m3 receptors muscarinic receptors will be expressed in Xenopus oocytes, and the effects of halothane, isoflurane, desflurane, ketamine, propofol and althesin on their functioning will be studied. Specific aims are (1) to compare these anesthetics as to their interactions with the muscarinic receptor subtypes; (2) to determine their sites of action in the muscarinic signaling cascade; (3) to localize using mutation studies the intramolecular sites of action of anesthetic drugs on muscarinic signaling; and (4) to compare the findings in Xenopus oocytes with those obtained in mammalian cells. The results of these studies will provide a better understanding of anesthetic-protein interactions in general, and of anesthetic interference with the clinically important muscarinic signaling system in particular.