The long term goal of this project is to characterize the molecular basis of nicotinic acetylcholine receptor function and two approaches will be used. The first approach is to extend ongoing site-directed mutagenesis and chemical modification studies to characterize the functional role of specific residues in regulating the ion channel properties of Torpedo californica acetylcholine receptor. The second approach is to construct and express biochemically useful amounts of functional acetylcholine receptors by exploiting the insect baculovirus system. For the mutagenesis experiments, particular attention will be given to Cys residues on the M4 transmembrane helix regions of the receptor since these regions appear to play a role in regulating channel gating. In addition to site-specific mutations, hybrid and chimeric receptors will be used to assess the functional role of specific receptor domains. Experiments designed to examine fatty acid acylation, pH effects, desensitization, time-dependent maturation of channel activity, and chemical reactivity of altered receptors will be used to test specific hypotheses related to both the functional role of cysteines and other domains. Functional effects will be monitored by binding assays and by voltage-clamp and single-channel analyses of AChRs expressed in Xenopus levels oocytes following injection with mRNAs for the subunits. Efforts to express fully functional receptors in insect cells will focus on neuronal acetylcholine receptors since these receptors have a simpler subunit structure. The development of a system for expressing large amounts of functional chimeric receptors consisting of a single subunit type will be a longer term goal designed to extend biochemical and biophysical approaches to recombinant receptors. This application proposes the continuation of a series of laboratory studies examining the mechanisms by which behavioral manipulations reduce pain. The project focuses on several components of behavioral pain control techniques, including mood alteration, distraction, imagery and expectancy. The proposed studies are based on the project's work to date, indicating that phasic pain (more sensory in nature) may best be controlled by tasks with high attention demand, while tonic laboratory pain (with more reactive components) is best controlled by such manipulations as improving mood. Specifically, we (a) examine the aspects of mood that alter pain tolerance, including interaction between mood and coping strategies, (b) examine components of the hypnotic induction, (c) examining the interaction between behavioral interventions and the administration of opioid analgesics, and (d) examine the generality of our previous findings to clinical pain of which is of greater severity and duration than is typical for laboratory studies. In examining these issues, we measure different types of pain response. Of critical importance are subjects' ratings of both pain and distress as well as behavioral measures such as pain tolerance levels. Additional physiological measures include evoked potentials, evoked magnetic fields, and background EEG. These measures supplement self-report and behavioral measures of pain by offering complimentary data regarding cortical processing of painful stimuli.
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