Figure 1. Effect of W583 mutations on TRPV5 function.

(a) Left panel depicts a ⁴⁵Ca²⁺ uptake assay of HEK293 cells transfected with either wild type (WT) TRPV5, I575A, M578A, H582A, or W583A. Data is shown as percentage of WT (left panel) (N = 9, three independent experiments performed in triplicate). Ruthenium red (RR), 10 μM, is used as control to inhibit the TRPV5-mediated Ca²⁺ uptake. The right panel shows a representative immunoblot using HA antibody for TRPV5 expression, and β-actin as loading control. (b) Multiple sequence alignment of the region surrounding W583 in different species of TRPV5 and amongst other TRPV family members. Light blue letters represent conserved amino acids and the red box indicates the conservation of W583. (c) ⁴⁵Ca²⁺ uptake assay of HEK293 cells transfected with either wild type (WT) TRPV5 or the indicated mutants, depicted as percentage of WT (upper panel) (N = 9, three independent experiments performed in triplicate). Ruthenium red (RR) is used as negative control. (d) Representative immunoblot of cell lysates of the respective Ca²⁺ uptake experiments with HA antibody for TRPV5 and β-actin as loading control. (e) Quantification of cell death by counting the number of trypan blue stained (death) HEK293 cells upon transfecting wild type (WT) TRPV5 or the indicated mutants, depicted as percentage of the total cell amount (N = 6, three independent experiments performed in duplicate). (f) 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 = 30–40, total from two independent experiments). Values are shown as mean ± SEM. Asterisk indicates statistical significance (p < 0.05) compared to WT. (g) ⁴⁵Ca²⁺ uptake assay of HEK293 cells transfected with wild type (WT) TRPV5 or the indicated mutants, depicted as percentage of WT and corrected for protein expression levels (upper panel) (N = 9, three independent experiments performed in triplicate). Ruthenium red (RR) is used as a negative control. (h) Cell surface biotinylation of HEK293 cells transfected with wild type (WT) TRPV5 or indicated mutants. The biotin fraction represents the TRPV5 present at the plasma membrane (top panel) and input demonstrates TRPV5 expression in total cell lysates (bottom panel). Representative immunoblot of three independent experiments is shown.