Transient receptor potential canonical type 6 (TRPC6) O-GlcNAcylation at Threonine-221 plays potent role in channel regulation
Sumita Mishra,
Junfeng Ma,
Desirae McKoy,
Masayuki Sasaki,
Federica Farinelli,
Richard C. Page,
Mark J. Ranek,
Natasha Zachara,
David A. Kass
Affiliations
Sumita Mishra
Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Junfeng Ma
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA
Desirae McKoy
Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Masayuki Sasaki
Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Federica Farinelli
Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Richard C. Page
Department of Chemistry and Biochemistry, Miami University, Oxford, OH, USA
Mark J. Ranek
Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Natasha Zachara
Department of Biological Chemistry, Department of Oncology, Johns Hopkins University, Baltimore, MD, USA
David A. Kass
Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Pharmacology and Molecular Sciences, Johns Hopkins University, Baltimore, MD, USA; Corresponding author
Summary: Transient receptor potential canonical type 6 (TRPC6) is a non-voltage-gated channel that principally conducts calcium. Elevated channel activation contributes to fibrosis, hypertrophy, and proteinuria, often coupled to stimulation of nuclear factor of activated T-cells (NFAT). TRPC6 is post-translationally regulated, but a role for O-linked β-N-acetyl glucosamine (O-GlcNAcylation) as elevated by diabetes, is unknown. Here we show TRPC6 is constitutively O-GlcNAcylated at Ser14, Thr70, and Thr221 in the N-terminus ankryn-4 (AR4) and linker (LH1) domains. Mutagenesis to alanine reveals T221 as a critical controller of resting TRPC6 conductance, and associated NFAT activity and pro-hypertrophic signaling. T→A mutations at sites homologous in closely related TRPC3 and TRPC7 also increases their activity. Molecular modeling predicts interactions between Thr221-O-GlcNAc and Ser199, Glu200, and Glu246, and combined alanine substitutions of the latter similarly elevates resting NFAT activity. Thus, O-GlcNAcylated T221 and interactions with coordinating residues is required for normal TRPC6 channel conductance and NFAT activation.
