G-protein coupled receptors (GPCRs) are the targets for multiple therapeutic agents. A complete understanding of the signaling mediated by these receptors is an important step in developing future therapeutic agents. The analysis of the kinetics of activation and inactivation of GPCRs has been limited, particularly in studies using endogenous receptors in brain slices. The photolysis of caged ligands, both agonists and antagonists, overcomes the diffusion barriers associated with dense tortuous tissues such as brain slices. This presentation will center on the use of caged-dopamine and -sulpiride, a D2-receptor antagonist, in a study on the kinetics of the activation of potassium conductance (GIRK) mediated by the D2-autoreceptor expressed on dopamine cells of the substantia nigra. Photolysis of dopamine activates the potassium conductance through the D2-receptor with at time course that is similar to the activation mediated by synaptically released endogenous dopamine. Photolysis of caged sulpiride rapidly blocked the D2-autoreceptor. The time course of residence of dopamine on the D2-receptor during synaptic activation was determined by varying the timing of photolysis relative to that of synaptically released endogenous dopamine. The results show that dopamine is bound to D2-autoreceptor for about 100 ms following the release of dopamine. The activation of the potassium conductance that has a 50 ms delay following the activation of D2-receptors, peaks in about 300 ms and decays with a time constant of about 250 ms. Thus dopamine receptor activation precedes the downstream functional action resulting in the activation of potassium conductance.

Studying G-protein coupled receptors using caged ligands

ACS Fall 2020 Virtual Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.