Five distinct muscarinic receptors have recently been identified (m1- m5) using molecular genetic techniques. Unfortunately, the genetically defined receptors, cannot be easily assigned to muscarinic effects previously defined by pharmacological agents, as the drugs currently available are unable to distinguish between all five of the receptor subtypes, identified genetically. Thus, the goal of this proposal is to determine the effects of the five muscarinic receptors on individual ionic conductances and the mechanisms involved, in an attempt to understand the role of each muscarinic receptor subtype in control of cellular activity. This information will aid the rational selection of specific receptor subtypes as targets for therapeutic development, and may lead to new treatments for disorders such as depression, schizophrenia, Parkinsons or Alzheimers disease. This proposal will focus on the effects of the muscarinic receptors on an inwardly rectifying potassium and calcium conductances. Based on the second messengers activated by the different receptor subtypes and the pertussis toxin sensitivity of the G-proteins mediating the response, we can make tentative predictions about the coupling selectivity of the muscarinic receptors for these ion channels. The proposal will therefore test three hypotheses. The first is that m2 and m4 will couple with stimulation of the inward rectifier. The second is that m1, m3 and m5 will activate calcium conductances, while the third predicts that calcium currents will be inhibited by m2 and m4. The use of neuronal preparations to test these hypotheses, is are confounded by the inability of currently available methods, to distinguish between the five receptor subtypes, for purposes of electrophysiological investigation. In the proposed studies, we plan to resolve this issue by transfecting cells, chosen specifically for their expression of an inward rectifier or calcium conductances, with the genes encoding each of the muscarinic receptors. In addition to determining the effects of the receptor subtypes, this approach allows an unprecedented opportunity for study of mechanism of action, due to the electrophysiological simplicity of the cells. The coupling selectivity of the receptors with each conductance and mechanism of action will be assessed using the single channel and whole cell patch clamp recording techniques. Promising data from initial electrophysiological recordings have shown, that stimulation of m2-transformed RBL cells resulted in an increase in the inwardly rectifying potassium conductance, while stimulation of m4-transfected 3T3 cells caused a reduction in calcium conductance. The information obtained with these studies, can be extrapolated to neurons, and will thus add to our understanding of how these different receptor subtypes modulate cellular activity.
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