The goal of this proposal is to develop a working understanding and ion permeation models that account for the structure and function of glutamate receptors (GluRs) and nicotinic acetylcholine receptors (nAChRs). The ongoing work is at different stages for the two receptor types. -1- For the peripheral (P) nAChR, biochemical, biophysical and ultrastructural data have provided a picture that relates structure and function. Our research has focused on how the open channel structure governs ion permeation, and for the first time, we presented an ion permeation model that describes permeation data, based explicitly on the general structure of the channel. The working understanding and physically based permeation model resulting from the research now serve as predictive guides into the study of synaptic processes. For example, the results and model guided our characterization of MK -801 inhibition of nAChRs, and provided a prediction of the synaptic Ca 2+ influx through PnAChRs. The fundamental biophysical work also will serve as a basis for the interpretation of results with neuronal (N) nAChRs. In vitro transcribed mRNAs from the cDNAs encoding different subunits of NnAChRs will be expressed in oocytes. The functions of different subunit combinations will be characterized, and then, they will be compared with our working picture of the PnAChR. The objective is to understand the functional diversity that can arise from different NnAChR subunit combinations. This work is important because the heterogeneous distribution of subunits in the CNS suggests that alterations in the NnAChR's compositions play a role in modulating cholinergic signals that influence synaptic transmission. -2- Structure-function studies of GluRs are proposed because glutamate synapses are the most important excitatory synapses in the brain, but there is little biochemical or biophysical information and practically no structural data for the channels. The proposed research is the first step along the same path we have taken with the PnAChR. The work will progress through a series of experiments that each contribute to the final goal of associating function to the general structure of the channel. Experiments on ion permeation, open channel blockade and ion-water interactions will provide the following information about the pore: the minimum cross section, the length of the minimum cross section, the size of the entrance vestibules, the general charge, the number and position of binding sites, and the volume change associated with channel gating. Because methods that resolve structures of water soluble proteins cannot be applied at high resolution to ion channels in membranes, the proposed research, at present, is the best way to answer questions about the open channel structure and function of the GluRs. If crystals for the channels become available, these basic structural results should guide the interpretation of the crystals. The results also are important for relating the amino acid sequences of the cloned channels to their functions. For example, these types of results guided the development of the currently accepted structural model based on the primary sequences for the PnAChR. As with the PnAChR, the permeation models and the general working knowledge gained from these experiments should lead to predictions that direct research into the GluRs' involvement in synaptic processes.
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