P2X4 subunits are part of P2X native channels in murine myenteric neurons

Abstract

The aim of the present study was to investigate if P2X4 receptors are expressed in murine myenteric neurons and if these receptors contribute to form functional channels in the neuronal membrane by using molecular and electrophysiological techniques. The whole-cell recording technique was used to measure membrane currents induced by ATP (IATP) in myenteric neurons. Compared with recombinant P2X4 receptor-channels (reported by others in a previous study), native myenteric P2X receptors have a relative lower sensitivity for ATP (EC50=102µM) and α,β methylene ATP (not effect at 30 or 100µM). BzATP was a weak agonist for native P2X receptors. KN-62 had no effect on myenteric P2X channels whereas PPADS (IC50=0.54µM) or suramin (IC50=134µM) were more potent antagonists than on P2X4 homomeric channels. IATP were potentiated by ivermectin (effect that is specific on P2X4 receptors) and zinc. Western blotting shows the presence of P2X4 protein and RT-PCR the corresponding mRNA transcript in the small intestine. Immunoreactivity for P2X4 receptors was found in most myenteric neurons in culture. Single-cell RT-PCR shows the presence of P2X4 mRNA in 90% of myenteric neurons. Our results indicate that P2X4 receptors are expressed in the majority of myenteric neurons, contribute to the membrane currents activated by ATP, and because most properties of IATP does not correspond to P2X4 homomeric channels it is proposed that P2X4 are forming heteromeric channels in these neurons. P2X4 subunits have a widespread distribution within the myenteric plexus and would be expected to play an important role in cell signaling.