In allosteric proteins like G-protein coupled receptors (GPCRs), the ligand binding site (LBD) and the effector site in GPCRs are far away from each other (tens of Å away). Further, there are other sites in the receptor where positive and negative allosteric modulators (PAM or NAM) bind to alter GPCR signaling activity. PAM or NAMs are emerging as promising therapeutic options for neuropsychiatry disorders. The million-dollar question is how signal from the LBD or these allosteric sites is transmitted to the G-protein coupled site. We are keen to understand the sequence of global conformational changes, known as gating, that ensues on ligand binding to GPCRs. In the lab, we use the M2-muscarinic acetylcholine receptor as a model GPCR and try to probe the molecular conformational dynamics by employing single channel patch-clamp, strategic mutagenesis, thermodynamics, and transition state studies.

This approach is innovative in 2 ways: 1. Since GPCRs cannot be studied directly by patch-clamp, we will use the G protein-coupled Inwardly Rectifying K+ channels (GIRK) as a reporter to study the allosteric conformational dynamics in GPCRs. Agonist binding activates GPCRs by dissociating the Gβγ complex from the inactive Gαβγ complex, which in turn activates the GIRK channel. Due to the direct coupling of GPCRs and GIRK channels, they are also termed as GPCR-gated channels. This provides a good system to understand GPCR allostery. 2. By using patch-clamp, it will be possible to study the allosteric changes happening at a time scale of 10s of s as compared to few 100 ms time scale in fluorescence-based methods.

Currently, we are in the process of measuring the various kinetics and energetic parameters of M2-muscarinic mAChRs.