Figure 1. Roles of the TRPP3 W81 (Pre-S1 Domain) and K568 (TRP-like Domain) Residues in the N-C Interaction and Channel Function.

(A) Left panel: representative whole-cell current traces obtained from Xenopus oocytes expressing human TRPP3 WT or mutant DW81-L95 (with the W81-L95 domain deletion), using the two-electrode voltage-clamp technique. Oocytes were voltage clamped at −50 mV, and currents were recorded using the Na⁺-containing extracellular solution (Na) (100 mM NaCl, 2 mM KCl, 1 mM MgCl₂, and 10 mM HEPES [pH 7.5]) or added with 5 mM CaCl₂ (Na⁺Ca). The Ca-activated current (i.e., current at “Na+Ca” – current at “Na”), indicated by the double-arrowed line, was measured to assess TRPP3 channel activity (Chen et al., 1999). Right panel: averaged Ca-activated currents from oocytes expressing TRPP3 WT or an indicated mutant or H₂O-injected oocytes (Ctrl) are shown (n = 17–22). Oocytes were from three batches. ***p < 0.001 (compared with WT).

(B) Averaged Ca-activated currents from oocytes expressing TRPP3 WT or an indicated point mutant. ***p < 0.001.

(C) Averaged Ca-activated currents from oocytes expressing TRPP3 WT or an indicated W81 point mutant. Currents were averaged from three independent experiments and normalized to that of WT.

(D) Amino acid sequence alignment of the TRPP3 pre-S1 helix from indicated species, with the conserved residue W highlighted red.

(E) Averaged Ca-activated currents obtained from oocytes expressing TRPP3 WT or an indicated point mutant in TRP-like domain.

(F) Averaged and normalized Ca-activated currents from oocytes expressing TRPP3 WT or an indicated K568 point mutant.

(G) Amino acid sequence alignment of the TRPP3 TRP-like domain from indicated species, with the conserved residue K highlighted blue.

(H) Representative immunoblots of the surface biotinylated (surface) and whole-cell (total) TRPP3 WT or indicated W81 or K568 point mutants.

(I) Left panel: W81-K568 interaction examined with co-IP assays using oocytes co-expressing full-length (FL) TRPP3 and His-tagged TRPP3 N-terminal peptide (His-P3NP; I40-L95). Right panel: data from experiments in left panel were quantified, averaged, and normalized. **p < 0.01; n = 3.

(J) W81-K568 interaction examined with His pull-down assays using the purified GST-tagged human TRPP3 N terminus (GST-P3N; M1-L95) and His-tagged human TRPP3 C-terminal peptide (His-CP; I560-K660) from E. coli. Ctrl, purified GST-human FUBP1 M1-V112 from E. coli.

(K) Colocalization of P3NP with FL TRPP3 examined with co-immunofluorescence assays using oocytes co-expressing FL TRPP3 and His-P3NP. The scale bar represents 50 μm.

(L) Effects of co-expressed P3NP or its point mutants on TRPP3 Ca-activated currents. None, no P3NP expression. Shown are normalized and averaged currents from three independent experiments (n = 15–20).

Data are presented as mean ± SEM. See also Figures S1 and S2.