Cross-inhibition between P2X and nAChR ligand-gated ion channels in the enteric nervous system

The enteric nervous system (ENS) resides in the gut wall and contains neurons that can control gastrointestinal function independently of the central nervous system. The ENS contains two main types of ligand-gated ion channels that are important mediators of fast excitatory synaptic transmission; P2X and the nicotinic acetylcholine receptors (nAChRs). Although these channels are structurally distinct and have been assumed to function independently, recent studies have shown that these receptors have a mutually inhibitory interaction. Whole-cell patch clamp techniques were used to assess the function of P2X2 and alpha3beta4 nAChRs in cultured myenteric neurons and P2X and nAChRs expressed separately and together in human embryonic kidney (HEK-293) cells, a heterologous system that permits molecular manipulations. These molecular aspects included a detailed look at interactions between these receptors in three different states; open, desensitized, and closed. This work provided evidence that P2X2 and nAChRs cross-inhibition occurs in all three of these states, including constitutive inhibition between these receptors in the closed state. The specificity of this interaction was investigated among the different subtypes of the P2X receptors. Cross-inhibition occurred between alpha3beta4 nAChRs and the P2X 3 and P2X4 receptor subtypes in open and desensitized states.;More specific molecular manipulations included the determination of the direct molecular mechanisms responsible for this interaction via the use of a truncated P2X2 receptor with a stop codon to shorten the C-terminal tail. Studies with this truncated receptor provided evidence that the C-terminal tail of the P2X2 receptor is important in mediating cross inhibition of alpha3beta4 nAChRs in the open and closed states. Furthermore, with the use of a purified protein construct corresponding to the C-terminal tail of the P2X2 receptor, competitive experiments were performed that provide evidence for a physical interaction between the P2X2 receptor and the alpha3beta4 nAChR. A better understanding of receptor interactions in enteric neurons would likely lead to novel drug targets that would be useful in the treatment of common gastrointestinal motility disorders such as irritable bowel syndrome.