Wang Z , Ng C , Liu X , Wang Y , Li B , Kashyap P , Chaudhry HA , Castro A , Kalontar EM , Ilyayev L , Walker R , Alexander RT , Qian F , Chen XZ , Yu Y .

P30DKO90868 HHS|NIH|National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), DK102092 HHS|NIH|National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), 230G18a PKD Foundation (PKDF), R01DK111611 HHS|NIH|National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), R01 DK111611 NIDDK NIH HHS , KFOC180027 Kidney Foundation of Canada (KFOC), DG RGPIN 401946 Gouvernement du Canada|Natural Sciences and Engineering Research Council of Canada (NSERC), DG RGPIN 05842 Gouvernement du Canada|Natural Sciences and Engineering Research Council of Canada (NSERC), P30 DK090868 NIDDK NIH HHS , R15 DK102092 NIDDK NIH HHS

	Graphical abstract
	Figure 1. PC1 and PC2 express in Xenopus oocytes but yield no channel current A. Transmembrane topology of PC1 and PC2 proteins. The two proteins associate at the C‐terminus through the coiled‐coil domains and the extracellular side via the TOP domains. The GAIN domain and the GPS site in PC1 and the EF‐hand motif in PC2 are indicated. The last six transmembrane domains of PC1 (shown in orange) share sequence similarity with PC2. B. Western blot of oocyte lysate (left) and biotinylation‐purified surface (right) samples showing the expression of PC1 and PC2 in Xenopus oocytes and enhanced surface trafficking of the PC1/PC2 complex compared to either protein expressed individually. Anti‐PC1 C‐terminus antibody 29 recognized both full‐length (asterisk) and GPS‐cleaved CTF (open circle) of PC1. A higher‐glycosylated 130 kDa PC2 (star) band was only seen when PC1 is coexpressed. C. Co‐IP followed by Western blot showing the association between PC1 and PC2 that were expressed in Xenopus oocytes. IP was done with an anti‐FLAG antibody. Bands of full‐length (asterisk) and GPS‐cleaved CTF (open circle) of PC1 are indicated. Both 120 and 130 kDa bands of PC2 were seen in the IPed product. D. Representative current–voltage relationship (I–V) curves (left) and a scatter plot and bar graph (right) showing coexpression of WT PC1 and PC2 produced no current in TEVC recording. The current of the GOF PC2_F604P is included as a control. Currents at +60 mV are shown in the bar graph. Each point represents the recorded current from one oocyte. Oocyte numbers for scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, ***P < 0.001, Student's t ‐test).
	Figure 2. PC2_L677A/N681A (AA) mutant is a new GOF channel A. Cryo‐EM structure of PC2. Left: Bottom view of PC2 homomeric tetramer showing the pore‐lining S5‐S6 from each subunit assembles into the channel pore. Right: Side view of S5‐S6 from two subunits showing the L677 and N681 (in red) contributes to the restriction at the lower gate. F604P (in cyan) on S5 and the three residues (in green) forming the selectivity filter are also indicated. The structure shown here is previously reported with Protein Data Bank (PDB) code 5T4D 20. A gray dashed line indicates the path of the ion flow. B. Representative I–V curves (left) and a scatter plot and bar graph (right) showing that the PC2_AA is a GOF mutant and gave rise to a larger current than PC2_F604P. Scatter plot and bar graph show the average current sizes at +60 mV. The cations included in the bath solution, 100 mM Na+ and 2 mM Ca2+ in this case, are indicated by the thick‐lined boxes here and in all the following figures. Oocyte numbers for bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t ‐test). C. Representative currents of PC2_F604P and PC2_AA mutants in the divalent ion‐free bath solution, which contains 100 mM Na+. D. Representative currents of indicated WT and mutants of PC2 in a bath solution containing 70 mM Ca2+. E. Calcium‐activated chloride channel (CaCC) blocker MONNA (10 μM) or niflumic acid (NFA) (1 mM) partially blocked the current recorded from the PC2_AA‐injected oocytes in the 70 mM Ca2+ bath solution.
	Figure 3. The PC1/PC2_AA complex channel has greater Ca2+ permeability than the homomeric PC2_AA channel A, B. Representative I–V curves (left) and scatter plot and bar graphs (right) showing the currents from oocytes expressing PC2_AA alone, PC1 with PC2_AA, and PC1 with WT PC2, in bath solutions containing 100 mM Na+ (A) and 100 mM Na+ and 2 mM Ca2+ (B). Scatter plot and bar graphs show currents at +80 mV and −80 mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, ***P < 0.001, Student's t ‐test). C. Representative I–V curves of PC1/PC2_AA recorded in a bath solution containing 100 mM Na+ and 2 mM Ca2+ solution in the absence or presence of 10 μM MONNA. D. Representative I–V curves (left) and scatter plot and bar graphs (right) showing the currents from oocytes expressing PC2_AA alone, PC1 with PC2_AA, and PC1 with WT PC2, in bath solutions containing 70 mM Ca2+. Scatter plot and bar graphs show currents at +80 mV and −70 mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t ‐test). E. Representative I–V curves of PC1/PC2_AA recorded in a bath solution containing 70 mM Ca2+ solution in the absence or presence of 10 μM MONNA. F. Representative currents of PC2_AA (left) and PC1/PC2_AA (right) in solutions containing the indicated Ca2+ concentrations. All solutions contain 20 μM of MONNA to block CaCC current. Corresponding concentrations of NMDG+ were added to compensate the osmolarity. See Fig EV2 for currents of the full voltage scale and the details of solutions used. G. Scatter plot and bar graph showing the higher radiolabeled 45Ca uptake rate of oocytes expressing the PC1/PC2_AA complex channel compared to that of oocytes expressing PC1 alone or PC2 alone. For oocytes injected with PC2 alone, five times more concentrated PC2 RNA was injected to increase its surface expression. The purple dashed line indicates the background 45Ca uptake set by the measurement with the water‐injected oocytes. Data were averaged from three independent experiments. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t ‐test). H. Surface biotinylation followed by Western blot showing the expression levels of the indicated proteins in lysate and plasma membrane of oocytes in 45Ca uptake experiments.
	Figure 4.Both the CTF and the TRP‐like domain (TLD) are sufficient for ion channel function of PC1 in the PC1/PC2 complex A. Transmembrane topology of PC1 and PC2 proteins, showing the NTF, CTF, and the TLD of PC1. The six transmembrane domains in TLD (indicated with the dashed line box) share sequence homology with the transmembrane domains of PC2. B–D. Representative I–V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full‐length PC1/PC2_AA with that of PC1‐CTF/PC2_AA in bath solutions containing 100 mM Na+ (B), 100 mM Na+ and 2 mM Ca2+ (C), or 70 mM Ca2+ (D). Currents at both +80 mV and −80 (or −70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, *P < 0.05; ***P < 0.001, Student's t ‐test). E. Scatter plot and bar graph showing that the current of PC1‐CTF/PC2_AA is completely abolished when WT PC2 was used, or after introducing R4090W mutation in the putative pore region of PC1‐CTF. Inserted are Western blot images showing the expression of the corresponding proteins. Top: anti‐PC1. Bottom: anti‐PC2. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t ‐test). F–H. Representative I–V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na+ and 2 mM Ca2+ (F), 100 mM Na+ (G), or 70 mM Ca2+ (H), showing PC1‐TLD/PC2_AA gave rise to current with similar properties as full‐length PC1/PC2_AA channel. I. Co‐IP followed by Western blot showing the association between PC2_AA and the indicated full‐length or fragment PC1. Bands of full‐length (asterisk), GPS‐cleaved CTF or expressed CTF fragment (open circle), and TLD (star) of PC1 are indicated. J. Surface biotinylation followed by Western blot showing the expression of the complexes formed between PC2_AA and full‐length PC1, PC1‐CTF, or PC1‐TLD at the oocyte surface. Besides the blots shown in the figure, surface samples were also blotted with an anti‐PC1 N‐terminus antibody, and the cleaved NTF fragment was found associated with the plasma membrane in the full‐length sample (shown in Appendix Fig S4). Bands of full‐length (asterisk), GPS‐cleaved CTF or expressed CTF fragment (open circle), and TLD (star) of PC1 are indicated.
	Figure 5.PC1‐CTF/PC2_AA heteromeric channel has a different ion permeability to that of the PC2_AA homomeric channel A. Representative I–V curves of the PC2_AA and PC1‐CTF/PC2_AA channels in divalent ion‐free bath solutions containing 100 mM of indicated ions, showing the differences in reversal potential. B. Bar graphs showing the markedly different reversal potentials of PC2_AA channel and the PC1‐CTF/PC2_AA channel in bath solutions containing 100 mM of the indicated ions. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, ***P < 0.001, Student's t ‐test). C, D. Bar graphs showing the permeability ratios of the indicated ions to that of Na+ (P x: P Na) (C) and NMDG+ (P x: P NMDG) (D). These ratios are strikingly different between the two channels. The ratios of the particular ions, which are the values of the bars, are indicted on top of the bars.
	Figure 6.Pore mutations in PC1 or PC2 led to changes in ion permeability of the PC1‐CTF/PC2_AA channel A. TOP: a bottom view of a previously reported cryo‐EM structure of PC1/PC2 channel (PDB code 6A70) 42, which is missing the NTF of PC1 and the intracellular C‐terminal tails from both proteins. S10‐S11 of one PC1 subunit and S5‐S6 from three PC2 subunits assembled to form the pore. Bottom: The mutated amino acids in this experiment (in purple) are mapped on S10‐S11 of one PC1 and S5‐S6 of one PC2 subunit in the complex. The dashed gray arrow indicates the putative ion flow path. Since human PC1 was used for structure determination, mouse amino acids mutated in this study are indicated in parentheses. Due to the low resolution of the PC1 pore region in the structure, side chains are not seen for the three mutated amino acids at the putative selectivity filter region. E4068 (E4078 in human) is not solved in the structure, and it is labeled to indicate its approximate location in the structure. B, C. Bar graphs showing the reversal potentials of PC1‐CTF/PC2_AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions. Two PC1 and two PC2 mutations caused dramatic changes in the reversal potential for almost all tested ions (B), indicating that these amino acids are essential for ion permeability. Another two PC1 mutations and one PC2 mutation only led to relatively small changes of reversal potential for some ions but not others (C), indicating a less important role of these amino acids in ion permeability. Statistical significance between reversal potentials of all pore mutants and that of the PC1‐CTF/PC2_AA channel is indicated. Representative I–V curves are shown in Fig EV5. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t ‐test). D, E. Bar graphs showing the permeability ratios of the indicated ions to that of Na+ (P x: P Na) (D) and NMDG+ (P x: P NMDG) (E). Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases.
	Figure 7.GPS cleavage of PC1 is not necessary for channel activity of the PC1/PC2_AA channel A. Transmembrane topology of PC1 and PC2 proteins, showing the cleavage at the N‐terminal GPS site (in circles), the “HLT” tripeptide where the cleavage happens, and the two mutations, L3040H and T3041V, that abolish GPS cleavage. B, C. Representative currents and scatter plot and bar graphs showing the currents of the full‐length WT and mutant PC1s associated with PC2_AA in bath solutions containing 100 mM Na+ and 2 mM Ca2+ (B) or 70 mM Ca2+ (C). In scatter plot and bar graphs, currents were normalized to the average current of PC1/PC2_AA recorded from the same batch of oocytes. Oocyte numbers in scatter plot and bar graphs are indicated in parentheses. Data are presented as mean ± SD in bar graphs (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t ‐test). D. Surface biotinylation followed by Western blot showing the surface expression of the PC1_T3041V/PC2_AA complex, but not the PC1_L3040H/PC2_AA complex, in Xenopus oocytes. PC2_AA is completely absent from the surface sample when PC1_L3040H was coexpressed, indicating that, in our experimental condition, almost all PC2_AA were in the complex with PC1_L3040H and trapped in the process of cell surface trafficking. Bands of full‐length (asterisk) and GPS‐cleaved CTF (open circle) of PC1 and 130 kDa PC2 (star) are indicated.
	Figure 8.Several key residues in the pore of the PC1/PC2 channel may play roles in ion conductance Structure of the PC1/PC2 complex showing the side view of S10‐S11 from the PC1 subunit and S5‐S6 from one PC2 subunit in the cryo‐EM structure of PC1/PC2 (PDB code 6A70) 42. Side chains of PC2‐L677A and N681 are shown in red and N674 in cyan. Side chains of amino acids on S11 of PC1 at this region were shown in purple for the three positively charged amino acids and in green for others. In PC1/PC2 complex, no bulky side chain from PC1 lines up with PC2‐L677A and N681 to form the restriction at this position, while R4090 of PC1 and N674 of PC2 narrow down the channel pore in the middle.
	Figure EV1. Coexpression of PC1 greatly inhibited the current of PC2_F604PLeft: representative current–voltage (I–V) curves of the indicated protein combinations. Right: Scatter plot and bar graph show the average currents at +60 mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (*P < 0.05, ***P < 0.001, Student's t‐test).Source data are available online for this figure.
	Figure EV2. Representative I–V curves of PC2_AA (left) and PC1/PC2_AA (right) in solutions with varying Ca2+ concentrationComponents of these solutions are shown in the table. 20 μM of MONNA was included in all solutions, and the osmolarity was compensated by adding corresponding concentrations of NMDG+, which is not permeable through these channels.
	Figure EV3. Expressing full‐length PC1, PC1‐CTF, or PC1‐TLD without PC2_AA in Xenopus oocytes did not give rise to significant currentScatter plot and bar graph show the average current sizes of oocytes expressing indicated full‐length or fragment PC1 proteins in a solution containing 100 mM Na+ and 2 mM Ca2+. Oocytes expressing both PC1‐CTF and PC2_AA were used as a positive control. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, ***P < 0.001, Student's t‐test).Source data are available online for this figure.
	Figure EV4. The different effects of three channel blockers on the currents of the PC2_AA and the PC1‐CTF/PC2_AA channels A–C.
	Figure EV5. Pore mutations change the ion permeability of the PC1/PC2_AA channelRepresentative I–V curves show the shifts of reversal potential caused by indicated mutations in bath solutions containing 100 mM of indicated ions. Compared to group 2 mutations, group 1 mutations cause more dramatic reversal potential shifts.

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