Serotonin 5-HT₇ receptor slows down the G_s protein: a single molecule perspective

The 5-hydroxytryptamine (serotonin) receptor type 7 (5-HT₇R) is a G protein-coupled receptor present primarily in the nervous system and gastrointestinal tract, where it regulates mood, cognition, digestion, and vasoconstriction. 5-HT₇R has previously been shown to bind to its cognate stimulatory G_s protein in the inactive state. This phenomenon, termed "inverse coupling," is thought to counteract the atypically high intrinsic activity of 5-HT₇R. However, it is not clear how active and inactive 5-HT₇ receptors affect the mobility of the G_s protein in the plasma membrane. Here, we used single-molecule imaging of the G_s protein and 5-HT₇R to evaluate G_s mobility in the membrane in the presence of 5-HT₇R and its mutants. We show that expression of 5-HT₇R dramatically reduces the diffusion rate of G_s. Expression of the constitutively active mutant 5-HT₇R (L173A) is less effective at slowing G_s diffusion presumably due to the reduced ability to form long-lasting inactive complexes. An inactive 5-HT₇R (N380K) mutant slows down G_s to the same extent as the wild-type receptor. We conclude that inactive 5-HT₇R profoundly affects G_s mobility, which could lead to G_s redistribution in the plasma membrane and alter its availability to other G protein-coupled receptors and effectors.