PKD2 is an essential ion channel subunit in the primary cilium of the renal collecting duct epithelium

Mutations in either Pkd1 or Pkd2 result in Autosomal Dominant Polycystic Kidney Disease (ADPKD). Although PKD2 is proposed to be an ion channel subunit, recordings of PKD2 ion channels conflict in their properties. Using a new ADPKD mouse model, we observe primary cilia are abnormally long in cells associated with cysts. Using primary cultures of collecting duct epithelial cells, we show that PKD2, but not PKD1, is a required subunit for primary cilia ion channel. The ciliary PKD2 channel conducts potassium and sodium ions, but little calcium. We also demonstrate that PKD2 is not constitutively active in the plasma membrane, but PKD2 channels are functional in primary cilia and are sensitized by high cilioplasmic [Ca2+]. We introduce a novel method for measuring PKD2 channels heterologously expressed in primary cilia of HEK-293 cells, which will have utility characterizing Pkd2 variants that cause ADPKD in their native ciliary membrane.


INTRODUCTION
Autosomal dominant polycystic kidney disease (ADPKD) is an adult-onset disease characterized 47 by focal cyst development resulting from heterozygous mutations in Pkd1 or Pkd2 (Brasier & 48 Henske, 1997; Grantham, 2001;Hughes et al., 1995;Mochizuki et al., 1996). While considered 49 a dominant monogenic disease, the prevailing two-hit model states that ADPKD is recessive at 50 the cellular level and that cysts develop from cells after acquiring a second somatic mutation to  with the progression of ADPKD (5.7 ± 0.4 µm for 2 months and 18.4 ± 1.2 µm for 4 months post-171 treatment) with Arl13B-EGFP tg :cPkd2 mice, whereas cilia length from control littermates did not 172 differ substantially over the same time course (3.8 ± 0.16 µm and 4.5 ± 0.2 µm, respectively).

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Also, we found that cilia length from tubule cells lining cysts were ~4-times longer than from 174 unaffected tubules from the same animals ( Figure 2C) (12 ± 1.1 µm and 3.1 ± 0.2 µm, 175 respectively). As for Arl13B-EGFP tg :cPkd1 mice, we observed an ~2.4-fold increase in cilia 176 length with the progression of ADPKD (4.1 ± 0.1 µm for control littermates and 9.9 ± 0.5 µm for 177 2 months post-treatment). These results demonstrate the neither PKD1 nor PKD2 expression is 178 required for primary ciliogenesis from the tubule epithelium, but implies that PKD1 or PKD2 179 expression is somehow related to cilia length. Since aberrant cilia morphology was mostly found 180 in cystic tissue epithelia compared to non-cystic tubules, ciliary PKD1 or PKD2 may regulate 181 continuing renal tubular cell differentiation. However, it is unclear if irregular cilia morphology is Using animals from the same study design, we harvested pIMCD cells from Arl13B-EGFP tg mice.

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The cell membrane of the dissociated cells retained anti-aquaporin 2 antibody reactivity and 187 Arl13B was found in the primary cilia of intact distal collecting ducts (Figure 3A-C). Using the 188 validated antibody described in Figure 1, we confirmed the lack of ciliary PKD2 from cultured 189 pIMCD cells from post-doxycycline-treated Arl13B-EGFP tg :cPkd2 mice ( Figure 3D). Importantly,  Ciliary PKD2 preferentially conducts K + and Na + over Ca 2+ ions 225 As discussed in the introduction, it is widely reported that calcium is a major charge carrier for 226 PKD2 under physiological conditions. However, we find that the collecting duct epithelia cilia 227 11 membrane is nearly 2.5 times more selective for potassium than sodium ions (relative 228 permeability P K /P Na = 2.4, Figure 5-figure supplement 1A), with little permeation by calcium 229 (P Ca /P Na = 0.06), barely different than presumably impermeant NMDG (P NMDG /P Na = 0.05). Here, 230 the relative permeability was estimated by the measured change in reversal potential when 231 sodium was replaced by each test cation (Table 3). These data also demonstrate that PKD2's 232 selectivity is distinct from that previously reported for PKD1-L1/2-L1 recorded in the cilia of RPE 233 and MEF cells, which is ~6x more selective for Ca 2+ over Na + and K + (DeCaen et al., 2013). 234 We tested the effect of changing external calcium ([Ca 2+ ] ex ) while maintaining a constant level from ciliary relative permeability, we also compared the single channel conductance of inward 241 Na + , K + , and Ca 2+ when they were exclusively present in the pipette (cilia-attached 242 configuration). Of the three ions tested, K + conducted through ciliary PKD2 channels with the 243 greatest inward conductance (g K = 142 ± 6 pS), followed by sodium (g Na = 89 ± 4 pS) and calcium 244 (g Ca = 4 ± 2 pS) ( Figure 5). Importantly, inward calcium single channel currents were only ion conductance by PKD2 agrees with the relative permeability of the cilia membrane, from which 252 we conclude that the major PKD2 conductance is monovalent, with very little inward Ca 2+ flux.

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PKD2 functions as a channel in primary cilia, but not in the plasma membrane 254 Since intracellular calcium has been reported to sensitize the ER-localized PKD2(Cai et al.,  cilia conductance is most selective for potassium but differ in estimates of sodium and calcium 365 permeability (P x /P K = 1; 0.14; 0.55 for K + , Na + and Ca 2+ , respectively)(S. J. Kleene & Kleene,366 2017) compared to P X /P K = 1: 0.4: 0.025 for K + , Na + and Ca 2+ , respectively, in this study.

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Apparent differences in the selectivity may arise from the differences in methodologies

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If Na + /Ca 2+ exchangers or Na + -dependent kinases are found in cilia, PKD2 activity could underlie 389 a slow, cumulative signal via calcium changes and/or kinase activity.

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Renal epithelial cilia are exposed to urine, which contains varying ion concentrations as a 391 function of position in the nephron. Human and murine distal collecting duct epithelial cells are  Ciliary PKD2 channels are activated by internal calcium 407 We have demonstrated that extraciliary Ca 2+ can block monovalent conductance through PKD2,   Here we showed that the ciliary pIMCD PKD2 channel is blocked by external Ca 2+ but is  Soon after these studies, PKD2 core structures were solved using single-particle electron cryo- filter replacement of the PKD2-L1 channel with that of PKD2 conferred monovalent selectivity to 468 the otherwise Ca 2+ -permeant PKD2-L1 channel. However, it is important to note that the native 469 ciliary PKD2 channel's ion selectivity, as described here, is not completely recapitulated in the 470 PKD2 filter chimera, where single channel Ca 2+ conductance was ~27-fold smaller than K + (g Ca 471 = 8 ± 2 pS, twice as large as the native cilia PKD2 channels g Ca = 4 ± 1 pS). Nonetheless, the 472 native PKD2 cilia channel and the PKD2 filter chimera share similar ion selectivity, where K + is 473 favored over Na + and Ca 2+ as reflected in the magnitudes of single channel conductance (pIMCD 474 cilia g K = 142 ± 6 pS, g Na = 89 ± 4 pS; PKD2 chimera g K = 218 ± 3 pS, g Na = 139 ± 3 pS) and 475 relative permeability (P x /P Na pIMCD cilia = 2.4: 1: 0.06 and PKD2 chimera = 2.2: 1: 0.5 for K + , 476 Na + and Ca 2+ respectively). The physiological implications for the differential cilia expression of 477 polycystins and attendant differences in ion selectivity is not clear. PKD2 is primarily a monovalent channel with selectivity K + > Na + , whose current is blocked by  Our genomic PCR data (Figure 1-figure supplement 2D)  interfere with the amino-terminal cilia-localization sequence (R 6 VXP) (Geng et al., 2006) and 549 likewise, the C-terminal GFP tag may interfere with the ER retention sequence found in the C-550 terminus (Cai et al., 1999). However, since we have demonstrated that native (untagged) PKD2 551 is functionally expressed in cilia of collecting duct epithelia, the C-terminally-tagged PKD2-GFP 552 over-expressed in HEK-293 cilia appears to properly localize. Like many overexpressed ion 553 channels, we observed a high amount of GFP fluorescence from N-and C-terminally tagged 554 PKD2 within intracellular compartments, such as the ER. PKD2 in the ER has been shown to be 555 sensitized by cytoplasmic calcium, triggering Ca 2+ -induced Ca 2+ release, possibly through direct 556 interaction with IP3R channel (Koulen et al., 2002;Vassilev et al., 2001). However, we did not 557 examine PKD2 function in the ER or how cilia PKD2 may alter intracellular store calcium release.  where E rev , a, R, T and F are the reversal potential, effective activity coefficients for the cations 587 (i, internal and e, external), the universal gas constant, absolute temperature, and the Faraday 588 constant, respectively. The effective activity coefficients (x) were calculated using the following Where, I min . and I max . are the minimum and maximum response, IC 50 is the half-maximum 619 inhibition and K is the slope factor.      We would like to thank the Somlo lab (Yale University) for providing us with the kidney specific,