Zheng W, Yang X, Hu R, Cai R, Hofmann L, Wang Z, Hu Q, Liu X, Bulkley D, Yu Y, Tang J, Flockerzi V, Cao Y, Cao E, Chen XZ.

R01 DK110575 NIDDK NIH HHS , R15 DK102092 NIDDK NIH HHS

           POLYCYSTIC KIDNEY DISEASE 2 WITH OR WITHOUT POLYCYSTIC LIVER DISEASE; PKD2

	Fig. 1. Identification of PKD2L1 hydrophobic gate in the S6 helix. a Left panel, membrane topology of polycystin channels. The TOP domain between S1 and S2 and the P-loop between S5 and S6 are indicated. Right panel, amino acid (aa) sequence alignment of S6 helix of human PKD2, PKD2L1 and PKD2L2. Each single underlined aa of PKD2L1 was mutated to asparagines (N). b Representative current traces obtained from oocytes injected with WT or a mutant (L557N or A558N) PKD2L1 cRNA, or water (Ctrl). Oocytes were voltage clamped at -50 mV in the presence of the extracellular solution containing (in mM) 100 N-methyl-d-glucamine (NMDG)-Cl, 2 KCl, 1 MgCl2, 10 HEPES at pH 7.5 (“NMDG”), or Na-containing solution (equimolar Na+ substituting NMDG) (“Na”). Dashed lines are the baselines from which plateau current values were determined. c Averaged currents as the difference between NMDG- and Na-containing solutions obtained at −50 mV, as in b, in oocytes expressing the single mutants, as indicated. For each mutant, currents were averaged from 10 to 17 oocytes from three batches. Data are presented as mean ± SEM. d Representative I–V curves from Ctrl (water-injected oocytes), PKD2L1 WT, L557N or A558N expressing oocytes obtained at current points indicated by the vertical bars in the traces in b, using the indicated voltage ramp protocol. e Western blot of surface biotinylated and total PKD2L1 WT or indicated mutants. f Whole-mount immunofluorescence showing the oocyte surface expression of PKD2L1 WT or mutants from e. Scale bar, 50 μm
	Fig. 2. Characterization of mutants at PKD2L1 sites L557 and A558. a L557 was replaced with different aa, as indicated. Shown are averaged currents obtained under the same experimental conditions as in Fig. 1b (n = 10–15 oocytes from 3 batches). Data are presented as mean ± SEM. b A558 was replaced with different aa, as indicated, and averaged currents were obtained as in Fig. 1b (n = 10–17 oocytes from 3 batches). Data are presented as mean ± SEM. c Left panel, representative I–V curves for three indicated A558 mutants generated under the same experimental condition as in Fig. 1d. Right panel, averaged outward currents at +80 mV for indicated A558 mutants (n = 10–14). Data are presented as mean ± SEM. d Representative I–V curves for three indicated L557 mutants obtained as in Fig. 1d. e Western blot of surface biotinylated and total protein of PKD2L1 WT or indicated mutants. f Whole-mount immunofluorescence showing the oocyte surface expression of PKD2L1 WT or mutants from e. Scale bar, 50 μm. g L557 and A558 were replaced with W or N. Left panel, representative I–V curves of PKD2L1 WT or indicated mutants obtained as in Fig. 1d. Right panel, averaged current at −50 mV for PKD2L1 WT or indicated mutants (n = 8–11). Data are presented as mean ± SEM
	Fig. 3. Identification and characterization of PKD2 hydrophobic gate. a Each single underlined aa of human PKD2 was mutated to N. Representative I–V curves were obtained in oocytes expressing WT or a mutant PKD2, as indicated, in the presence of the divalent free Na-containing solution (in mM): 100 NaCl, 2 KCl, 10 HEPES at pH 7.5. The gain-of-function mutant F604P (in the S5 helix) serves as a positive control and water-injected oocytes as a negative control (Ctrl). b Averaged currents at +80 mV obtained under the same experimental conditions as in a in expressing or control oocytes, as indicated. Currents were averaged from 10–13 oocytes from at least three batches. Data are presented as mean ± SEM. c Upper panel, averaged currents obtained as in panel a for PKD2 WT, A678 mutants, L677N or F604P (n = 8–13). Data are presented as mean ± SEM. Lower panel, western blot of surface biotinylated and total protein of PKD2 WT or indicated mutants. d Upper panel, the PKD2 L677 was replaced with different aa as indicated. Shown are averaged currents obtained as in a, with F604P as a positive control (n = 10–14). Data are presented as mean ± SEM. Lower panel, western blot showing total protein of PKD2 WT or indicated mutants present in the injected oocytes. e Representative I-V curves for F604P/L677 double mutants, as indicated, under the same condition as in a. f Averaged currents at +80 mV recorded from oocytes injected with cRNA of the indicated PKD2 WT or F604P/L677 double mutants (n = 8–12). Data are presented as mean ± SEM. g Averaged currents at +80 mV obtained from oocytes expressing PKD2 WT, or indicated single or double mutants (n = 9–12). Data are presented as mean ± SEM. h Averaged currents at +80 mV obtained from oocytes expressing PKD2 WT, L677N or L677N/N681L mutant. Data are presented as mean ± SEM
	Fig. 4. Rescue of PKD2-associated zebrafish phenotypes by PKD2 gate mutants. a Representative zebrafish embryos showing different severities in tail curling induced by PKD2 MO knockdown. Normal (no curvature), Moderate (significant curvature, <90°), Substantial (90°–180°), and Severe (>180°). b Averaged percentages of embryos with Normal, Moderate, Substantial and Severe tail curling at 3dpf. Embryos were co-injected with 2.5 ng PKD2 MO together with none or 100 pg mRNAs of WT or mutant PKD2, as indicated. Ctrl, uninjected embryos. Data were from three independent experiments with the indicated total numbers of embryos. Statistic significance was determined by the χ2 test. NS, no significance; *P < 0.05; **P < 0.01. PKD2 F604P mutant serves as a positive control. c Water-injected (Normal) and PKD2 MO-injected (Cyst) zebrafish embyros at 3 dpf showing curly tail and pronephric cyst formation (arrows) which is confirmed by a histologic section that also displayed dilated pronephric tubules (asterisks). G, glomerulus; Pt/Pd, pronephric tubule/duct; Nc, notochord. d Averaged percentages of embryos exhibiting pronephric cysts under the same conditions as those in b. Data are presented as mean ± SEM. Statistic significance was determined by Student’s t-test. NS, no significance; **P < 0.01; ***P < 0.001
	Fig. 5. Comparison of structure of PKD2 F604P with that of WT channel. a, b Side view of superposition of tetrameric (a) and subunit (b) structures of PKD2 F604P (orange) and WT (blue). c Comparison between S5 helix in PKD2 F604P and WT channels. The F604 and P604 residues are shown. d Solvent-accessible pathway along the pore mapped using the HOLE program for PKD2 WT and F604P structures. Residues forming the selectivity filter and lower constriction points are indicated. e Comparison between the pore radii, calculated with the program HOLE, for the PKD2 WT and F604P structures. f Upper panel, comparison between the S4-S5 linker in the PKD2 WT and F604P structures. The R581 at the beginning of the linker is shown. Lower panel, sequence alignment of S4-S5 linker of human PKD2, PKD2L1 and PKD2L2. The conserved cationic residues were highlighted in bold and magenta. g Left panel, Representative I-V curves for PKD2 WT, single mutant F604P and double mutant F604P/R581A obtained under the same condition as in Fig. 3a. Right panel, averaged currents at +80 mV recorded from oocytes injected with cRNA of PKD2 WT or mutant F604P or F604P/R581A. Data are presented as mean ± SEM. ***P < 0.001 by Student’s t-test. h Left panel, representative whole-cell current traces obtained from control oocytes (Ctrl, water-injected) or oocytes injected with cRNA of human PKD2L1 WT or mutant K568A. Oocytes were voltage clamped at −50 mV and currents were recorded using Na-containing solution (see Fig. 1b) without (Na) or with addition of 5 mM CaCl2 (Na + Ca). The Ca-activated peak current, indicated by the double arrowed line, was measured to assess PKD2L1 channel activation. Right panel, averaged Ca-activated peak currents from oocytes expressing PKD2L1 WT or K461A mutant or water-injected oocytes (Ctrl). Data are presented as mean ± SEM. ***P < 0.001 by Student’s t-test
	Fig. 6. PKD2 S6 conformational changes induced by F604P mutation. a Side view of superposition of S6 in the PKD2 WT (blue) and F604P (orange). The π-helix in the WT structure is indicated by an arrow. The F667 residue, located in the π-helix, and gate residue L677 are shown. b Top view of the pore lined by S6 in the PKD2 WT and F604P structures showing twisting and bending movements of distal S6 induced by the transition of π-helix to α-helix. c, d Averaged currents at +80 mV obtained under the same experimental conditions as in Fig. 3a in oocytes injected with cRNA of PKD2 WT, L677N/F676 (c) or F604P/F676 (d) double mutant. Currents were averaged from 10–13 oocytes. Ctrl, water-injected oocytes. Data are presented as mean ± SEM. Statistic significance was determined by Student’s t-test. ***P < 0.001. e Comparison between S6 in the closed (blue) and open (orange) states of PKD2, TRPV1, TRPML1 or TRPML3. The aromatic residue in the π-helix and hydrophobic gate residue were shown for each channel. PKD2 WT (PDB: 5T4D); TRPV1 apo (unliganded and closed state, PDB: 3J5P), Cap (capsaicin-bound state, PDB: 3J5R); TRPML1 apo (PDB: 5WJ5), ML-SA1 (agonist ML-SA1-bound state, PDB: 5WJ9); TRPML3 apo (PDB: 6AYE), ML-SA1 (6AYF). f Proposed mechanistic model for PKD2 activation induced by F604P mutation. The black arrows indicate proposed movements during the activation

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