The epithelial sodium channel (ENaC) mediates amiloride-sensitive Na⁺ transport in epithelial cells. It is responsible for salt and fluid reabsorption in the distal collecting duct of the kidney, distal colon, lung and the excretory ducts of the exocrine glands. ENaC’s function contributes to Na⁺ and K⁺ homeostasis, regulating blood pressure and maintaining the fluid layer lining the lungs. Diseases associated with defective regulation of ENaC include cystic fibrosis and respiratory infections such as those caused by respiratory syncytial virus¹ and pseudomonas aeruginosa². It has been suggested that the purinergic receptor agonists, UTP and ATP, are released during infections and feedback onto purinergic receptors to cause ENaC inhibition. Consistent with these findings, extracellular application of purinergic receptor agonists also reduces the activity of ENaC. The aim of our studies has been to decipher the key components of the signalling pathway by which purinergic receptor stimulation regulates ENaC activity. Fisher Rat Thyroid (FRT) cells expressing ENaC were used as a model cell line for our studies. Suspected genes involved with the signalling pathway were manipulated using cell transient transfection with plasmid DNA or siRNA. Short circuit currents were monitored using Ussing Chamber electrophysiology. We have found that the purinergic signalling mechanism which regulates ENaC activity involves the activation of apical and basolateral P2Y₂ purinergic receptors, activation of the βγ-subunits of a pertussis toxin sensitive G-protein, and activation of phospholipase Cβ-4. In addition we found an increase in intracellular calcium and basolateral transport of potassium and chloride play a role in this mechanism.