The heteromeric PC-1/PC-2 polycystin complex is activated by the PC-1 N-terminus

Mutations in the polycystin proteins, PC-1 and PC-2, result in autosomal dominant polycystic kidney disease (ADPKD) and ultimately renal failure. PC-1 and PC-2 enrich on primary cilia, where they are thought to form a heteromeric ion channel complex. However, a functional understanding of the putative PC-1/PC-2 polycystin complex is lacking due to technical hurdles in reliably measuring its activity. Here we successfully reconstitute the PC-1/PC-2 complex in the plasma membrane of mammalian cells and show that it functions as an outwardly rectifying channel. Using both reconstituted and ciliary polycystin channels, we further show that a soluble fragment generated from the N-terminal extracellular domain of PC-1 functions as an intrinsic agonist that is necessary and sufficient for channel activation. We thus propose that autoproteolytic cleavage of the N-terminus of PC-1, a hotspot for ADPKD mutations, produces a soluble ligand in vivo. These findings establish a mechanistic framework for understanding the role of PC-1/PC-2 heteromers in ADPKD and suggest new therapeutic strategies that would expand upon the limited symptomatic treatments currently available for this progressive, terminal disease.


Introduction
The most common monogenetic disease in humans is ADPKD; a major nephropathy characterized by renal cysts that leads to urinary tract infection, hypertension, aneurysm, and ultimately end-stage renal disease (ESRD) (Kathem et al., 2014;Harris and Torres, 2009). Mutations in one of two ciliary proteins, PC-1 (encoded by PKD1) and PC-2 (also known as TRPP1 and encoded by PKD2), account for 85% and 15% of ADPKD-causing mutations, respectively Cornec-Le Gall et al., 2014).
PC-1 is an 11 transmembrane (TM) protein of 4303 amino acids with a long extracellular N-terminus that contains multiple cell adhesion domains (Yoder et al., 2002;Hughes et al., 1995). PC-1 and its family members (PC-1L1, PC-1L2, and PC-1L3) share the same 11-TM topology but vary in the size and adhesion domain composition of their N-termini (Ishimaru et al., 2006). A unique feature of PC-1 family members, shared only by adhesion G protein-coupled receptors (GPCRs), is the GPCR autoproteolysis inducing (GAIN) domain. This domain catalyzes peptide bond hydrolysis at a GPCR proteolysis site (GPS) that is positioned proximal to the first TM helix (TM1) (Araç et al., 2012;Prö mel et al., 2013;Trudel et al., 2016). Autoproteolysis generates two fragments: an N-terminal fragment (NTF) and a multi-TM core (Qian et al., 2002). The NTF contains multiple domains, including leucine-rich repeats, a C-type lectin (CTL) domain, immunoglobulin (Ig)-like polycystic kidney disease (PKD) repeat domains, a single low-density lipoprotein (LDL) receptor motif, and the receptor for egg jelly (REJ) domain (Hughes et al., 1995;Babich et al., 2004;Moy et al., 1996).
The function of the multi-TM core remains poorly defined. Autoproteolysis occurs early in the ER secretory pathway such that the N-terminal fragment remains non-covalently tethered (Lin et al., 2004;Wei et al., 2007). While mutations within the GPS site of PC-1 result in renal cyst formation, the full functional consequences of autoproteolysis are poorly understood (Qian et al., 2002;Yu et al., 2007).
Attempts to exogenously express and record from PC-1 and/or PC-2 in whole cell recordings of mammalian cells (Hanaoka et al., 2000;Delmas et al., 2004;Cai et al., 2004) and reconstituted lipid bilayers González-Perrett et al., 2001 have yielded divergent results, likely due to subcellular enrichment of PC-1 and PC-2 (for a detailed discussion see Liu et al., 2018;Kleene and Kleene, 2017). In addition, despite recent advances in measuring ion channel activity in small subcellular organelles such as the cilium (Kleene and Kleene, 2012;DeCaen et al., 2013;Dong et al., 2008), it remains technically challenging to study ion channel activity in primary cilia due to their small size (10 À15 L, approximately the size of a bacterium). This technical hurdle has hindered investigations of the physiological regulation of polycystins and how they lead to renal disease when mutated. The unknown role of PC-1 within the channel complex, in particular, has hindered progress in the field. Some studies suggest that PC-1 acts as a dominant-negative subunit of the heteromeric channel (Su et al., 2018), whereas others suggest that it is a pore-forming subunit that fine-tunes ion selectivity . Moreover, PC-1 has been shown to be dispensable for polycystin currents in ciliary membranes . Here we develop cellular assays that allow us to probe the eLife digest On the surface of most animal and other eukaryotic cells are small rod-like protrusions known as primary cilia. Each cilium is encased by a specialized membrane which is enriched in protein complexes that help the cell sense its local environment. Some of these complexes help transport ions in out of the cell, while others act as receptors that receive chemical signals called ligands. A unique ion channel known as the polycystin complex is able to perform both of these roles as it contains a receptor called PC-1 in addition to an ion channel called PC-2.
Various mutations in the genes that code for PC-1 and PC-2 can result in autosomal dominant polycystic kidney disease (ADPKD), which is the most common monogenetic disease in humans. However, due to the small size of primary cilia -which are less than a thousandth of a millimeter thick -little is known about how polycystin complexes are regulated and how mutations lead to ADPKD. To overcome this barrier, Ha et al. modified kidney cells grown in the lab so that PC-1 and PC-2 form a working channel in the plasma membrane which surrounds the entire cell. As the body of a cell is around 10,000 times bigger than the cilium, this allowed the movement of ions across the polycystin complex to be studied using conventional techniques.
Experiments using this newly developed assay revealed that a region at one of the ends of the PC-1 protein, named the C-type lectin domain, is essential for stimulating polycystin complexes. Ha et al. found that this domain of PC-1 is able to cut itself from the protein complex. Further experiments showed that when fragments of PC-1, which contain the C-type lectin domain, are no longer bound to the membrane, they can activate the polycystin channels in cilia as well as the plasma membrane. This suggests that this region of PC-1 may also act as a secreted ligand that can activate other polycystin channels.
Some of the genetic mutations that cause ADPKD likely disrupt the activity of the polycystin complex and reduce its ability to transport ions across the cilia membrane. Therefore, the cell assay created in this study could be used to screen for small molecules that can restore the activity of these ion channels in patients with ADPKD. function of polycystin complexes in the plasma membrane and compare them to polycystin channels in their native primary cilia. Our heterologous system recapitulates the biophysical properties of cilia-localized polycystin channels and demonstrates how distinct PC-1 family members contribute to ion permeation. Importantly, we reveal that the N-terminal domain of PC-1 functions as a soluble ligand and is an indispensable activator of the polycystin complex, providing further evidence for chemosensory regulation of the polycystin complex (Delling et al., 2016).

Results
PC-1 and PC-2 form functional channels in the plasma membrane Because robust assays to investigate the heteromeric polycystin complex are lacking, we developed an assay system to determine the contribution of PC-1 family members to polycystin function. We were able to target PC-1 to the plasma membrane of HEK293 cells ( Figure 1B) and mIMCD3 cells (Figure 1-figure supplement 1E) by replacing its endogenous signal peptide with a strong Ig kchain secretion sequence followed by an HA tag (sPC-1; Figure 1A, Table 1; Salehi-Najafabadi et al., 2017;Zhu et al., 2011). The HA tag allowed us to quantify sPC-1 plasma membrane expression by measuring surface staining following addition of anti-HA antibodies to non-permeabilized cells. Co-expression of sPC-1 and PC-2 resulted in strong HA surface staining, whereas expression of sPC-1 alone resulted in only marginal surface staining, in both HEK293 cells ( Figure 1B and C) and IMCD3 cells (Figure 1-figure supplement 1D and E). Co-expression of PC-2 and PC-1 with an extracellular HA tag but without the endogenous leader sequence ( HA PC-1) failed to generate comparable HA surface staining, supporting the notion that an Ig k-secretion sequence promotes membrane localization of the complex ( Figure 1B). To test whether PC-2 facilitates sPC-1 membrane localization by promoting ER exit or by co-migration to the plasma membrane, we inserted a FLAG epitope into the extracellular tetragonal opening for polycystins (TOP) domain of PC-2 (PC-2 FLAG ) (Figure 1-figure supplement 1A, Table 1). Robust surface HA and FLAG staining only occurred when both sPC-1 and PC-2 FLAG were co-expressed, suggesting that they are shuttled simultaneously to the plasma membrane (Figure 1-figure supplement 1B and C). Similarly, coexpression of sPC-1 with PC-2 containing a recently in an oocyte expression system characterized gain of function mutation of PC-2 (PC-2 F604P , Table 1; Arif Pavel et al., 2016) resulted in comparable HA surface expression ( Figure 1B and C).
Collectively, these results demonstrate that PC-1/PC-2 subunits form a functional channel when reconstituted in mammalian cells and support the notion that PC-1 subunits form part of the core channel complex.

Polycystin activation depends on CTL and TOP domains
To gain a more detailed understanding of the role played by the N-terminus in polycystin complex activation, we replaced smaller regions of the PC-1L3 N-terminus with those from PC-1. One chimera replaced the 108 AA CTL domain of PC-1L3 with the CTL of PC-1 (sPC-1L3 1CTL ; Figure 2A, Table 1). sPC-1L3 1CTL /PC-2 F604P generated robust outward currents (87.5 ± 9.0 pA/pF, n = 5) ( Figure 2C and G) whereas the inverse chimera (sPC-1 1L3CTL ) produced only minimal currents (9.1 ± 1.9 pA/pF, n = 10). These data strongly suggest that the PC-1 CTL domain is essential to activate the polycystin complex.
In mammalian cells, the PC-1 ectodomain undergoes at least one proteolytic cleavage event at the GPS site, suggesting that the N-terminus might be liberated as a secreted ligand. Indeed, it has been reported that PC-1 N-terminus-containing exosomes interact with primary cilia in kidney nephrons (Hogan et al., 2009). We therefore investigated whether fragments of the PC-1 N-terminus can regulate the polycystin complex. We purified various PC-1 NTFs from HEK cell supernatant ( In both cases, the currents were smaller than those for sPC-1/PC-2 F604P ( Figure 1F). Boiling NTF 263-535 at 95˚C for 10 min rendered it inactive ( Figure 3B). Next, we perfused HEK cells overexpressing PC-1 DNT /PC-2 F604P heteromers with NTF 263-535 and measured acute changes in current density. While PC-1 DNT / PC-2 F604P channels remained inactive in the absence of NTF 263-535 , acute application increased current density by 765.1 ± 185.05% (from 5.9 ± 1.3 pA/pF to 59.1 ± 19.3 pA/pF, n = 7) within 10 s of application (Figures 3D, F and G). However, non-transfected cells did not respond to NTF 263-535 under comparable condition (4.6 ± 1.6 pA/pF to 6.8 ± 1.4 pA/pF, n = 9) ( Figure 3E and G). We then asked whether the removal of PC-1 N-terminus inactivates the polycystin heteromer. To test this possibility, we perfused sPC-1/PC-2 F604P overexpressing HEK cells with 0.125% trypsin, assuming that proteolytic digestion of extracellular proteins also removes critical regions within the PC-1 N-terminus. As shown in Figure 3H perfusion with 0.125% trypsin decreased sPC-1/PC-2 F604P Currents are recorded at +180 mV (black) and À100 mV (red). Orange bar indicates addition of trypsin. (J) I-V relationships for sPC-1/PC-2 F604P before (black) and after (red) 0.125% trypsin application. (K) Current densities at +100 mV potential from for sPC-1/PC-2 F604P (n = 6) and untransfected cells (n = 5) before (blank) or after (red) 0.125% trypsin application. dependent currents by 78.4 ± 4.9% (from 52.0 ± 17.1 pA/pF to 8.9 ± 1.4 pA/pF, n = 6, Figure 3H and J, and K). By contrast, untransfected cells did not respond to 0.125% trypsin (7.2 ± 1.9 pA/pF versus 4.6 ± 0.8 pA/pF, n = 5). Taken together, these data suggest that the N-terminus including the CTL of PC-1 is necessary and sufficient to activate the polycystin complex. Next, we asked how the extracellular CTL domain in PC-1 might regulate activation of the entire channel complex. We reasoned that the TOP domain in PC-2 is well positioned to function as a surface receptor as it forms a bridge between the pore domain and voltage sensor-like domain, and is critical for channel activation (Shen et al., 2016;Vien et al., 2020). In addition, different glycosylation patterns of the TOP domain might underlie conformational changes in the homomeric PC-2 complex (Wilkes et al., 2017). We observed prominent glycosylation of the TOP domain in PC-2 at position N375, N362, and N328 ( Figure 4A). Moreover, although mutation of N375 to glutamine did not affect surface localization of sPC-1/PC-2 F604P N375Q ( Figure 4B and C), it rendered the channel inactive ( Figure 4D and E). Thus, glycosylation of the TOP domain in PC-2 appears to be required for CTL-dependent polycystin activation.

N-terminal PC-1 fragments activate ciliary polycystin complexes
Although the results so far strongly suggest that the PC-1 CTL domain is critical for activating heterologous polycystin complexes, native polycystin complexes reside in the specialized membranes of primary cilia Kleene and Kleene, 2017;Vien et al., 2020). We, therefore, investigated whether the PC-1 N-terminus is able to activate polycystin complexes in the cilia of polarized mouse epithelial (mIMCD-3) cells using excised ciliary membrane patch clamp recordings ( Figure 5figure supplement 1B). Without any NTFs, we readily detected single channel openings at potentials more positive than +60 mV and more negative than À80 mV ( Figure 5A) in 7 out of 38 cilia. After ablating PC-1 expression in mIMCD3 cells using CRISPR/CAS9 ( Figure 5-figure supplement 1A) ciliary channels remained readily detectable in 4 of 13 cilia ( Figure 5B), in agreement with previous reports . In addition, single channel conductance was comparable between wt mIMCD3 (g = 79.5 ± 0.1 pS) and PC-1 knockout cilia (g = 82.3 ± 0.0 pS) ( Figure 5-figure supplements 1G, H, 2A and B). However, we noted that the open probability of channels at negative membrane potentials in PC-1 knock out primary cilia (0.58 ± 0.01) was higher than in wt IMCD3 cilia (0.08 ± 0.01) ( Figure 5G). These data suggest that PC-2 forms a homomeric complex in the absence of PC-1 subunits with a higher open probability than that of PC-1/PC-2 heteromers. The results also support the idea that PC-1 subunits impede cation flux across the membrane, in agreement with our PC-1 pore mutant data ( Figure 1I-K). Ablation of PC-2 expression using CRISPR/CAS9 rendered primary cilia electrically silent (n = 11), confirming that PC-2 subunits are an essential component of polycystin channels ( Figure 5C; Liu et al., 2018;Kleene and Kleene, 2017).
To test whether NTFs can activate the native polycystin complex, we recorded single channels in inside out patches pulled from primary cilia in the presence of 0.7 mg/mL (equal to~50 nM final concentration) of NTF 263-535 in the recording pipette. Although the percentage of electrically active cilia (n = 4/13) remained largely unaffected, NTF 263-535 increased the probability of single channels openings by~five-fold and significantly prolonged the duration of channel openings at negative membrane potentials ( Figure 5E-G).
Collectively, these findings demonstrate that PC-2 subunits are essential for functional polycystin channels in primary cilia, that PC-1 subunits also participate in the native heteromeric complex, and that NTF 263-535 can activate this complex under physiological conditions.

NTF 263-535 activates Ca 2+ influx into primary cilia
Having established that NTF 263-535 can activate ciliary polycystin channels, we asked whether it could also trigger Ca 2+ entry through the channel. We initially attempted single channel recordings from mIMCD-3 wt cilia under conditions in which Ca 2+ is the sole charge carrier (see Materials and methods). We were unable to measure currents, even at very negative membrane potentials (À140 mV) ( Figure 6A), in agreement with previously published results . Surprisingly, however, the application of NTF 263-535 revealed channel openings close to physiological membrane potentials ( Figure 6B). Single channel conductance was smaller when using Ca 2+ instead of Na + and K + as the charge carrier (47.5 pS, Figure 6C). Voltage-dependent open probability and open time remained unchanged ( Figure 6D and E).

Discussion
We have demonstrated that PC-1 and related family members form a functional ion channel with PC-2. These heteromeric complexes exhibit distinct biophysical properties, suggesting that PC-1 members are critical for adjusting the electrical excitability of primary cilia in response to the local environment. By expressing membrane-targeted PC-1 or PC-1L3 with PC-2 in mammalian cells, we show that the PC-1 N-terminus is essential for the activation of complexes containing PC-2 subunits, including those carrying the gain of function mutation F604P. More specifically, we show that the CTL domain within the N-terminus of PC-1 is essential for channel activation and that it might function by interacting with the TOP domain of PC-2. In cilia, we provide evidence that an N-terminal fragment containing the CTL domain is sufficient to activate endogenous Ca 2+ -permeable polycystin channels and elevate [Ca 2+ ] cilium . Thus, we suggest a novel autoregulation mechanism in which the N-terminus of PC-1 activates the channel it is attached to or channels on adjacent cilia following cleavage to become a soluble ligand.

The PC-1 ectodomain is critical for polycystin complex activation
Our data identify PC-1's CTL domain as an essential component for polycystin activation. Interestingly, the CTL domain is a hotspot for ADPKD-causing mutations (Dong et al., 2019), supporting the critical importance of this domain (Figure 2-figure supplement 1B). Although C-type lectins are the most diverse family of mammalian carbohydrate-binding proteins (Keller and Rademacher, 2020), little is known about the function of the CTL motif within PC-1 (Sandford et al., 1997), but it has been reported to bind to carbohydrates in a calcium-dependent manner (Weston et al., 2001). All PC-1 family members except PC-1L1 contain an N-terminal CTL, yet PC-1L3 CTL fails to activate the complex. Because the CTL amino acid sequence diverges between different PC-1s, we speculate that each binds to different carbohydrates. It is worth noting that other domains within the N-terminus are also implied in cell-cell recognition, including leucine-rich repeats (LRRs), cell wall integrity and stress response component (WSC), and REJ domain. Future studies addressing the specificity and mechanism of CTL binding, especially to carbohydrate moieties, will be of interest.
Both PC-1 and PC-2 have a large extracellular TOP domain, which contains prominent glycosylation sites. We speculate that the CTL domain in the native PC-1/PC-2 complex interacts with glycans in the TOP domain, which allosterically regulates the ion permeation pathway and/or gating apparatus of the heteromeric polycystin channel (Vien et al., 2020; Figure 7). In particular, the N-glycan attached to N375 of PC-2 is adjacent to a structure that extends above TM3 and TM4 of the voltage sensor-like domain, so it is conceivable that binding of the CTL domain to this glycan will initiate conformational changes in the voltage sensor-like domain, which could be transmitted to the pore to regulate ion permeation. Indeed, ablation of this glycan by mutation (N375Q) results in a complex that can traffic to the plasma membrane but that remains inactive.

Plasma membrane-targeted polycystins recapitulate biophysical features of ciliary currents
Signal peptides are cleaved from the nascent polypeptide early in the biosynthetic pathway, thus sPC-1, which is ectopically targeted to the plasma membrane, closely resembles wt PC-1 (Salehi-Najafabadi et al., 2017). Likewise, the effective membrane targeting sequence found in the N-terminus of P2Y12 has been successfully used to target adhesion GPCRs to the plasma membrane without compromising functionality (Liebscher et al., 2015). The PC-2 F604P mutant has recently been characterized as a gain of function mutant in a Xenopus oocyte overexpression model (Arif Pavel et al., 2016). It is thought that the F604P substitution within S5 induces conformational changes in the S4-S5 linker (Zheng et al., 2018) that lock PC-2 in an open state. Indeed, proline substitutions in the S5 segment of several other TRP family members result in constitutive channel activity (Grimm et al., 2007;Xu et al., 2007;Zhou et al., 2017). A careful comparison of our ciliary recordings of endogenous polycystin channels with membrane-targeted polycystins reveals that both channel populations exhibit comparable biophysical characteristics, lending weight to the effectiveness of our plasma membrane expression system for studying polycystins.  Our results are in agreement with previous reports of channel activity in the ciliary membrane of renal epithelial cells Kleene and Kleene, 2017;DeCaen et al., 2013;Flannery et al., 2015). Because no measurable electrical activity remains in mIMCD-3 cells following CRISPR ablation of PC-2, we confirm that the PC-2 subunit is an indispensable component of ciliary currents in kidney epithelial cells Kleene and Kleene, 2017). We also find that primary cilia of mIMCD-3 cells still contain functional channels after CRISPR ablation of PC-1. We speculate that currents recorded from PC-1 knockout cilia are due to homotetrameric PC-2 channels, although we cannot exclude a contribution from other PC-2-containing heteromeric channels such as PC-1L1/PC-2, PC-1L3/PC-2, or PC-2/TRPM3 (Field et al., 2011;DeCaen et al., 2013;Flannery et al., 2015;Bai et al., 2008;Kleene et al., 2019;Zhang et al., 2013). We noted two striking differences between channels in wt and PC-1 knockout mIMCD-3 cilia at voltages close to the resting membrane potential . First, the native PC-1/PC-2 heteromeric channel in wt cilia showed very short channel openings compared to the presumably homotetrameric PC-2 channel, supporting the hypothesis raised by cryo-EM data that PC-1 reduces ion flux (Su et al., 2018). Second, wt IMCD-3 cilia were electrically silent between membrane potentials of À60 mV and +40 mV whereas channels in PC-1 knockout cilia opened at À40 mV.

The PC-1 N-terminus likely underlies the molecular mechanism of chemosensation
In our experiments, soluble fragments containing the CTL motif not only restore channel function in polycystin complexes lacking the PC-1 N-terminus but also lengthen the open time duration of endogenous polycystins at negative membrane potentials. This strongly suggests that the PC-1 N-terminus is an agonist for polycystin activation. Our results provide the first insights into why ADPKD-causing mutations are enriched within both CTL and GAIN domains, the latter of which is critical for proteolytic processing and subsequent shedding of the CTL-containing ectodomain. Our results show that soluble fragments of PC-1 potentiate endogenous polycystin channels that already contain one PC-1 N-terminus. Given the predicted three PC-2:1 PC-1 stoichiometry of the heteromeric polycystin complex, we hypothesize that possibly all three PC-2 TOP domains need to be simultaneously engaged by CTL domains in order to trigger a concerted conformational change in their respective voltage sensor-like domains and therefore full channel activation. Alternatively, the tethered PC-1 N-terminus in a native heteromeric polycystin complex may be sterically constrained to participate in intramolecular interactions with its core complex, and thus might engage in intermolecular interactions, either between polycystin complexes on one cilium or as a cilia-cilia proximity sensor.
Alternatively, as-yet-unknown proteins and/or small molecule ligands can bind to modules (such as LRRs) that are in proximity to or in the CTL domain itself. This might mask the carbohydrate-binding site on the CTL domain and thus add another layer of regulation to the polycystin complex. While our results show that CTL is required for channel activation, we cannot exclude the possibility that other more potent agonists of the polycystin complex exist. Future work will determine how the PC-1 N-terminus functions as a polycystin agonist in vivo, either as a soluble ligand or bound to vesicles such as exosomes. We speculate that transport of such ligands by fluids may underline the molecular chemosensory mechanism of primary cilia.
Our data reveal that activation of endogenous polycystins by N-terminal fragments of PC-1 increases ciliary [Ca 2+ ] in~30% of cilia imaged. This is in agreement with previously published work in which only a fraction of cilia shows electrically active polycystin channels. It remains to be Figure 6 continued probabilities obtained at membrane potentials between À60 mV and À120 mV (n = 3/16). (E) Open time histogram of channel opening events after NTF 263-535 application at À100 and 120 mV. Small bar graph indicates time constants obtained from one decay fits of the histogram (n = 3/16). (F) Representative TIRF images after addition of 50 nM NTF 263-535 to mIMCD-3 cells stably expressing Arl13B-scarlet-GCaMP6. GCaMP images are pseudo-colored based on pixel intensity. Scale bar: 5 mm. (G) Representative traces of changes in fluorescence (DF/F) during TIRFM imaging. Image acquisition every 250 ms. (H) Quantification of maximum peak area of fluorescence (DF/F*s) for control, PC-2 knock out cells + NTF 263-535 , wt mIMCD3 cilia + boiled NTF 263-535 , and wt mIMCD3 cilia + NTF 263-535 . (I) Quantification of number of calcium spikes observed in TIRFM imaging for control, PC-2 knock out cells + NTF 263-535 , wt mIMCD3 cilia + boiled NTF 263-535 , and wt mIMCD3 cilia + NTF 263-535 . Quantification is from three independent experiments, **p<0.01. Figure 7. Graphical abstract PC-1 N-terminus with its C-type lectin domain interacts with carbohydrates in the PC-2 TOP domain and activates the channel complex in the cilium. PC-1 N-terminus may either activate the channel as a soluble ligand or remain tethered to the channel complex and undergo intermolecular binding to TOP domains on neighboring channels. Channel activity can be different in the presence of N-terminal fragments of PC-1. determined whether and how agonistic ciliary second messengers, which may only be stochastically present during ciliary recordings, add another layer of regulation to ciliary polycystin channels. Alternatively, there may be an as-yet-unidentified inhibitory signal in cilia that prevents activation of the polycystin complex in some ciliary recordings. Molecular biology hArl13B-EGFP was described previously. Arl13b-scarlet-GCaMP6s is an updated version of our previously characterized ciliary Ca 2+ sensor Arl13b-mCherry-GECO1.2 in which mcherry and GECO1.2 have been replaced with mScarlet and GCaMP6s, respectively. k IgG HA-PC-1L3 (sPC-1L3) has been described previously (Salehi-Najafabadi et al., 2017). sPC-1 was generated by using a unique BglII site within hPKD1 to replace the endogenous signal peptide with the 12 aa k IgG leader sequence followed by HA tag using PCR amplification and Gibson assembly (NEB). HA PC-1 was generated by inserting an HA tag between the predicted endogenous signal peptide and mature polypeptide chain following the same protocol. For the construction of FLAG-PC-2, several positions within the extracellular domains of PC-2 with negatively charged amino acids were tested. We identified one region at position 372 that tolerated the insertion of the FLAG sequence, based on surface trafficking. Chimera constructs of PC-1 and PC-1L3 were generated by substituting the ectodomain at the GPS cleavage site with the corresponding orthologue using PCR and Gibson assembly. All PC-1 and PC-2 combinations were cloned into pTRE3G-Bi vector (Takara Bio). The MCSI site was used for PC-1 variants while the MCSII site was used for PC-2 variants. This ensured simultaneous translation of PC-1 and PC-2 from the same plasmid. All DNA sequences were confirmed by sequencing (ELIM Bio).

Immunocytochemistry and confocal microscopy
Cells were fixed with 4% formaldehyde, permeabilized with 0.2% Triton X-100, and blocked by 2% FBS, 2% BSA, and 0.2% fish gelatin in PBS. Cells were labeled with the indicated antibody and secondary goat anti-rabbit, anti-rat or anti-mouse fluorescently labeled IgG (Thermo Fisher) and Hoechst 33342 (Thermo Fisher). Confocal images were obtained using a Nikon spinning disk with a 63Â oil immersion, 1.2 N.A. objective, or a 100Â oil immersion, 1.4 N.A. objective at the UCSF Nikon Imaging Core. Images were further processed using ImageJ (NIH).
Cell culture and quantification of plasma membrane PC-1 and PC-2 Tet inducible mIMCD3 and HEK293 cells were generated from parental lines (obtained from ATCC) according to the manufacture's protocol (Takara Bio). HEK or IMCD3 cells expressing the tet activator were transiently co-transfected with the indicated pTRE3G-Bi vector (Takara Bio) and the EGFP vector using Lipofectamine LTX for mIMCD3 or Lipofectamine2000 for HEK293 (Thermo Fisher). Surface trafficking of PC-1 and PC-2 was measured by quantifying the amount of anti-HA or anti-FLAG antibody bound to the plasma membrane, respectively. 24 hr after transfection 1 ug/mL doxycycline was added and cells were incubated for an additional 24 hr. For surface staining, adherent live cells were incubated with opti-MEM containing either anti-HA antibody (1:100) or anti-FLAG antibody (1:100) for 20 min at room temperature to avoid internalization of antibodies. Cells were washed twice with opti-MEM and fixed with 4% PFA. After incubation with blocking buffer, cells were labeled with secondary goat anti-rat or goat anti-mouse antibodies conjugated to Alexa 647. Several Z stacks of indicated groups were acquired using identical settings and 647 fluorescence was quantified on all GFP positive cells using ImageJ. This approach allowed an unbiased quantification of membrane inserted HA. In addition, expression of the proteins was confirmed in total staining using permeabilized cells. mIMCD3 cells with ablated PC-2 expression were described previously (Kleene and Kleene, 2017). Cells were tested for mycoplasma contamination regularly (Lonza). Data are representative of at least three independent experiments.

Electrophysiology
Recordings were performed using a multiclamp 200B (Axon Instruments), digitized using a digidata 1324A (Axon Instruments) and recorded using pClamp software (Axon Instruments). Whole cell configuration patch clamp data were filtered at 1 kHz and sampled at 10 kHz. Unless stated otherwise, the voltage step pulse was applied from À100 mV to 180 mV in 20 mV increments during 150 ms and holding potential was given at À60 mV. For the voltage ramp pulse, the same range of voltage steps was applied with 500 ms duration. The resistances of pipettes for whole cell and ciliary patch clamp were 6-8 MW and 18-24 MW, respectively. The tip of the pipette was further polished using Narishige MF-830 microforge equipped with a 100Â Nikon objective. For patch clamp experiment, the extracellular solution consisted of (mM): 145 Na-gluconate, 5 KCl, 2 CaCl 2 , 5 MgCl 2 , 10 HEPES, and adjusted to pH 7.4 using NaOH. The intracellular solution was used as follows: 90 NaMES, 10 NaCl, 2 MgCl 2 , 10 HEPES, 5 EGTA, 100 nM free calcium adjusted by CaCl 2 , and adjusted to pH 7.4 using NaOH. The free calcium was calculated using CaBuf software (G.Droogmans, Leuven, Belgium). For calcium permeability test, the extracellular solution consisted of (mM): 2 mM CaCl 2 and 10 HEPES adjusted to pH 7.4 using Trizma base and adjusted to 295mOsm using mannitol. The intracellular solution consisted of (mM): 10 HEPES and 1 EGTA adjusted to pH 7.4 Trizma base.

Statistical and data analysis for electrophysiology
Ciliary excised patch clamp data and whole cell configuration patch clamp were analyzed with Clampfit10.6 (Axon Instruments/Molecular Devices), Origin8 (Originlab), and Prism10.0 (Graphpad). Data are shown as mean ± SEM, and n represents independent experiments for the number of tested cells in electrophysiology. For relative open probability of sPC-1-PC-2 F604P , the data obtained at À80 mV tail pulse were fitted to a Boltzmann distribution using Origin8 (Originlab). P o (V) = P -100 +(P +180 ÀP -100 )/(1+exp[(V 1/2 ÀV)/k]) where P -100 and P +180 are the open probabilities of the channel at the most negative potential (À100 mV) and the most positive potential (+180 mV), respectively. V indicates the membrane potential, V 1/2 is the half-maximal activation potential, and k is the slope factor. For single channel open probability, P open was calculated by: where the total time that the channel presented in the open state and T is the total observation time. If a patch contains more than one of the same type of channel, Popen was computed by: where, N indicates the number of channels in the patch. We used the following equation to populate data.
To ¼ X Lto; where, L indicates the level of the channel opening. The absolute probability of the channel being open NPo is computed by: where, Tc indicates the total closed time Ca 2+ -imaging in primary cilia IMCD3 cells expressing Arl13b-mScarlet-GCaMP6 in primary cilia were imaged as described previously (Ishikawa and Marshall, 2015). In brief, cells were grown in the bottom side of 24 mm transwell insert with 8 um mesh size allowing rapid exchange of fluids across the membrane. Cilia were observed under an inverted Nikon microscope equipped with an TIRF imaging setup. Images were acquired 1 min after applying 1 mg/mL PC-1 NTF into the transwell. This assay allowed fast imaging along the entire length of the cilium. In most cases, cilia were localized by 561 nm (mScarlet) excitation and imaged in 488 nm (GCaMP6) and using the full CCD chip (200 ms exposure; five fps). Fluorescence was quantified and processed using ImageJ and Python.

Expression of N-terminal fragments of PC-1
Constructs were designed to produce various PC-1 N-terminal fragments as secreted proteins in HEK293S GnT1 -/cells using the BacMam expression system. In brief, the endogenous signal sequence of PC-1 was replaced with the strong IgG kappa leader peptide followed by either a M1-FLAG epitope or a maltose-binding protein fusion that allows for affinity purification using anti-M1 Flag antibody or amylose resin, respectively. HEK293S GnT1-/-cells were transduced with baculoviruses when cell density reaches 1 -2 Â 10 6 cells/mL. Sodium butyrate was added to at a final concentration of 5 mM to enhance protein expression 8-12 hr post-transduction. Temperature was reduced to 30˚C and the supernatant was harvested 5 days post-transduction. Various PC-1 N-terminal fragments were purified by affinity chromatography whereby secreted PC-1 fragments were captured by passing supernatant through affinity resins using gravity. The purified PC-1 fragments were then exchanged to a buffer containing 20 mM HEPES, 150 mM NaCl, pH 7.4 via extensive dialysis at 4C. PC-1 N-terminal fragments were then flash-frozen in liquid nitrogen and stored as aliquots at À80˚C until use.

Data analysis
Group data are presented as mean ± SEM. Statistical comparisons were made using unpaired student t-tests for electrophysiology and quantification of surface expression. The Mann-Whitney U-test was used for comparing peak area of Ca 2+ signals and Fisher's exact test was used for comparing # of Ca 2+ peaks (Prism). Statistical significance is denoted with an asterisk (*p<0.05; **p<0.01).