Targeting mechanisms activating ion-channel for preventing acute lung injury

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ABSTRACT Endothelial injury occurring during bacterial and viral infections results in uncontrolled accumulation of protein- rich fluid and inflammatory cells in the underlying tissue, hallmarks of acute lung injury (ALI), and acute respiratory distress syndrome (ARDS). Despite remarkable advances in supportive care, patient survival in the setting of ALI and ARDS remains near 40%. We have demonstrated a crucial role of transient receptor potential channel 6 (TRPC6) mediated Ca2+ entry in initiating inflammatory signaling that causes ALI. However, we also showed that mutation of isoleucine (I)111 for its isomer leucine (L)111 in TRPC6 or block of TRPC6 at isoleucine111 using a novel peptide allows the channel to gain new functions independent of Ca2+ entry for programming EC from inflammatory into the regenerative lineage, thereby resolving lung injury. Thus, understanding the mechanisms of action of I111 in inducing channel activity and the therapeutic value of blocking I111 to promote EC regeneration hold the key to preventing these diseases. We show that: 1) substitution of I111 for its isomer L111 in the Ist ankyrin repeat domain (ARD) of TRPC6 blocks Ca2+ entry; 2) I111L mutation initiates allosteric transitions in TRPC6 leading to loss of channel function, based on nuclear magnetic resonance (NMR) studies; 3) I111L-TRPC6 induces EC regenerative signaling during injury as evidenced by expression of ERG, a transcription factor maintaining EC homeostasis, and EC proliferation, leading to rapid lung repair after injury; 4) rescue of WT-TRPC6 but not the I111L-TRPC6 mutant in EC of Trpc6-/- mice reinstates LPS-induced lung vascular hyperpermeability by suppressing the expression of ERG but augmenting NFB-expression and inflammatory signaling; 5) inducing conditional deletion of ERG in EC impaired EC proliferation and induced lung injury, and, 6) a TRPC6 blocking peptide spanning I111-TRPC6 suppresses Ca2+ entry in EC but promotes EC proliferation and resolution of lung inflammatory injury. Epigenetic changes in chromatin accessibility enable signal- dependent activation of transcription factors that bind gene promoters and enhancers to dictate cell functions. Intriguingly, ATAC-seq and Chip-seq of EC sorted from control versus injured lungs suggest that WT or mutated channel selectively activates the EC epigenome either in favor of NFB or ERG transcriptional activities to switch EC phenotype, thereby dictating the outcome of lung injury. Based on these exciting findings, in Aim#1, we will determine the novel mechanisms induced by isoleucine111 in regulating TRPC6 structural organization and functions. In Aim#2, we will test the hypothesis that in contrast to WT-TRPC6, the I111L TRPC6 mutant gains new functions independent of channel activity to program the EC epigenome to adopt a regenerative lineage and therapeutically blocking this residue function will therefore repair the vascular injury in the pre-clinical models of lung injury. Studies will use multipronged approaches, including molecular modeling, multi-omics, and 2- photon imaging of lung EC, along with an I111-TRPC6 blocking peptide to accomplish these aims. We believe these studies to be translational for developing specific TRPC6 antagonists to prevent ARDS.