ASIC3 is an acid-sensing ion channel expressed in sensory neurons, where it participates in acidic and inflammatory pain. In addition to the "classical" transient current, ASIC3 generates a sustained current essential for pain perception. Using chimeras between the ASIC3 and ASIC1a channels we show that the first transmembrane domain (TM1), combined with the N-terminal domain, is the key structural element generating the low pH (<6.5)-evoked sustained current. The TM1 domain also modulates the pH-dependent activation of the fast transient current thus contributing to a constitutive window current, another type of sustained current present near physiological pH. The C-terminal and the TM2 domains negatively regulate both types of sustained current, and the extracellular loop affects its kinetics. These data provide new information to aid understanding the mechanisms of the multifaceted pH gating of ASIC3. Together with the peak current, both components of the sustained current (window and sustained at pH <6.5) allow ASIC3 to adapt its behavior to a wide range of extracellular pH variations by generating transient and/or sustained responses that contribute to nociceptor excitability.Ischemia, inflammation, tumors, or tissue damages are associated with a decrease in extracellular pH (1). Activation of nociceptive sensory nerve endings by tissue acidosis correlates with pain sensations in human and rodents (1-4). The acidsensing ion channel 3 (ASIC3) 2 is predominantly expressed in sensory neurons and has been recently shown to be a sensor of acid-mediated and primary inflammatory pain in rats (4). ASIC3 belongs to the acid-sensing ion channel family, which comprises at least seven isoforms encoded by four different genes (5-7). These isoforms, which comprise cytosolic N and C termini, two transmembrane helices, and a disulfide-rich, multidomain extracellular region, can associate into homo-or heterotrimers (8, 9). ASICs are amiloride-sensitive voltage-independent cationic channels that are activated by a decrease in extracellular pH. Protons trigger a transient inward current that desensitizes rapidly in all of the ASICs except ASIC3, which carries in addition to the transient current a sustained current that does not fully inactivate while the pH remains acidic (10 -12). For moderate extracellular acidification (between pH 7.3 and 6.7 for the rat ASIC3 channel), the sustained current results from the window of overlap between inactivation and activation of the fast transient current (4, 13), whereas the sustained current that is activated by lower pH seems to involve a different, uncharacterized mechanism. Despite the important role proposed for the ASIC3-sustained current in pain perception (4, 13, 14), very little is known about the molecular mechanisms underlying this current. This paper identifies the key structural elements involved in the generation and in the modulation of the window current (I window ) observed at modest acidifications near the physiological pH and of the low pH (Ͻ6.5)-evoked sustained cur...