Previous studies on the rat P2X2 receptor demonstrated that structural modifications to amino acid side chains within the second transmembrane domain lead to receptor activation in the absence of exogenously applied ATP (Rassendren et al, 1997. Cao et al, 2007. Cao et al, 2009). Present work has been aimed towards the characterization of these apparently ATP-independent currents, to investigate the molecular mechanism underlying this phenomenon using a combination of computer modeling, amino acid substitution, heterologous expression in HEK293 cells, the real time modification of engineered cysteines by MTS compounds and electrophysiological techniques. A screen of cysteine substituted receptors at TM2 positions (from G323 to T354) with the membrane permeable MTS compound, MTSP, found that the compound evoked substantial currents from cells expressing P2X2[I328C] receptors, but not from cells expressing other TM2 cysteine substituted, nor wild type receptors. MTSP-evoked currents had similar properties to ATP currents in terms of rectification, NMDG+ permeability and unitary currents. Further investigation indicated that hydrophobic, unbranched modifications to the side chain at position 328 were the most effective. Overall, the results from this work demonstrate that increasing the length and hydrophobicity of an unbranched side-chain at position 328 leads to full receptor activation without the requirement for ATP. These results suggest that the highly conserved native Ile at position 328 stabilizes the closed of the receptor due to its branched nature.