Figure 5. Rescue the TRPV5 CaM binding mutants by the G579A mutation.

(a) ⁴⁵Ca²⁺ uptake assay of HEK293 cells transfected with either wild type (WT) TRPV5 or the indicated mutants (G579A, W583A, S698X, W702A, G579-W583A, G579A-S698X, and G579A-W702A), depicted as percentage of WT (upper panel) (N = 9, three independent experiments performed in triplicate). Ruthenium red (RR) is used as a negative control. (b) Cell lysates of the respective experiments were immunoblotted with HA antibody, and using β-actin as loading control. A representative immunoblot is shown. (c) The basal intracellular Ca²⁺ levels are shown as fura-2 ratio in arbitrary units (a.u.) for HEK293 cells expressing either wild type (WT) TRPV5 or the indicated mutants (N = 11–66, total from two independent experiments). Values are shown as mean ± SEM. Asterisk indicates statistical significance (p < 0.05) compared to WT. (d) CaM binding assay of HEK293 cells transfected with wild type (WT) or indicated mutants of TRPV5. Samples were analyzed by immunoblotting with HA antibody. The CaM beads fraction represents the TRPV5 bound to the CaM agarose beads (top panel) and input demonstrates TRPV5 expression in total cell lysates (bottom panel). Representative immunoblot of three independent experiments is depicted.