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

doi:10.1016/j.isci.2023.106294

Transient receptor potential canonical type 6 (TRPC6) O-GlcNAcylation at Threonine-221 plays potent role in channel regulation

iScience. 2023 Feb 28;26(3):106294. doi: 10.1016/j.isci.2023.106294. eCollection 2023 Mar 17.

Authors

Sumita Mishra¹, Junfeng Ma², Desirae McKoy¹, Masayuki Sasaki¹, Federica Farinelli¹, Richard C Page³, Mark J Ranek¹, Natasha Zachara⁴, David A Kass^{1

5}

Affiliations

¹ Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
² Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA.
³ Department of Chemistry and Biochemistry, Miami University, Oxford, OH, USA.
⁴ Department of Biological Chemistry, Department of Oncology, Johns Hopkins University, Baltimore, MD, USA.
⁵ Department of Pharmacology and Molecular Sciences, Johns Hopkins University, Baltimore, MD, USA.

Abstract

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.

Keywords: Biochemistry; Cell biology; Cellular physiology.

Abstract

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