Yu Y , Ulbrich MH , Li MH , Dobbins S , Zhang WK , Tong L , Isacoff EY , Yang J .

GM085234 NIGMS NIH HHS , NS035549 NINDS NIH HHS , NS045383 NINDS NIH HHS , R01 NS035549 NINDS NIH HHS , R01 GM085234 NIGMS NIH HHS , R01 NS045383 NINDS NIH HHS

	Figure 2. Both PKD1L3 and TRPP3 line the pore(a) Amino acid sequence alignment of the putative pore region of human (h) and mouse (m) TRPP3 and PKD1L3. Red: identical; green: conserved. Residues marked by arrows were critical for ion selectivity. (b–d) Representative I–V curves of acid-induced currents of WT PKD1L3/TRPP3 in a bath solution containing Na+, dimethylamine+, trimethylamine+, or Mg2+. The two curves in each panel were obtained from the same oocyte. (e–j) Bar graph showing the reversal potential of currents recorded from WT (grey bars) or mutant (red bars) PKD1L3/TRPP3 complexes (mutation indicated at the top of each panel) in a bath solution containing dimethylamine+, trimethylamine+, or Mg2+. All pair-wise data were obtained from the same batch of oocytes and all pair-wise comparisons are significantly different (P<0.05 as determined by unpaired two-tailed Student's t-test), except the middle panel in j. Representative I–V curve for each condition is shown in Supplementary Fig. S2. Results are shown as mean and standard deviation. n = number of measurements.(Yang)
	Figure 3. Homomeric TRPP3 complexes on the plasma membrane of Xenopus oocytes contain 3 TRPP3 subunits(a) TIRF image from an oocyte expressing TRPP3-EGFP. Immobile spots were selected (circles) for photobleaching analysis. Scale bar, 2 μm. (b) Time course of photobleaching of two representative spots in a, showing three EGFP bleaching steps (arrows). Green bar indicates EGFP excitation. (c) Distribution of observed and calculated bleaching steps for TRPP3-EGFP fluorescence spots. The 1–3 step distribution (green bars) is well fit by a binomial distribution (white bars) that assumes that each spot contains 3 EGFPs and that the probability of EGFP to be fluorescent is 70%.(Yang)
	Figure 4. The PKD1L3/TRPP3 heteromeric channel has a 1:3 subunit stoichiometry(a and d) TIRF images of EGFP fluorescence from an oocyte expressing the indicated constructs, showing spots that exhibited EGFP and mCherry dual fluorescence and were immobile (circles). Other spots do not fit analysis criteria. Scale bar, 2 μm. (b and e) Time course of photobleaching of two representative dual-fluorescence spots of either TRPP3-EGFP + PKD1L3-mCherry (b) or PKD1L3-EGFP + TRPP3-mCherry (e), showing EGFP bleaching steps (arrows). EGFP and mCherry excitation is indicated by green and red bar, respectively. (c and f) Distribution of observed EGFP bleaching steps (green bars) for dual-fluorescence spots of either TRPP3-EGFP + PKD1L3-mCherry (c) or PKD1L3-EGFP + TRPP3-mCherry (f). In c, observed EGFP bleaching steps are compared to a binomial distribution assuming that 3 EGFP molecules exist in each spot and based on 78% of EGFPs being fluorescent (white bars). (g) Schematic diagram of the heteromeric PKD1L3/TRPP3 channel viewed from the side. P: channel pore. Green and red loops depict the putative selectivity filter formed by the pore loops shown in Fig. 2a. The inner pore is presumed to be lined by the 11th transmembrane segment (S11) of PKD1L3 and the 6th transmembrane segment (S6) of TRPP3.(Yang)
	Figure 5. The TRPP3 C-terminus forms a trimer(a) Putative transmembrane topology of TRPP2 and TPPP3. The C-terminus of both proteins contains a coiled-coil domain (red bar). (b) Amino acid sequence alignment of TRPP2 and TRPP3 coiled-coil domains. Red: identical; green: conserved. Asterisks indicate hydrophobic residues at the 1st and 4th positions in characteristic heptad repeats of canonical coiled-coil domains. (c) Crystal structure of TRPP3-G699-W743 fragment, showing that the TRPP3 coiled-coil domain forms a trimer. (d) Side-chains, shown in sticks, of hydrophobic residues involved in the formation of the coiled-coil domain trimer. The underlined amino acids were mutated to alanine to generate mut6. (e) Superposition of the structures of TRPP3 and TRPP2 coiled-coil domains (PDB: 3HRN). (f) Gel filtration profile of MBP-tagged WT TRPP3 C-terminal fragment (TRPP3_CT) and TRPP3_CT_mut6. The right shift of the latter indicates a decrease in molecular mass. (g) Bar graph comparing the calculated and measured molecular masses of MBP-TRPP3_CT and MBP-TRPP3_CT_mut6. Measured molecular masses were obtained by static light scattering. Calculated molecular masses were obtained assuming that the protein is a monomer (for mut6), a trimer (for WT), or a tetramer (for WT).(Yang)
	Figure 6. The TRPP3 coiled-coil domain trimer is important for the assembly and surface expression of homomeric TRPP3 complexes(a) HA- and FLAG-tagged full-length TRPP3, as indicated on top, were expressed in Xenopus oocytes and immunoprecipitated (IP) with anti-FLAG antibody coated beads. All proteins had comparable expression as can be seen from the lysate samples (lower two gels). Reduced amount of HA-tagged TRPP3 was co-IPed with FLAG-tagged TRPP3 mutants that lack the coiled-coil trimer interaction (upper gel, lanes 3 and 4). IB: immunoblot. On SDS-PAGE, WT TRPP3 gave rise to a monomeric band at ~90 kDa (arrows) and two oligomeric bands with molecular masses of ~200 kDa and >250 kDa (asterisks). The oligomeric complexes appeared even at more severe denaturing and reducing conditions (Supplementary Fig. S8). The two FLAG-tagged TRPP3 mutants showed reduced amount of both oligomeric bands, suggesting a weakened homomeric assembly of TRPP3 (upper gel immunobloted with anti-Flag antibody, lanes 3 and 4; also see Fig. 7c, d). Bar graph at right shows the normalized ratio of the intensity of HA-TRPP3 (WT or mutants as indicated at bottom)/Flag-TRPP3. The sum of the intensity of all monomeric and oligomeric bands for each TRPP3 protein was used in the calculation. A smaller ratio suggests a weakened association between HA-TRPP3 and Flag-TRPP3. (b) TIRF images of EGFP fluorescence from oocytes expressing the indicated constructs. Upper panel was a duplication of Fig. 3a. Scale bar, 2 μm.(Yang)
	Figure 7. The TRPP3 coiled-coil domain trimer is essential for the surface expression of the PKD1L3/TRPP3 complex(a) Bar graph comparing acid-induced whole-cell currents from oocytes expressing the indicated constructs. (b) TIRF images of both EGFP and mCherry fluorescence from oocytes expressing the indicated constructs. The images for both combinations were obtained from oocytes injected with the same amount of cRNAs and on the same day after injection, and are representative of >6 different oocytes. Scale bar, 2 μm. (c) Western blot following SDS-PAGE showing TRPP3 and PKD1L3 expressed on the plasma membrane (upper gels) and in the lysate (lower gels) of oocytes expressing the indicated constructs. The actin control shows that only surface proteins were biotinylated. Monomeric and oligomeric TRPP3 are indicated by arrows and asterisks. PKD1L3 showed a full-length band (>250 kDa, arrow heads) and a cleaved band (~180 kDa, open circles) (lower gel, lanes 3, 4, 6 and 7). Disruption of the TRPP3 coiled-coil domain trimer reduced the surface expression of TRPP3 (upper gel, lanes 5–7) and PKD1L3 (upper gel, lanes 6 and 7). Bar graphs at the bottom show the normalized intensity of surface TRPP3 or PKD1L3 proteins when PKD1L3 was coexpressed with WT or the indicated mutant TRPP3. Data were obtained from lines 4, 6 and 7 on the top two gels. The sum of the intensity of all monomeric and oligomeric bands for each protein was used in the calculation. (d), Western blot following SDS-PAGE showing that PKD1L3 (HA-tagged) can be co-immunoprecipitated with all indicated TRPP3 constructs (FLAG-tagged). Monomeric and oligomeric TRPP3 are indicated by arrows and asterisks. Full-length and cleaved PKD1L3 are indicated by arrow heads and open circles. Bar graph at the bottom shows the normalized ratio of the intensity of HA-PKD1L3/FLAG-TRPP3 (WT or the indicated mutants).(Yang)

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