Reduced affinity of calcium sensing-receptor heterodimers and reduced mutant homodimer trafficking combine to impair function in a model of familial hypocalciuric hypercalcemia type 1
Fig 4
[Ca2+]o-effect relationship for wild-type and mutant CaSR expressed alone or together.
A, the time courses of increases in [Ca2+]i, following the increase of [Ca2+]o from 1 to 10 mM, in HEK cells transfected with pEGFP/CaSR, pcDNA-myc/CaSR, pEGFP/mCaSR, pEGFP/CaSR and pcDNA-myc/mCaSR, or pEGFP/mCaSR and pcDNA-myc/CaSR or untransfected controls. [Ca2+]i changes, shown as F/F0, was measured in cells grown on glass coverslips and loaded with 2 μM X-rhod1 in Tyrode’s solution. The gray traces are fluorescent signals for single cells in the field of view, while the average values (black) indicate the mean responses (n = 12–27). B, concentration-response relationship of [Ca2+]o-dependent mobilization of [Ca2+]I in HEK 293 cells expressing wild-type and/or mutant CaSR. Each point reflects the average peak response above F0 (see A) from a single coverslip and three separate coverslips were used for each [Ca2+]o value. Values are means ± SEM. Curves were drawn according to equation1: for CaSR-EGFP, CaSR-myc, CaSR-EGFP/mCaSR-myc, mCaSR-EGFP/CaSR-myc, untransfected HEK, mCaSR-EGFP respectively: Fmax = 0.998 ± 0.021, n = 2.163 ± 0.171, and EC50 = 4.552 ± 0.218; Fmax = 0.979 ± 0.015, n = 2.31 ± 0.231, and EC50 = 4.603 ± 0.154; Fmax = 0.803 ± 0.008, n = 1.910 ± 0.086, and EC50 = 6.021 ± 0.153; Fmax = 0.793 ± 0.012, n = 1.889 ± 0.114, and EC50 = 5.845 ± 0.171; Fmax = 0.274 ± 0.089, n = 0.931 ± 0.266, and EC50 = 42.722 ± 33.6; Fmax = 0.162 ± 0.010, n = 2.039 ± 0.43, and EC50 = 19.195 ± 2.49.