Fig. 6: Negative charges in the lower interface of the VFT limit CaSR activation but is not involved in basal activity.
From: Absence of calcium-sensing receptor basal activity due to inter-subunit disulfide bridges
a Electrostatic potential map showing charged residues in CaSR LB2 interface in the active state (PDB: 5FDK). b Sequence alignment of the LB2 interface of the human CaSR and rat mGluRs using Clustal Omega and ESPript 3. CaSR is used as reference for residue numbering and the blue boxes indicated the conserved residues. c Scheme showing constructs of where the negatively charged residues of LB2 interface were mutated into alanine (13 A) in the background of the WT and CSCS. 13 A includes mutations at residues E224, E228, E229, E231, E232, D234, D238, E241, D248, E249, E250, E251, and E257. d Basal IP1 accumulation for the CaSR the WT and indicated mutants (n = 6). e Intracellular calcium release induced by CaCl2 in the WT and indicated mutants and the corresponding pEC50 (n = 7). f Intracellular calcium release measured for the WT and indicated mutants stimulated by PAM R568 in the absence of ligands (n = 4). Data above are mean ± SEM of at least four independent experiments performed in triplicates and normalized to the mock (d) or the WT (e, f). Significance was analyzed using one-way ANOVA with Dunnett’s multiple comparisons with ****P ≤ 0.0001 and ns for P > 0.05 versus the mock (d) or the WT (e), and ###P ≤ 0.001 versus the CSCS.
