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AP2M1 induced internalization of TRPC6 regulates calcium influx
Author(s) -
Piplani Honit,
Sukriti Sukriti,
Sharma Tiffany,
Meyer Otto,
Mehta Dolly
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.627.10
Subject(s) - trpc6 , trpc , ankyrin , transient receptor potential channel , microbiology and biotechnology , hek 293 cells , chemistry , ankyrin repeat , internalization , receptor , biochemistry , biology , gene
Transient receptor canonical channel (TRPC)‐6, activated by second messenger DAG, induces calcium entry in endothelial cells independent of ER store depletion. We recently showed that deletion of TRPC6 in mice prevented lung endothelial permeability and inflammation in response to thrombin and LPS indicating targeting TRPC6 will be valuable in preventing uncontrolled increase in endothelial permeability. Here we determine the pathway that inhibits TRPC6 channel activity under physiological setting. Amino acid sequence analyses of TRPC6 reveals the presence of 4 ankyrin‐like repeat domains which are known to be important for protein‐protein interactions and activity. Thus, we performed mutational analysis on these repeats and assess channel activity. We found that deletion of Ist ankyrin repeat in TRPC6 blocked calcium influx in HEK cells. Further mutational analysis identified that 96‐111 residues within Ist ankyrin repeat was required for TRPC6 cell surface retention and calcium influx. Sequence analysis using ELM database showed that this domain has consensus site for AP2M1 (Adaptor‐related protein complex 2, mu 1 subunit) binding. AP2M1 is known to internalize transmembrane proteins via clathrin dependent endocytosis. Overexpression of AP2M1 with wt‐TRPC6 in HEK cells decreased calcium influx as well as expression level of TRPC6. Thus, designing a peptide against AP2M1 binding motif in TRPC6 will help in identifying targets for inhibiting TRPC6 activity.