title: Fast endocytic recycling determines TRPC1-STIM1 clustering in ER-PM junctions and plasma membrane function of the channel.
abstract: Stromal interaction molecule 1 (STIM1) senses depletion of ER-Ca2+ store and clusters in ER-PM junctions where it associates with and gates Ca2+ influx channels, Orai1 and TRPC1. Clustering of TRPC1 with STIM1 and Orai1 in these junctions is critical since Orai1-mediated Ca2+ entry triggers surface expression of TRPC1 while STIM1 gates the channel. Thus, plasma membrane function of TRPC1 depends on the delivery of the channel to the sites where STIM1 puncta are formed. This study examines intracellular trafficking mechanism(s) that determine plasma membrane expression and function of TRPC1 in cells where Orai1 and TRPC1 are endogenously expressed and contribute to Ca2+ entry. We report that TRPC1 is internalized by Arf6-dependent pathway, sorted to Rab5-containing early endosomes, and trafficked to ER-PM junctions by Rab4-dependent fast recycling. Overexpression of Arf6, or Rab5, but not the respective dominant negative mutants, induced retention of TRPC1 in early endosomes and suppressed TRPC1 function. Notably, cells expressing Arf6 or Rab5 displayed an inwardly rectifying ICRAC current that is mediated by Orai1 instead of TRPC1-associated ISOC, demonstrating that Orai1 function was not altered. Importantly, expression of Rab4, but not STIM1, with Rab5 rescued surface expression and function of TRPC1, restoring generation of ISOC. Together, these data demonstrate that trafficking via fast recycling endosomes determines TRPC1-STIM1 clustering within ER-PM junctions following ER-Ca2+ store depletion which is critical for the surface expression and function of the channel. Ca2+ influx mediated by TRPC1 modifies Ca2+-dependent physiological response of cells.
authors.affiliation: Secretory Physiology Section, Molecular Physiology and Therapeutics Branch, NIDCR, NIH, MD 20892, USA.
authors.forename: Lorena Brito
authors.initials: LB
authors.lastname: de Souza
authors.affiliation: Secretory Physiology Section, Molecular Physiology and Therapeutics Branch, NIDCR, NIH, MD 20892, USA.
authors.forename: Hwei Ling
authors.initials: HL
authors.lastname: Ong
authors.affiliation: Secretory Physiology Section, Molecular Physiology and Therapeutics Branch, NIDCR, NIH, MD 20892, USA.
authors.forename: Xibao
authors.initials: X
authors.lastname: Liu
authors.affiliation: Secretory Physiology Section, Molecular Physiology and Therapeutics Branch, NIDCR, NIH, MD 20892, USA. Electronic address: indu.ambudkar@nih.gov.
authors.forename: Indu S
authors.initials: IS
authors.lastname: Ambudkar
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D000071740:ORAI1 Protein; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*; D020695:rab4 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000089982:ADP-Ribosylation Factor 6; D015220:Calcium Channels; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; C427755:RAB5C protein, human; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; D020695:rab4 GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D002118:Calcium
keywords: Endocytic recycling; Orai1; STIM1; Store-operated calcium entry; Surface expression; TRPC1