title: SNF8, a member of the ESCRT-II complex, interacts with TRPC6 and enhances its channel activity.
abstract: BACKGROUND
  Transient receptor potential canonical (TRPC) channels are non-selective cation channels involved in receptor-mediated calcium signaling in diverse cells and tissues. The canonical transient receptor potential 6 (TRPC6) has been implicated in several pathological processes, including focal segmental glomerulosclerosis (FSGS), cardiac hypertrophy, and pulmonary hypertension. The two large cytoplasmic segments of the cation channel play a critical role in the proper regulation of channel activity, and are involved in several protein-protein interactions.
  
  
  RESULTS
  Here we report that SNF8, a component of the endosomal sorting complex for transport-II (ESCRT-II) complex, interacts with TRPC6. The interaction was initially observed in a yeast two-hybrid screen using the amino-terminal cytoplasmic domain of TRPC6 as bait, and confirmed by co-immunoprecipitation from eukaryotic cell extracts. The amino-terminal 107 amino acids are necessary and sufficient for the interaction. Overexpression of SNF8 enhances both wild-type and gain-of-function mutant TRPC6-mediated whole-cell currents in HEK293T cells. Furthermore, activation of NFAT-mediated transcription by gain-of-function mutants is enhanced by overexpression of SNF8, and partially inhibited by RNAi mediated knockdown of SNF8. Although the ESCRT-II complex functions in the endocytosis and lysosomal degradation of transmembrane proteins, SNF8 overexpression does not alter the amount of TRPC6 present on the cell surface.
  
  
  CONCLUSIONS
  SNF8 is novel binding partner of TRPC6, binding to the amino-terminal cytoplasmic domain of the channel. Modulating SNF8 expression levels alters the TRPC6 channel current and can modulate activation of NFAT-mediated transcription downstream of gain-of-function mutant TRPC6. Taken together, these results identify SNF8 as a novel regulator of TRPC6.
authors.affiliation: Division of Nephrology, Beth Israel Deaconess Medical Center, Research North 304B, 99 Brookline Ave, Boston, MA 02215, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Carrasquillo
authors.forename: Dequan
authors.initials: D
authors.lastname: Tian
authors.forename: Sneha
authors.initials: S
authors.lastname: Krishna
authors.forename: Martin R
authors.initials: MR
authors.lastname: Pollak
authors.forename: Anna
authors.initials: A
authors.lastname: Greka
authors.forename: Johannes
authors.initials: J
authors.lastname: Schlöndorff
mesh_terms: D056827:Endosomal Sorting Complexes Required for Transport / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D009154:Mutation; D050778:NFATC Transcription Factors / Q000378:metabolism; D011485:Protein Binding; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*; D000074287:TRPC6 Cation Channel; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D050778:NFATC Transcription Factors; D034741:RNA, Small Interfering; C525859:SNF8 protein, human; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human