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

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      Summary

      A primary cilium is a solitary slender non-motile protuberance of structured microtubules (9+0) enclosed by plasma membrane 1 . Housing components of the cell division apparatus between cell divisions, they also serve as specialized compartments for calcium signaling 2 and Hedgehog (Hh) signaling pathways 3 . Specialized sensory cilia such as retinal photoreceptors and olfactory cilia employ diverse ion channels 4- 7 . An ion current has been measured from primary cilia of kidney cells 8 but the responsible genes have not been identified. The polycystin proteins (PC, PKD), identified in linkage studies of polycystic kidney disease 9 , are candidate channels divided into two structural classes: 11-transmembrane (TM) proteins (PKD1, PKD1-L1 and PKD1-L2) remarkable for a large extracellular N-terminus of putative cell adhesion domains and a GPCR proteolytic site, and the 6-TM channel proteins (PKD2, PKD2-L1, PKD2-L2; TRPPs). Evidence suggests that the PKD1s associate with the PKD2s via coiled-coil domains 10- 12 . Here, we employ a transgenic mouse in which only cilia express a fluorophore and employ it to directly record from primary cilia and demonstrate that PKD1-L1 and PKD2-L1 form ion channels at high densities in several cell types. In conjunction with the companion manuscript 2 , we show that the PKD1-L1/PKD2-L1 heteromeric channel establishes the cilia as a unique calcium compartment within cells that modulates established Hedgehog pathways.

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      Vertebrate Smoothened functions at the primary cilium.

      The unanticipated involvement of several intraflagellar transport proteins in the mammalian Hedgehog (Hh) pathway has hinted at a functional connection between cilia and Hh signal transduction. Here we show that mammalian Smoothened (Smo), a seven-transmembrane protein essential for Hh signalling, is expressed on the primary cilium. This ciliary expression is regulated by Hh pathway activity; Sonic hedgehog or activating mutations in Smo promote ciliary localization, whereas the Smo antagonist cyclopamine inhibits ciliary localization. The translocation of Smo to primary cilia depends upon a conserved hydrophobic and basic residue sequence homologous to a domain previously shown to be required for the ciliary localization of seven-transmembrane proteins in Caenorhabditis elegans. Mutation of this domain not only prevents ciliary localization but also eliminates Smo activity both in cultured cells and in zebrafish embryos. Thus, Hh-dependent translocation to cilia is essential for Smo activity, suggesting that Smo acts at the primary cilium.
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        ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures.

        Mammals detect temperature with specialized neurons in the peripheral nervous system. Four TRPV-class channels have been implicated in sensing heat, and one TRPM-class channel in sensing cold. The combined range of temperatures that activate these channels covers a majority of the relevant physiological spectrum sensed by most mammals, with a significant gap in the noxious cold range. Here, we describe the characterization of ANKTM1, a cold-activated channel with a lower activation temperature compared to the cold and menthol receptor, TRPM8. ANKTM1 is a distant family member of TRP channels with very little amino acid similarity to TRPM8. It is found in a subset of nociceptive sensory neurons where it is coexpressed with TRPV1/VR1 (the capsaicin/heat receptor) but not TRPM8. Consistent with the expression of ANKTM1, we identify noxious cold-sensitive sensory neurons that also respond to capsaicin but not to menthol.
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          Piezos are pore-forming subunits of mechanically activated channels

          Mechanotransduction plays a crucial role in physiology. Biological processes including sensing touch and sound waves require yet unidentified cation channels that detect pressure. Mouse piezo1 (mpiezo1) and mpiezo2 induce mechanically activated cationic currents in cells; however, it is unknown if piezos are pore-forming ion channels or modulate ion channels. We show that Drosophila piezo (dpiezo) also induces mechanically activated currents in cells, but through channels with remarkably distinct pore properties including sensitivity to the pore blocker ruthenium red and single channel conductances. mpiezo1 assembles as a ~1.2 million-Dalton tetramer, with no evidence of other proteins in this complex. Finally, purified mpiezo1 reconstituted into asymmetric lipid bilayers and liposomes forms ruthenium red-sensitive ion channels. These data demonstrate that piezos are an evolutionarily conserved ion channel family involved in mechanotransduction.
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            Author and article information

            Affiliations
            [1 ]Howard Hughes Medical Institute, Department of Cardiology, Children's Hospital Boston, 320 Longwood Avenue, Boston, MA 02115, USA
            [2 ]Department of Neuroscience, School of Medicine, Tufts University, Boston, MA 02111, USA
            [3 ]Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA
            Author notes
            []Correspondence and requests for materials should be addressed to D.E.C. ( dclapham@ 123456enders.tch.harvard.edu )
            [*]

            These authors contributed equally to this work

            Journal
            0410462
            6011
            Nature
            Nature
            Nature
            0028-0836
            1476-4687
            5 May 2014
            12 December 2013
            27 June 2014
            : 504
            : 7479
            : 315-318
            24336289 4073646 10.1038/nature12832 HHMIMS539239

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