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Alanine mutations were introduced into the human wild-type CaSR gene by using QuickChange Lightning Site-directed mutagenesis kit (Stratagene). Mutations were confirmed by the Genomic Technologies Core Facility at The University of Manchester. HEK-293 cells were then transiently transfected with wild-type or mutant receptors by using FuGENE-6 (Roche Diagnostics). Cells were used experimentally within 72 h of transfection. For stable transfection, vectors were first linearized (Ssp1) and then CaSR-expressing cells were selected by using hygromycin (400 μg/ml).
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