Cocaine has become a major health problem because of its highly addictive nature and widespread use. It is believed that cocaine's euphoria and reinforcing property results from its inhibition of dopamine (DA) uptake into nerve terminals in the mesocorticolimbic system. The DA hypothesis will be tested by isolating the DA transporter and reconstituting the cocaine-sensitive DA uptake function into synthetic lipids, then comparing the potencies of cocaine analogs on these functions with their known in vivo potencies. This hypothesis is challenged by the fact that other potent DA transport inhibitors (e.g. benztropine, nomifensine and mazindol) do not have the reinforcing properties of cocaine. The overall objective is to understand the molecular mechanism of action of cocaine. This will be achieved by isolating the cocaine receptor (i.e. DA transporter) from bovine brain, determining its molecular properties and examining how cocaine differs from other DA uptake inhibitors in its action. The techniques to be used are biochemical and pharmacological.
The specific aims are: 1- To synthesize cocaine analogs for covalent labeling of the receptor and affinity chromatography and radiolabeled photoaffinity probes. 2- To determine the mechanism of allosteric regulation of the cocaine-sensitive [3H]DA transport by ATP, cations and anions. 3- To purify the receptor protein, determine its molecular weight, subunit structure, amino acid sequence and hydropathy profile. 4- To reconstitute the cocaine-sensitive DA uptake function in liposomes using impure and pure proteins. 5- To compare the actions of cocaine, mazindol, benztropine, nomiphensine and methamphetamine on the DA transporter binding and function. 6- To investigate the effects of chronic cocaine treatment of the DA transporter activity and its response to ATP and ions. The data will help understand molecular structure and function of the cocaine receptor and its involvement in cocaine addiction.
Showing the most recent 10 out of 13 publications
