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. 2013 Dec 12;504(7479):315-8.
doi: 10.1038/nature12832.

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

Paul G DeCaen  1 , Markus Delling  1 , Thuy N Vien  2 , David E Clapham  3
Affiliations

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

Paul G DeCaen et al. Nature. .

Erratum in

  • Nature. 2014 Sep 11;513(7517):266

Abstract

A primary cilium is a solitary, slender, non-motile protuberance of structured microtubules (9+0) enclosed by plasma membrane. Housing components of the cell division apparatus between cell divisions, primary cilia also serve as specialized compartments for calcium signalling and hedgehog signalling pathways. Specialized sensory cilia such as retinal photoreceptors and olfactory cilia use diverse ion channels. An ion current has been measured from primary cilia of kidney cells, but the responsible genes have not been identified. The polycystin proteins (PC and PKD), identified in linkage studies of polycystic kidney disease, 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 domains. 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 manuscript, 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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Figures

Figure 1
Figure 1. A calcium-selective ion channel is richly expressed in primary cilia
(a) Confocal image of an hRPE Smo-EGFP cell and patch clamp electrode. (b) Whole-cell leak-subtracted currents elicited by 1 s depolarizing pulses from -100 to 100 mV in +5 mV increments recorded from the cell body, primary cilia and an excised primary cilia (recorded from the same cilium). (c) Whole-cell currents activated by ramp voltage protocols from -100 to +100 mV measured from the primary cilia where extracellular Na+-based saline was replaced by the cation indicated. (d) Single channel currents activated by 1.5 s depolarizations to the indicated potentials (left) and average current amplitudes (right; ± SEM, n = 8 cilia). (e) Estimated endogenous cilia ion channel densities compared to those from other biological preparations,-.
Figure 2
Figure 2. Primary cilia currents measured from four different cell types
Averaged cilia current traces in control and bath-applied 100 μM ATP or 10 μM Gd3+ from: (a) human RPE cell line stably expressing Smoothened-EGFP; (b) Primary mRPE cells from the Arl13B-EGFPtg mouse; (c) Kidney IMCD cell line stably expressing Arl-EGFP and (d) Primary embryonic fibroblasts from the Arl13B-EGFPtg mouse. (e) Average single channel current/voltage relation. The slope is used to estimate conductance (± SEM, n = 4-7 cilia). (f) Average open times in the presence and absence of ATP at -100 and +100 mV potentials measured from the cilia of RPE Smo-EGFP cells (± SEM, n = 6 cilia).
Figure 3
Figure 3. MEF primary cilium currents compared from wt and PKD2-L1 null animals
Left; Cilium currents recorded from MEFs isolated from (a) wt and (b) PKD2-L1-/- animals. Currents were elicited by a series of ramps from -100 to +100 mV in control conditions (black traces) or in the presence of 10 μM calmidazolium (CMZ, red trace). Right; Resulting current amplitudes (-100 mV, grey circles; +100 mV, black circles) plotted as a function of time. Red bar indicates application of extracellular 10 μM CMZ. (c) Scatter and whisker (± SD) plots of current magnitudes at +100 mV and -100 mV from MEF cilia. Individual cilia are represented as connected circles in control (black) and after calmidazolium (red). Averages are indicated by the dark horizontal lines. Student's t-test results: * denotes P-value < 0.05; n.s. denotes P-value > 0.05; n = 9-11 cilia.
Figure 4
Figure 4. Plasma membrane expressed PKD1-L1/PKD2-L1 channels match those of Icilia
(a, b) Whole-cell currents recorded from cells transfected with (a) PKD2-L1 alone or (b) PKD1-L1 and PKD2-L1, where extracellular 1 μM calmidazolium (CMZ, red trace) was applied and Na+-based saline was exchanged by the cations indicated. (c) Single channel currents measured from the cell membrane of cells transfected by PKD2-L1 (black traces), or PKD1-L1 and PKD2-L1 (blue traces). (d) Resulting average single channel current amplitudes plotted against voltage measured from transfected cells (± SEM, n = 4-5 cells).
Extended Data Figure 1
Extended Data Figure 1. Ion selectivity and pharmacology of ciliary hRPE current
(a) Diagram of the primary cilia depicting the EGFP-labeled Smoothened protein (green), transition fibers (black line), 9+0 axoneme (purple), basal body (pink) and centriole (gray). (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; ± SEM, 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 (gray circles) and +100 mV (black circles).
Extended Data Figure 2
Extended Data Figure 2. ATP indirectly activates the cilia conductance from four different cell types
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 (a) human RPE Smo-GFP cell lines, (b) mouse RPE Arl-GFP primary cells, (c) mouse MEF Arl-GFP primary cells, (d) mouse kidney IMCD Arl-EGFP cells (scale = 10 pA and 200 ms). Bottom, corresponding open probability histograms measured in control (grey) and in the presence of 100 μM ATP (red; ± SEM, n = 4-6 cilia, asterisks indicates 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
Extended Data Figure 3. Anti-PKD1-L1 and -PKD2-L1 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 PKD1-L1 or PKD2-L1. (c) Box (± SEM) and whisker (± SD) plots of cilia total outward (+100 mV) and inward (-100 mV) current measured 72 h after double-siRNA treatment. PKD-L 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: * denotes P-value
Extended Data Figure 4
Extended Data Figure 4. Heterologous PKD1-L1 and PKD2-L1 form an ion channel
(a) Immunoprecipitation of Flag– and HA–tagged PKD1-L1 and PKD2-L1 heterologously expressed in HEK293T cells. (b) Box (± SEM) and whisker (± SD) plots of the current densities measured from PKDx-L1 family-transfected HEK cells at -100 mV (bottom) and +100 mV (top). Averages are indicated by the red lines. Statistical significance from Student's t-test comparing transfected to untransfected cells are indicated by asterisks (P-value +-based extracellular solution (average खErev) and the estimated relative permeability (Px/PNa) for HEK cells transfected with PKD2-L1 alone or with PKD2-L1 and PKD1-L1 (± SEM, n= 4-6 cells).
Extended Data Figure 5
Extended Data Figure 5. The PKD1-L1/PKD2-L1 channel is mechanosensitive only at high pressures
(a, b) Results of pressure clamp (0-100 mm Hg, red line) on PKD1-L1/PKD2-L1 single channel events recorded from (a) RPE primary cilia and (b) HEK-293T cells transfected with PKD1-L1 and PKD2-L1. (c, d) Left, Expanded times scales from (a) and (b). Right, corresponding averaged normalized amplitude histograms are plotted for the indicated applied pipette (± SEM, 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
Extended Data Figure 6. The PKD1-L1/PKD2-L1 channel is highly temperature sensitive
(a, b) The effects of repeated temperature stimulations from 22 to 37°C on the PKD1-L1/PKD2-L1 current recorded from (a) RPE primary cilia and (b) HEK-293T cells (plasma membrane) transfected with PKD1-L1 and PKD2-L1. 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 PKD1-L1/PKD2-L1 currents recorded from (left) RPE primary cilia and (right) when heterologously expressed in HEK-293T cells. Q10 values were derived from 3 linear fits of the average normalized current magnitude from 3 phases of the thermal response (21-24°C; 24-32°C; 32-38°C; ± SEM; n = 4 cilia or 4 cells).
See this image and copyright information in PMC

Comment in

  • Calcium: an ion channel for cilia.
    Du Toit A. Du Toit A. Nat Rev Mol Cell Biol. 2014 Feb;15(2):78. doi: 10.1038/nrm3736. Epub 2013 Dec 27. Nat Rev Mol Cell Biol. 2014. PMID: 24370826 No abstract available.

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