Nicotine is a habit forming drug that is widely used in our society. The widespread distribution of the nicotinic acetylcholine receptors (AChRs) in the brain suggests that the dynamics of nicotine use in humans is likely to be complex. Understanding mechanisms underlying nicotines action in the CNS must begin with the elucidation of the functions of various CNS-AChR subtypes. The findings that many AChRs are highly permeable to calcium makes them candidates for mediating many essential calcium-dependent events in neurons by altering intracellular calcium concentrations ([Ca]i). The main aim of this proposal is to examine the calcium signalling properties of a subtype of hippocampal AChRs that bind the snake venom toxin alpha-bungarotoxin (a-Bgt-AChRs). Calcium permeability of the a-Bgt-AChR channel will be determined from cultured hippocampal neurons using direct calcium fluororescence measurements using electrodes filled with the fluo-3 pentapotassium salt coupled with whole cell voltage clamp determinations. The ability of aBgt-AChRs to alter [Ca]i in response to nicotine application in intact neurons will be examined by calcium imaging from cells loaded with fluo-3 AM. The temporal patterns of calcium signals generated by the various cholinergic receptors on these neurons and their interactions with other sources contributing to [Ca]i will be examined in detail. This will elucidate the role aBgt-AChRs play in maintaining calcium homeostasis in hippocampal neurons. The ability of aBgt-AChRs to regulate calcium dependent events like activation of phospholipase A2, CaM kinases and calmodulin sensitive adenylate cyclases will be studied using established biochemical procedures and spatial requirements of calcium increases for their activation determined. These studies will establish aBgt-AChRs as candidates for mediating calcium dependent events in the hippocampus. Further the ability of calcium to directly modulate aBgt-AChR function and thus maintain a rapid and tight control on cellular calcium levels will be examined using whole cell voltage clamp measurements. The structural determinants of calcium dependent regulation of aBgt-AChR function will be examined by site-directed mutagenesis using the alpha7 gene and the Xenopus oocyte expression system. The effects of altering a putative EF-hand domain on the function of the gene product will be examined by 2-electrode voltage clamp techniques. These studies will be the first detailed study of CNS aBgt-AChRs at the molecular and cellular level. Results obtained in this project will suggect roles for aBgt-AChRs in regulating hippocampal functions.
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