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Switching off TRPC6 Signaling: A new anti‐edemagenic strategy
Author(s) -
Amin Md Ruhul,
Piplani Honit,
Sharma Tiffany,
Mehta Dolly
Publication year - 2017
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.31.1_supplement.676.4
Subject(s) - trpc6 , trpc , microbiology and biotechnology , transient receptor potential channel , hek 293 cells , chemistry , ankyrin , biology , receptor , biochemistry , gene
Transient receptor canonical channel (TRPC)‐6, ubiquitously expressed nonselective cation channel activated by second messenger DAG, induces calcium entry in endothelial cells independent of ER store depletion. Recently, we have shown 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. TRPC6 activity has been implicated in acute lung injury sepsis, pulmonary hypertension and focal segmental glomerulosclerosis (FSGS). Here we identified key motif in Ankyrin 1 domain of TRPC6 that regulates gating of the channel. Amino acid sequence analyses of TRPC6 revealed the presence of 4 ankyrin‐like repeat domains which are known to be important for protein‐protein interactions, channel assembly 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 predicted YGNI (aa108‐aa111) motif as the one likely regulating TRPC6 activity based on resemblance with adaptor protein complex, mu subunit (AP2M1), an important signal for endocytosis. Mutation of YGNI to AAAA blocked Ca 2+ ‐entry in response to Hyperforin and OAG. Based on above studies, we have developed specific peptides which efficiently blocked Ca 2+ ‐entry in response to Hyp9 in both HEK and HPAE cells. Ongoing studies are underway to establish the clinical relevance of TRPC6 activity blocking peptides in animal model of lung injury.