Activation of the alpha1b-adrenergic receptor is initiated by disruption of an interhelical salt bridge constraint.

Rhodopsin receptor activation involves the disruption of a salt bridge constraint between glutamic acid 113 on transmembrane 3 and a lysine 296 in transmembrane 7, which forms a Schiff's base with retinal. Light-induced isomerization of cis-retinal to the all trans form breaks this rhodopsin salt bridge leading to receptor activation. The analogous residues in alpha1b-adrenergic receptors, aspartic acid 125 and lysine 331, also have the potential of forming a constraining salt bridge holding the receptor to an inactive protein configuration. This alpha1b-adrenergic receptor salt bridge constraint is then released upon binding by the receptor agonist. To test this hypothesis, site-directed mutagenesis was used to eliminate the positive charge at position 331 by substitution of an alanine. The expressed alpha1b-adrenergic receptor mutant demonstrated a 6-fold increased epinephrine binding affinity with no alterations of affinity values for selective adrenergic receptor antagonists. Furthermore, an increased epinephrine potency for total soluble inositol phosphate production along with an elevated basal inositol triphosphate level was observed in COS-1 cells transfected with mutant versus wild-type alpha1b-adrenergic receptors. Similar results were obtained for a lysine to a glutamic acid alpha1b-adrenergic receptor mutation. In addition, increased basal inositol triphosphate levels were also observed for two aspartic acid 125 alpha1b-adrenergic receptor mutations, consistent with this residue's role as the counterion of the salt bridge. Taken together, these alpha1b-adrenergic receptor mutations suggest a molecular mechanism by which the positively charged lysine 331 stabilizes the negatively charged aspartic acid 125 via a salt bridge constraint until bound by the receptor agonist.