The goal of this proposal is to apply a new technology employing a novel silicon biosensing device to the study of neurotransmitter receptors. The biosensor to be used is a prototype silicon based microphysiometer which potentiometrically measures extracellular pH changes associated metabolic activity resulting from receptor activation. The microphysiometer performs nondestructive measurements of a cells metabolic activity in real time. We will study the catabolic events associated with dopamine D1 and D2 receptor activation. This will be carried out using stable transfectants expressing known levels of cloned dopamine receptors linked to well defined second messenger and effector responses. We will correlate various effector system activity with the amplitude and t1/2 of the metabolic records obtained with the microphysiometer. By relating the kinetics of the metabolic response with effector activity in a variety of well understood cellular environments we will assign a metabolic signature to Particular receptor/effector combinations. The techniques developed and information learned in this study of D1 and D2 receptors will subsequently be brought to bear on our studies of new G protein-coupled receptors. We will take advantage of the ability of the silicon microphysiometer to detect a broad range of cellular events to rapidly screen an assortment of cloned but as yet uncharacterized receptors. Using the microphysiometer we should be able to screen an assortment of stable transfectants expressing novel receptors with a large number of pharmacological agents. The kinetics of the microphysiometric measurements should suggest details about the effector mechanisms associated with the new receptors. All information concerning ligand specificity and second messenger involvement obtained with the silicon microphysiometer will be corroborated by conventional methods to document the accuracy of the method.
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