A region in the seven-transmembrane domain of the human Ca²⁺ receptor critical for response to Ca²⁺

Of 12 naturally occurring, activating mutations in the seven-transmembrane (7TM) domain of the human Ca²⁺ receptor (CaR) identified previously in subjects with autosomal dominant hypocalcemia (ADH), five appear at the junction of TM helices 6 and 7 between residue Ile⁸¹⁹ and Glu ⁸³⁷. After identifying a sixth activating mutation in this region, V836L, in an ADH patient, we studied the remaining residues in this region to determine whether they are potential sites for activating mutations. Alanine-scanning mutagenesis revealed five additional residues in this region that when substituted by alanine led to CaR activation. We also found that, whereas E837A did not activate the receptor, E837D and E837K mutations did. Thus, region Ile⁸¹⁹-Glu⁸³⁷ of the 7TM domain represents a "hot spot" for naturally occurring, activating mutations of the receptor, and most of the residues in this region apparently maintain the 7TM domain in its inactive configuration. Unique among the residues in this region, Pro⁸²³, which is highly conserved in family 3 of the G protein-coupled receptors, when mutated to either alanine or glycine, despite good expression severely impaired CaR activation by Ca²⁺. Both the P823A mutation and NPS 2143, a negative allosteric modulator that acts on the 7TM through a critical interaction with Glu⁸³⁷, blocked activation of the CaR by various ADH mutations. These results suggest that the 7TM domain region Ile⁸¹⁹-Glu⁸³⁷ plays a key role in CaR activation by Ca²⁺. The implications of our finding that NPS 2143 corrects the molecular defect of ADH mutations for treatment of this disease are also discussed.