The nervous system operates by conducting impulses from one neuron to another by synaptic transmission. The essential events in synaptic transmission are the release of neurotransmitter from a presynaptic neuron and the binding of this neurotransmitter to receptors on the membrane of a postsynaptic cell. Binding of a neurotransmitter to a receptor eventually leads to a change in the ionic conductance of the postsynaptic cell. The biochemical processes that lead from membrane receptors to changes in ionic conductances are called signal transduction pathways. Many of the details of these signal transduction processes are not well understood, even though these processes underlie nervous system function in both normal and disease states. There are two major problems that will be investigated in this proposal: (1) the intracellular biochemical mechanisms involved in the transduction of neurotransmitter signals between membrane receptors and ionic channels and (2) the relationship between the molecular structure of the membrane receptors for neurotransmitters and their functions. The techniques to be applied in the proposed studies will provide precise control of intracellular and extracellular solutions, as well as of membrane potential. With accurate control over experimental conditions, it should be possible to study the signal transduction pathways with precision that has not been applied to these problems previously. Sympathetic neurons will serve as the model system to study the molecular mechanisms involved in signal transduction between membrane receptors and ion channels. The sympathetic ganglia provide a valuable preparation for the analysis of the actions of drugs and neurotransmitters. Studies of sympathetic neurons have provided considerable insight into the mechanisms of similar synaptic events that occur in the brain and spinal cord. Signal transduction pathways similar to the ones to be studied here are found throughout the nervous system and in cardiac and smooth muscle. This proposal will examine how intracellular application of various portions of the substance P receptor affects the response to substance P. Biochemical studies indicate that receptor desensitization may involve phosphorylation of the carboxyl tail of the receptor, but supporting evidence for a role of the receptor molecule in desensitization in intact neurons is lacking. Furthermore, the portions of the receptor involved in desensitization are not known. To determine which parts of the membrane receptors mediate various functions of the receptor, peptide fragments of small portions of the receptor will be applied intracellularly.
