Abstract
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-N-acetylglucosaminyltransferase (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.
Highlights
Transient receptor potential cation channel subfamily C member 6 (TRPC6) is a widely expressed ion channel
We considered that overexpression of TRPC6 with gain– of–function (GOF) mutations is detrimental to cellular survival or proliferation in light of unsuccessful attempts at generating stable cell lines constitutively expressing these mutant TRPC6 forms
Unlike TRPC6 with immature N-glycans expressed in MGAT1-knockout cells, a significantly lower ratio of TRPC6 lacking N-glycosylation sites was available for surface biotinylation. This was not due to the R895C mutation itself, as the NN 3 QQ mutation diminished surface expression of TRPC6 R895C and WT TRPC6 to similar extents (Fig. 7C). These results suggest that some form of N-linked glycosylation is required for TRPC6 surface expression, whereas MGAT1-dependent hybrid or complex N-glycans are required for proper TRPC6 channel activity, but not for surface expression
Summary
Transient receptor potential cation channel subfamily C member 6 (TRPC6) is a widely expressed ion channel. 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 TRPC6associated cytotoxicity, we identified several proteins essential for TRPC6 protein expression, including the endoplasmic reticulum (ER) membrane protein complex transmembrane insertase. Deletion of the mannosyl (␣-1,3-)-glycoprotein -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. 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. TRPC6 expression requires the activities of two transmembrane insertases, whereas complex N-glycosylation of TRPC6 is required for TRPC6 channel function but not for its surface expression
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