Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10801966 | Biochimica et Biophysica Acta (BBA) - Molecular Cell Research | 2015 | 46 Pages |
Abstract
Stromal interaction molecule 1 (STIM1) senses depletion of ER-Ca2Â + store and clusters in ER-PM junctions where it associates with and gates Ca2Â + influx channels, Orai1 and TRPC1. Clustering of TRPC1 with STIM1 and Orai1 in these junctions is critical since Orai1-mediated Ca2Â + 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 Ca2Â + 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-Ca2Â + store depletion which is critical for the surface expression and function of the channel. Ca2Â + influx mediated by TRPC1 modifies Ca2Â +-dependent physiological response of cells.
Keywords
TRPC1cytosolic Ca2 + concentrationTIRFMOrai1IsocPIP2ADP-ribosylation factor 6ARF6CAV1Dynamin-2TGNEESBFASOCESTIM1CRACIP3RThapsigarginIP3Inositol 1,4,5-triphosphate[Ca2 +]iearly endosomesEndocytic recyclingbrefeldinSurface expressionTrans-Golgi networkendoplasmic reticulumPlasma membranephosphatidylinositol 4,5-bisphosphateStromal interaction molecule 1Total internal reflection fluorescence microscopyStore-operated calcium entrycaveolin 1IP3 receptor
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Authors
Lorena Brito de Souza, Hwei Ling Ong, Xibao Liu, Indu S. Ambudkar,