A primary cilium is a solitary, slender, non-motile protuberance of structured microtubules (9+0) enclosed by plasma membrane1. Housing components of the cell division apparatus between cell divisions, primary cilia also serve as specialized compartments for calcium signalling2 and hedgehog signalling pathways3. Specialized sensory cilia such as retinal photoreceptors and olfactory cilia use diverse ion channels4,5,6,7. An ion current has been measured from primary cilia of kidney cells8, but the responsible genes have not been identified. The polycystin proteins (PC and PKD), identified in linkage studies of polycystic kidney disease9, are candidate channels divided into two structural classes: 11-transmembrane proteins (PKD1, PKD1L1 and PKD1L2) remarkable for a large extracellular amino terminus of putative cell adhesion domains and a G-protein-coupled receptor proteolytic site, and the 6-transmembrane channel proteins (PKD2, PKD2L1 and PKD2L2; TRPPs). Evidence indicates that the PKD1 proteins associate with the PKD2 proteins via coiled-coil domains10,11,12. Here we use a transgenic mouse in which only cilia express a fluorophore and use it to record directly from primary cilia, and demonstrate that PKD1L1 and PKD2L1 form ion channels at high densities in several cell types. In conjunction with an accompanying manuscript2, we show that the PKD1L1–PKD2L1 heteromeric channel establishes the cilia as a unique calcium compartment within cells that modulates established hedgehog pathways.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Bornens, M. The centrosome in cells and organisms. Science 335, 422–426 (2012)
Delling, M., DeCaen, P. G., Doerner, J. F., Febvay, S. & Clapham, D. E. Primary cilia are specialized calcium signalling organelles. Nature http://dx.doi.org/10.1038/nature12833 (this issue).
Corbit, K. C. et al. Vertebrate Smoothened functions at the primary cilium. Nature 437, 1018–1021 (2005)
Hardie, R. C. & Minke, B. The trp gene is essential for a light-activated Ca2+ channel in Drosophila photoreceptors. Neuron 8, 643–651 (1992)
Shin, J. B. et al. Xenopus TRPN1 (NOMPC) localizes to microtubule-based cilia in epithelial cells, including inner-ear hair cells. Proc. Natl Acad. Sci. USA 102, 12572–12577 (2005)
Stortkuhl, K. F., Hovemann, B. T. & Carlson, J. R. Olfactory adaptation depends on the Trp Ca2+ channel in Drosophila. J. Neurosci. 19, 4839–4846 (1999)
Story, G. M. et al. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 112, 819–829 (2003)
Kleene, N. K. & Kleene, S. J. A method for measuring electrical signals in a primary cilium. Cilia 1, 1–17 (2012)
Arnaout, M. A. Molecular genetics and pathogenesis of autosomal dominant polycystic kidney disease. Annu. Rev. Med. 52, 93–123 (2001)
Celic, A., Petri, E. T., Demeler, B., Ehrlich, B. E. & Boggon, T. J. Domain mapping of the polycystin-2 C-terminal tail using de novo molecular modeling and biophysical analysis. J. Biol. Chem. 283, 28305–28312 (2008)
Yu, Y. et al. Structural and molecular basis of the assembly of the TRPP2/PKD1 complex. Proc. Natl Acad. Sci. USA 106, 11558–11563 (2009)
Zhu, J. et al. Structural model of the TRPP2/PKD1 C-terminal coiled-coil complex produced by a combined computational and experimental approach. Proc. Natl Acad. Sci. USA 108, 10133–10138 (2011)
Gherman, A., Davis, E. E. & Katsanis, N. The ciliary proteome database: an integrated community resource for the genetic and functional dissection of cilia. Nature Genet. 38, 961–962 (2006)
Andrade, Y. N. et al. TRPV4 channel is involved in the coupling of fluid viscosity changes to epithelial ciliary activity. J. Cell Biol. 168, 869–874 (2005)
Raychowdhury, M. K. et al. Characterization of single channel currents from primary cilia of renal epithelial cells. J. Biol. Chem. 280, 34718–34722 (2005)
Yoshiba, S. et al. Cilia at the node of mouse embryos sense fluid flow for left-right determination via Pkd2. Science 338, 226–231 (2012)
Hille, B. Ion Channels of Excitable Membranes 3rd Edn (Sinauer Associates, 2001)
Chen, X. Z. et al. Polycystin-L is a calcium-regulated cation channel permeable to calcium ions. Nature 401, 383–386 (1999)
Praetorius, H. A. & Spring, K. R. A physiological view of the primary cilium. Annu. Rev. Physiol. 67, 515–529 (2005)
Besch, S. R., Suchyna, T. & Sachs, F. High-speed pressure clamp. Pflugers Arch. 445, 161–166 (2002)
Coste, B. et al. Piezo proteins are pore-forming subunits of mechanically activated channels. Nature 483, 176–181 (2012)
Sukharev, S. I., Blount, P., Martinac, B., Blattner, F. R. & Kung, C. A large-conductance mechanosensitive channel in E. coli encoded by mscL alone. Nature 368, 265–268 (1994)
Xu, H. et al. TRPV3 is a calcium-permeable temperature-sensitive cation channel. Nature 418, 181–186 (2002)
Benham, C. D., Gunthorpe, M. J. & Davis, J. B. TRPV channels as temperature sensors. Cell Calcium 33, 479–487 (2003)
Voets, T. et al. The principle of temperature-dependent gating in cold- and heat-sensitive TRP channels. Nature 430, 748–754 (2004)
Kottgen, M. et al. TRPP2 and TRPV4 form a polymodal sensory channel complex. J. Cell Biol. 182, 437–447 (2008)
Cang, C. et al. mTOR regulates lysosomal ATP-sensitive two-pore Na+ channels to adapt to metabolic state. Cell 152, 778–790 (2013)
Fieni, F., Lee, S. B., Jan, Y. N. & Kirichok, Y. Activity of the mitochondrial calcium uniporter varies greatly between tissues. Nature Commun. 3, 1317 (2012)
Shenkel, S. & Sigworth, F. J. Patch recordings from the electrocytes of Electrophorus electricus. Na currents and PNa/PK variability. J. Gen. Physiol. 97, 1013–1041 (1991)
Brisson, A. & Unwin, P. N. Quaternary structure of the acetylcholine receptor. Nature 315, 474–477 (1985)
P.G.D. was supported by NIH T32 HL007572. Animal work was, in part, supported by NIH P30 HD18655 to the IDDRC of Boston Children’s Hospital. We thank B. Navarro, N. Blair, J. Doerner, S. Febvay, and the members of the Clapham laboratory for advice and assistance.
The authors declare no competing financial interests.
Extended data figures and tables
a, Diagram of the primary cilia depicting the EGFP-labelled smoothened protein (green), transition fibres (black line), 9+0 axoneme (purple), basal body (pink) and centriole (grey). b, Table listing the average reversal potential change relative to the standard Na+-based extracellular solution (average ΔErev) and the estimated relative permeability (Px/PNa; ±s.e.m., n = 4 cilia). c–e, Left: representative currents from control (black traces), activation by 100 μM ATP, 30 µM ADP, or 10 μM UDP (red traces) and block by 10 μM Gd3+, 30 μM Ruthenium red and 10 μM La3+ (green, violet and grey traces, respectively). Right: corresponding time course of peak current recorded at −100 mV (grey circles) and +100 mV (black circles).
Extended Data Figure 2 ATP indirectly activates the cilia conductance from four different cell types.
a–d, Top: single-channel currents activated by 1.5-s depolarizations to the indicated potentials in control (black traces) and 100 μM extracellular ATP (red traces) recorded from primary cilia derived from human RPE SMO–GFP cell lines (a); mouse RPE ARL–GFP primary cells (b); mouse MEF ARL–GFP primary cells (c); and mouse kidney IMCD ARL–EGFP cells (d) (scale = 10 pA and 200 ms). Bottom: corresponding open probability histograms measured in control (grey) and in the presence of 100 μM ATP (red; ±s.e.m., n = 4–6 cilia, asterisks indicate P < 0.005). e, Average open dwell times measured from the cilia of these four cell types in control and ATP conditions.
Extended Data Figure 3 Anti-PKD1L1 and anti-PKD2L1 siRNA treatment attenuates the RPE ciliary current.
a, Table of primers used to detect transcript levels present in human RPE cells. b, Table of siRNAs and their knockdown efficiencies used to identify channel candidates. c, Example ciliary current measured from cells treated with siRNAs specific for PKD1L1 or PKD2L1. d, Box (±s.e.m.) and whisker (±s.d.) plots of cilia total outward (+100 mV) and inward (−100 mV) current measured 72 h after double-siRNA treatment. PKDL mRNAs were targeted by two siRNAs specific for two different regions of the target transcript. Averages are indicated by the red lines. Student’s t-test P values comparing treatment groups to scrambled siRNA; *P < 0.05; n = 8–12 cilia.
a, Immunoprecipitation of Flag– and HA–tagged PKD1L1 and PKD2L1 heterologously expressed in HEK293T cells. b, Box (±s.e.m.) and whisker (±s.d.) plots of the current densities measured from PKD1L1 and PKD2L1 family-transfected HEK cells at −100 mV (bottom) and +100 mV (top). Averages are indicated by the red lines. Statistical significances from Student’s t-test comparing transfected to untransfected cells are indicated by asterisks (P < 0.005; n = 10–23 cells) and those comparing PKD2L1 to the pore mutants are indicated by double asterisks (*P < 0.005 compared to untransfected cells; **P < 0.005 compared to PKD1L1/PKD2L1 transfected cells; n = 9–11 cells). c, An alignment of the PKD2L1 and PKD2 (polycystin 2) pore helix and selectivity filter with glutamate residues D523, D525 and D530 indicated. d, Table listing the average reversal potential change relative to the standard Na+-based extracellular solution (average ΔErev) and the estimated relative permeability (Px/PNa) for HEK cells transfected with PKD2L1 alone or with PKD2L1 and PKD1L1 (±s.e.m., n = 4–6 cells).
a, b, Results of pressure clamp (0–100 mm Hg, red line) on PKD1L1–PKD2L1 single channel events recorded from RPE primary cilia (a) and HEK293T cells (b) transfected with PKD1L1 and PKD2L1. c, d, Left: expanded times scales from a and b. Right: corresponding averaged normalized amplitude histograms are plotted for the indicated applied pipette (±s.e.m., n = 5 cilia and 6 cells). Cilia or cells were held at +100 mV and pressure changes were applied at 5-s intervals. Asterisks indicate a significant (P > 0.05) increase in channel opening events relative to the zero pressure condition.
a, b, The effects of repeated temperature stimulations from 22 to 37 °C on the PKD1L1–PKD2L1 current recorded from RPE primary cilia (a) and HEK293T cells (b) (plasma membrane) transfected with PKD1L1 and PKD2L1. Left: currents elicited by a series of 1-Hz voltage ramps from −100 to +100 mV from 21 to 38 °C in control conditions (red traces) or in the presence of 30 μM Gd3+ (grey trace). Right: resulting current amplitudes (−100 mV, grey circles; +100 mV, black circles) and cilia temperature (green circles) are plotted as a function of time. Grey bar indicates the duration of extracellular 30 μM Gd3+ application. c, Arrhenius plots of the PKD1L1–PKD2L1 currents recorded from (left) RPE primary cilia and (right) when heterologously expressed in HEK293T cells. Q10 values were derived from three linear fits of the average normalized current magnitude from three phases of the thermal response (21–24 °C; 24–32 °C; 32–38 °C; ±s.e.m.; n = 4 cilia or 4 cells).
An hRPE cell cilia expressing Smo-GCaMP3 is patch clamped by applying the glass pipette to the tip of the cilia and establishing a giga-seal by gentle suction (frames 4-13). Intentional movement of the pipette pulls the attached cilium (frames 14-18). The ciliary membrane was held at -60 mV and ruptured, establishing a ‘whole-cilium’ patch configuration in which the entire cilia membrane is voltage-clamped and the inside of the cilia perfused by the patch pipette. DIC (left), fluorescence (right), and merged (center) images were acquired in an inverted confocal microscope using a 60x objective. Break-in to the whole-cilium is accompanied by an increase in GCaMP fluorescence (frames 22-29). The video playback speed is 7 frames/s. (MOV 6049 kb)
RPE cells expressing Smo-GFP enable visualization of the primary cilium under confocal microscopy. Once the cilium was patched in the ‘whole-cilium’ configuration, the cilia-pipette connection is demonstrated by intentional movement of the pipette from side to side (frames 1-2). Part of the cilia was then detached from the cell by lifting the electrode (frames 3-4). The focus plane was raised and lowered (gap in fluorescence), demonstrating that the cilium has separated from the cell body (frames 4-9). The video playback speed is 7 frames/s. (MOV 952 kb)
About this article
Cite this article
DeCaen, P., Delling, M., Vien, T. et al. Direct recording and molecular identification of the calcium channel of primary cilia. Nature 504, 315–318 (2013) doi:10.1038/nature12832
Journal of Biological Chemistry (2019)
Direct visualization of cAMP signaling in primary cilia reveals up-regulation of ciliary GPCR activity following Hedgehog activation
Proceedings of the National Academy of Sciences (2019)
The Korean Journal of Physiology & Pharmacology (2019)
Loss of primary cilia increases polycystin-2 and TRPV4 and the appearance of a nonselective cation channel in the mouse cortical collecting duct
American Journal of Physiology-Renal Physiology (2019)