The exact subunit combinations of functional native acid-sensing ion channels (ASICs) have not been established yet, but both homomeric and heteromeric channels are likely to exist. To determine the ability of different subunits to assemble into heteromeric channels, a number of ASIC1a-, ASIC1b-, ASIC2a-, ASIC2b-, and ASIC3-containing homo-and heteromeric channels were studied by whole-cell patch clamp recordings with respect to pH sensitivity, desensitization kinetics, and level of sustained current normalized to peak current. Analyzing and comparing data for these three features demonstrated unique heteromeric channels in a number of co-expression experiments. Formation of heteromeric ASIC1a؉2a and ASIC1b؉2a channels was foremost supported by the desensitization characteristics that were independent of proton concentration, a feature none of the respective homomeric channels has. Several lines of evidence supported formation of ASIC1a؉3, ASIC1b؉3, and ASIC2a؉3 heteromeric channels. The most compelling was the desensitization characteristics, which, besides being proton-independent, were faster than those of any of the respective homomeric channels. ASIC2b, which homomerically expressed is not activated by protons per se, did not appear to form unique heteromeric combinations with other subunits and in fact appeared to suppress the function of ASIC1b. Co-expression of three subunits such as ASIC1a؉2a؉3 and ASIC1b؉2a؉3 resulted in data that could best be explained by coexistence of multiple channel populations within the same cell. This observation seems to be in good agreement with the fact that ASIC-expressing sensory neurons display a variety of acid-evoked currents.It is well established that tissue acidification, which may be present in inflammatory and ischemic conditions, causes pain (1-3). In line with this, peripheral sensory neurons exhibit sensitivity toward acid by activating several types of depolarizing currents (4 -6). Although the repertoire of ion channels responsible for these currents is not fully known, the family of acid-sensing ion channels (ASICs) 1 is believed to be an important constituent. The ASIC family is a member of the ENaC/ DEG superfamily, which also includes the amiloride-sensitive epithelial sodium channels (ENaCs), the mechanically gated degenerins of Caenorhabditis elegans (DEGs), and a neuropeptide-gated channel of Helix aspersa (FaNaC). The membrane topology of all channels within this superfamily comprises two transmembrane domains, intracellular N and C termini and a large extracellular loop with a number of conserved cysteine residues (7). Currently, four genes encoding six ASIC transcripts have been cloned and characterized from mammalian organisms. ASIC1a (BNaC2) and ASIC1b (ASIC1␤) are the products of alternatively spliced transcripts of the ASIC1 gene that differ in the N-terminal region, including the first transmembrane domain and the proximal part of the large extracellular domain (8 -10). ASIC2a (BNaC1, MDEG) and ASIC2b (MDEG2) are alternatively spliced forms of ...