Acid sensing ion channels (ASIC) are cation channels activated by external protons and play roles in nociception, synaptic transmission and in the physiopathology of ischemic stroke. Using luminescence resonance energy transfer (LRET), we show that upon proton binding there is a conformational change that increases LRET efficiency between the probes at the thumb and finger sub-domains in the extracellular domain of ASIC. Additionally we show that this conformational change is lost upon mutating the residues D238, E239, D260 lining the finger domains to alanines. Electrophysiological studies show that the single mutant D260A shifts the EC50 by 0.2 pH units, the double mutant D238A/E239A shifts the EC50 by 2.5 pH units, and the triple mutant D238A/E239A/D260A, exhibits no response to protons despite surface expression. The LRET experiments on the triple mutant D238A/E239A/ D260A shows no changes in the LRET efficiency upon reduction in pH from 8 to 6. The LRET and electrophysiological studies thus suggest that the three carboxylates, two of which form carboxy-carboxylate pairs, are essential for proton induced conformational changes in the extracellular domain, which in turn is necessary for receptor activation.