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bioRxiv · 10.1101/2022.06.15.496344

Constitutive Inhibition of Transient Receptor Potential CanonicalType 6 (TRPC6) by O-GlcNAcylation at Threonine-221

Kass, D. A.·Mishra, S.·Ma, J.·Sasaki, M.·Farinelli, F.·Page, R. C.·Ranek, M. J.·Zachara, N.
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Abstract

Transient receptor potential canonical type 6 (TRPC6) is a non-voltage gated cation channel that principally conducts calcium to regulate signaling in cardiac, vascular, neuronal and other cells. Abnormally increased TRPC6 expression/conductance and genetic gain of function mutations contribute to fibrosis, hypertrophy, proteinuria, and edema, notably linked to its stimulation of nuclear factor of activated T-cells (NFAT) signaling. Hyperglycemia (HG) also activates TRPC6/NFAT as a cause of diabetic renal disease. While prior work linked HG-TRPC6 activation to oxidant stress, the role of another major HG modification - O-GlcNAcylation, is unknown. Here we show TRPC6 is constitutively O-GlcNAcylated, TRPC6 and O-GlcNAc transferase proteins interact, this modification potently suppresses basal channel conductance and NFAT activity, and it is unaltered by HG. Proteomics identifies O-GlcNAcylation at Ser14, Thr70, and Thr221 in the N-terminus ankyrin-4 (AR4) and neighboring linker (LH1) domains of TRPC6. Of these, T221 is most impactful as a T221A mutation increases basal NFAT activity 11-fold, TRPC6 conductance 75-80% vs wild-type, and when expressed in cardiomyocytes amplifies NFAT-pro-hypertrophic gene expression. T221 is highly conserved and mutating homologs in TRPC3 and TRPC7 also markedly elevates basal NFAT activity. Molecular models predict electrostatic interactions between T221 O-GlcNAc and Ser199, Glu200, and Glu246, and we find similarly elevated NFAT activity from alanine substitutions at these coordinating sites as well. Thus, O-GlcNAcylation at T221 and its interaction with coordinating residues in AR4-LH1 is required for basal TRPC6 channel conductance and regulation of NFAT.

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Kass, D. A., Mishra, S., Ma, J., Sasaki, M., Farinelli, F., Page, R. C., Ranek, M. J., Zachara, N.. 2022-06-15. Constitutive Inhibition of Transient Receptor Potential CanonicalType 6 (TRPC6) by O-GlcNAcylation at Threonine-221. https://doi.org/10.1101/2022.06.15.496344

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