Our results demonstrate that these connexins play a dual crucial role in inner ear Ca 2؉ signaling: as hemichannels, they promote ATP release, sustaining long-range ICS propagation; as GJ channels, they allow diffusion of Ca 2؉ -mobilizing second messengers across coupled cells.deafness ͉ mouse models ͉ P2x7 receptor ͉ pannexin ͉ biosensor cells

Connexins are membrane proteins that assemble into gap-junction channels and are responsible for direct, electrical and metabolic coupling between connected cells. Here we describe an investigation of the properties of a recombinantly expressed recessive mutant of connexin 26 (Cx26), the V84L mutant, associated with deafness. Unlike other Cx26 mutations, V84L affects neither intracellular sorting nor electrical coupling, but specifically reduces permeability to the Ca(2+)-mobilizing messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)). Both the permeability to Lucifer Yellow and the unitary channel conductance of V84L-mutant channels are indistinguishable from those of the wild-type Cx26. Injection of Ins(1,4,5)P(3) into supporting cells of the rat organ of Corti, which abundantly express Cx26, ensues in a regenerative wave of Ca(2+) throughout the tissue. Blocking the gap junction communication abolishes wave propagation. We propose that the V84L mutation reduces metabolic coupling mediated by Ins(1,4,5)P(3) to an extent sufficient to impair the propagation of Ca(2+) waves and the formation of a functional syncytium. Our data provide the first demonstration of a specific defect of metabolic coupling and offer a mechanistic explanation for the pathogenesis of an inherited human disease.

Upon activation, neutrophils undergo histone citrullination by protein arginine deiminase (PAD)4, exocytosis of chromatin and enzymes as neutrophil extracellular traps (NETs), and death. In diabetes, neutrophils are primed to release NETs and die by NETosis. Although this process is a defense against infection, NETosis can damage tissue. Therefore, we examined the effect of NETosis on the healing of diabetic foot ulcers (DFUs). Using proteomics, we found that NET components were enriched in nonhealing human DFUs. In an independent validation cohort, a high concentration of neutrophil elastase in the wound was associated with infection and a subsequent worsening of the ulcer. NET components (elastase, histones, neutrophil gelatinase-associated lipocalin, and proteinase-3) were elevated in the blood of patients with DFUs. Circulating elastase and proteinase-3 were associated with infection, and serum elastase predicted delayed healing. Neutrophils isolated from the blood of DFU patients showed an increased spontaneous NETosis but an impaired inducible NETosis. In mice, skin PAD4 activity was increased by diabetes, and FACS detection of histone citrullination, together with intravital microscopy, showed that NETosis occurred in the bed of excisional wounds. PAD4 inhibition by Cl-amidine reduced NETting neutrophils and rescued wound healing in diabetic mice. Cumulatively, these data suggest that NETosis delays DFU healing.