Student Theses and Dissertations

Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Greengard Laboratory


Signal transduction, the process by which an extracellular signal is translated into an intracellular message, underlies all brain function. This thesis examines the role of protein phosphorylation in mediating three different molecular mechanisms by which neurotransmitter receptors transduce their signals: chemically-gated ion channels, receptor-linked ion channels and receptor-linked ion pumps. The nicotinic acetylcholine receptor, a neurotransmitter-gated ion channel, is phosphorylated by a protein tyrosine kinase in postsynaptic membranes in vitro and in vivo. Purified nicotinic receptor molecules from Torpedo electroplaques can be phosphorylated to known stoichiometries and reconstituted into lipid vesicles. Tyrosine phosphorylation increases the rate of the rapid phase of desensitization of the receptor as measured by single channel recording but does not alter other channel properties. These data provide direct evidence for the regulation of ion channel properties by tyrosine phosphorylation and suggest that tyrosine-specific protein phosphorylation, in addition to regulating cell transformation and proliferation, may regulate neuronal signal transduction. Medium spiny neurons, the principal cell type of the neostriatum, contain D1 and D2 dopamine receptors. D2 receptor activation is coupled in dissociated neurons to an outward, voltage-dependent, transient K+conductance, probably lA, as measured by whole-cell patch clamping. This D2 coupled current can be inhibited by micromolar concentrations of forskolin suggesting that the receptor-channel linkage is regulated by protein phosphorylation. This inhibition of the D2 signal transduction pathway by an adenylate cyclase-mediated process may underlie the electrophysiological antagonism of D1 and D2 receptor activation in striatum. The activity of Na+, K+-ATPase, an ion pump which couples the hydrolysis of ATP to the countertransport of Na+ and K+ ions across the plasma membrane, can be measured in permeabilized dissociated neostriatal neurons. The neurotransmitter dopamine, through a synergistic effect on D1 and D2 receptors, inhibits the Na++,K+-ATPase activity of isolated striatal neurons. This mechanism appears to involve protein phosphorylation since cAMP-dependent protein kinase activation mimics D1 receptor stimulation and phorbol ester treatment mimics the effect of dopamine application. These data provide unequivocal evidence for regulation by a neurotransmitter of a neuronal ion pump. They demonstrate that synergism between D1 and D2 receptor activation, which underlies many of the electrophysiological and behavioral effects of dopamine in the mammalian brain, can occur on the same neuron. In addition, the data suggest that dopamine, in addition to its regulation of ion channels, regulates cell excitability through the novel mechanism of pump inhibition.


A thesis submitted to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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