Direct recording and molecular identification of the calcium channel of primary cilia

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  • A Corrigendum to this article was published on 10 September 2014

Abstract

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.

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Figure 1: A calcium-selective ion channel is richly expressed in primary cilia.
Figure 2: Primary cilia currents measured from four different cell types.
Figure 3: MEF primary cilium currents compared from wild-type and Pkd2l1 null animals.
Figure 4: Plasma membrane expressed PKD1L1–PKD2L1 channels match those of Icilia.

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Acknowledgements

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.

Author information

All authors designed or conducted experiments and wrote the manuscript.

Correspondence to David E. Clapham.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Ion selectivity and pharmacology of ciliary hRPE current.

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). ce, 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.

ad, 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.

Extended Data Figure 4 Heterologous PKD1L1 and PKD2L1 form an ion channel.

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).

Extended Data Figure 5 The PKD1L1–PKD2L1 channel is mechanosensitive only at high pressures.

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.

Extended Data Figure 6 The PKD1L1–PKD2L1 channel is highly temperature sensitive.

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).

Supplementary information

Obtaining a ‘whole-cilium’ patch using confocal microscopy

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)

Obtaining an ‘excised-cilia’ patch from an RPE Smo-EGFP cell.

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)

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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

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