TY - JOUR
T1 - Transmembrane insertases and N-glycosylation critically determine synthesis, trafficking, and activity of the nonselective cation channel TRPC6
AU - Talbot, Brianna E.
AU - Vandorpe, David H.
AU - Stotter, Brian R.
AU - Alper, Seth L.
AU - Schlondorff, Johannes S.
N1 - Publisher Copyright:
© 2019 Talbot et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2019/8/23
Y1 - 2019/8/23
N2 - Transient receptor potential cation channel subfamily C member 6 (TRPC6) is a widely expressed ion channel. Gain- of-function mutations in the human TRPC6 channel cause autosomal-dominant focal segmental glomerulosclerosis, but the molecular components involved in disease development remain unclear. Here, we found that overexpression of gain- of-function TRPC6 channel variants is cytotoxic in cultured cells. Exploiting this phenotype in a genome-wide CRISPR/Cas screen for genes whose inactivation rescues cells from TRPC6- associated cytotoxicity, we identified several proteins essential for TRPC6 protein expression, including the endoplasmic reticulum (ER) membrane protein complex transmembrane insertase. We also identified transmembrane protein 208 (TMEM208), a putative component of a signal recognition particle-independent (SND) ER protein-targeting pathway, as being necessary for expression of TRPC6 and several other ion channels and transporters. TRPC6 expression was also diminished by loss of the previously uncharacterized WD repeat domain 83 opposite strand (WDR83OS), which interacted with both TRPC6 and TMEM208. Additionally enriched among the screen hits were genes involved in N-linked protein glycosylation. Deletion of the mannosyl (α-1,3-)-glycoprotein β-1,2-Nacetylglucosaminyltransferase (MGAT1), necessary for the generation of complex N-linked glycans, abrogated TRPC6 gain-of-function variant-mediated Ca2+ influx and extracellular signal-regulated kinase activation in HEK cells, but failed to diminish cytotoxicity in cultured podocytes. However, mutating the two TRPC6 N-glycosylation sites abrogated the cytotoxicity of mutant TRPC6 and reduced its surface expression. These results expand the targets of TMEM208-mediated ER translocation to include multipass transmembrane proteins and suggest that TRPC6 N-glycosylation plays multiple roles in modulating channel trafficking and activity.
AB - Transient receptor potential cation channel subfamily C member 6 (TRPC6) is a widely expressed ion channel. Gain- of-function mutations in the human TRPC6 channel cause autosomal-dominant focal segmental glomerulosclerosis, but the molecular components involved in disease development remain unclear. Here, we found that overexpression of gain- of-function TRPC6 channel variants is cytotoxic in cultured cells. Exploiting this phenotype in a genome-wide CRISPR/Cas screen for genes whose inactivation rescues cells from TRPC6- associated cytotoxicity, we identified several proteins essential for TRPC6 protein expression, including the endoplasmic reticulum (ER) membrane protein complex transmembrane insertase. We also identified transmembrane protein 208 (TMEM208), a putative component of a signal recognition particle-independent (SND) ER protein-targeting pathway, as being necessary for expression of TRPC6 and several other ion channels and transporters. TRPC6 expression was also diminished by loss of the previously uncharacterized WD repeat domain 83 opposite strand (WDR83OS), which interacted with both TRPC6 and TMEM208. Additionally enriched among the screen hits were genes involved in N-linked protein glycosylation. Deletion of the mannosyl (α-1,3-)-glycoprotein β-1,2-Nacetylglucosaminyltransferase (MGAT1), necessary for the generation of complex N-linked glycans, abrogated TRPC6 gain-of-function variant-mediated Ca2+ influx and extracellular signal-regulated kinase activation in HEK cells, but failed to diminish cytotoxicity in cultured podocytes. However, mutating the two TRPC6 N-glycosylation sites abrogated the cytotoxicity of mutant TRPC6 and reduced its surface expression. These results expand the targets of TMEM208-mediated ER translocation to include multipass transmembrane proteins and suggest that TRPC6 N-glycosylation plays multiple roles in modulating channel trafficking and activity.
UR - http://www.scopus.com/inward/record.url?scp=85071421495&partnerID=8YFLogxK
U2 - 10.1074/jbc.RA119.008299
DO - 10.1074/jbc.RA119.008299
M3 - Article
C2 - 31266804
AN - SCOPUS:85071421495
SN - 0021-9258
VL - 294
SP - 12655
EP - 12669
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 34
ER -