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

Activation of the rhodopsin-like 7-transmembrane (7-TM) receptors requires switching interhelical constraints that stabilize the inactive state to a n ew set of contacts in the activated state, which binds the cognate G-protei n. 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 so ught specific interhelical constraint contacts, using the M-1, muscarinic a cetylcholine receptor as a model. Histidine substitutions of particular gro ups of amino acids, in transmembrane domains 3, 6, and 7, created high-affi nity Zn2+ binding sites, demonstrating the close proximity of their side ch ains in the inactive state. Alanine point substitutions have shown the effe ct of weakening the individual intramolecular contacts. In each case, the a cetylcholine affinity was increased, implying promotion of the activated st ate. These amino acids are highly conserved throughout the 7-TM receptor su perfamily We propose that they form an important part of a network Of conse rved interhelical contacts that defines the off-state of a general transmem brane switch mechanism.