A network of conserved intramolecular contacts defines the off-state of the transmembrane switch mechanism in a seven-transmembrane receptor

Abstract

Activation of the rhodopsin-like 7-transmembrane (7-TM) receptors requires switching interhelical constraints that stabilize the inactive state to a new set of contacts in the activated state, which binds the cognate G- protein. The free energy to drive this is provided by agonist binding, which has higher affinity to the active than to the inactive conformation. We have sought specific interhelical constraint contacts, using the M1 muscarinic acetylcholine receptor as a model. Histidine substitutions of particular groups of amino acids, in transmembrane domains 3, 6, and 7, created high- affinity Zn2+ binding sites, demonstrating the close proximity of their side chains in the inactive state. Alanine point substitutions have shown the effect of weakening the individual intramolecular contacts. In each case, the acetylcholine affinity was increased, implying promotion of the activated state. These amino acids are highly conserved throughout the 7-TM receptor superfamily. We propose that they form an important part of a network of conserved interhelical contacts that defines the off-state of a general transmembrane switch mechanism.