Extracellular purines elicit strong signals in the nervous system. Adenosine-5'-triphosphate (ATP) does not spontaneously cross the plasma membrane, and nervous cells secrete ATP by exocytosis or through plasma membrane proteins such as connexin hemichannels. Using a combination of imaging, luminescence and electrophysiological techniques, we explored the possibility that Connexin 32 (Cx32), expressed in Schwann cells (SCs) myelinating the peripheral nervous system could be an important source of ATP in peripheral nerves. We triggered the release of ATP in vivo from mice sciatic nerves by electrical stimulation and from cultured SCs by high extracellular potassium concentration-evoked depolarization. No ATP was detected in the extracellular media after treatment of the sciatic nerve with Octanol or Carbenoxolone, and ATP release was significantly inhibited after silencing Cx32 from SCs cultures. We investigated the permeability of Cx32 to ATP by expressing Cx32 hemichannels in Xenopus laevis oocytes. We found that ATP release is coupled to the inward tail current generated after the activation of Cx32 hemichannels by depolarization pulses, and it is sensitive to low extracellular calcium concentrations. Moreover, we found altered ATP release in mutated Cx32 hemichannels related to the X-linked form of Charcot-Marie-Tooth disease, suggesting that purinergic-mediated signaling in peripheral nerves could underlie the physiopathology of this neuropathy.

The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in the respiratory system and the gastrointestinal tract. Accordingly, human CLCA1 is a critical mediator of hypersecretory diseases that manifest mucus obstruction, such as asthma, COPD, and cystic fibrosis. It has been reported that hCLCA1 modulates calcium-activated chloride channels (CaCCs) in mammalian cell lines (Hamann et al., J Physiol 587: 2255-74; 2009), and that CLCAs are proteolytically processed during secretion (Patel et al., Annu Rev Physiol 71: 425-49; 2009); however, the precise molecular mechanisms of CLCAs remain unclear. To address this, we used a combination of sequence analysis, structure prediction, proteomics, and biochemical, biophysical and electrophysiological assays in HEK293 cells expressing several human and murine CLCA isoforms. We found that CLCAs are metalloproteases capable of both self-cleavage and cross-cleavage of other family members. We identified a novel zincin metalloprotease domain in the N-terminus of CLCA itself that is responsible for the self-proteolysis, and defined a consensus cleavage motif unique to the CLCA family. The activating effect of hCLCA1 on endogenous CaCCs was abolished in cells transfected with mutations that disrupt the metalloprotease activity or the cleavage site, and was recovered in cells transfected with the N-terminal fragment of the proteolysis, but not with the C-terminal fragment. Together, our data indicate that this unique CLCA self-cleavage event is required to unmask the N-terminal fragment of the protein, which is then responsible for the modulation of CaCCs.Our study provides a functional basis for CLCA1 self-cleavage, and a novel mechanism for regulation of chloride channel activity.