Tracking Voltage-Sensitive Movements of the M2 Muscarinic Acetylcholine Receptor

Numerous G protein-coupled receptors (GPCRs) have been shown to change their activity levels in response to changes in membrane potential. More recently, it has been directly demonstrated that these changes in activity reflect underlying changes in conformational state. However, the nature of the motion undergone by G protein-coupled receptors during voltage changes remains poorly understood. By combining site-directed fluorometry of the M2 muscarinic acetylcholine receptor with site-directed mutagenesis of residues that may serve as quenchers of the fluorescent signal, we attempt to refine our understanding of the voltage-dependent conformational changes that occur in a GPCR. When using tetramethylrhodamine-5-maleimide (TMRM), voltage-sensitive fluorescence changes display an electrochromic response in addition to the component that provides information on the voltage-sensitive conformational changes of the receptor. Interestingly, some mutations of potential quenchers of the fluorescent signal also alter the electrochromic signal, suggesting that alterations at these sites may result in a change in the landscape of the electric field within the protein. This work was supported by R01-GM030376 and F31-NS081954.