Eshcol JO, Harding AM, Hattori T, Costa V, Welsh MJ, Benson CJ. Acid-sensing ion channel 3 (ASIC3) cell surface expression is modulated by PSD-95 within lipid rafts. Am J Physiol Cell Physiol 295: C732-C739, 2008. First published June 25, 2008 doi:10.1152/ajpcell.00514.2007.-Acid-sensing ion channel 3 (ASIC3) is a H ϩ -gated cation channel primarily found in sensory neurons, where it may function as a pH sensor in response to metabolic disturbances or painful conditions. We previously found that ASIC3 interacts with the postsynaptic density protein PSD-95 through its COOH terminus, which leads to a decrease in ASIC3 cell surface expression and H ϩ -gated current. PSD-95 has been implicated in recruiting proteins to lipid rafts, which are membrane microdomains rich in cholesterol and sphingolipids that organize receptor/ signaling complexes. We found ASIC3 and PSD-95 coimmunoprecipitated within detergent-resistant membrane fractions. When cells were exposed to methyl--cyclodextrin to deplete membrane cholesterol and disrupt lipid rafts, PSD-95 localization to lipid raft fractions was abolished and no longer inhibited ASIC3 current. Likewise, mutation of two cysteine residues in PSD-95 that undergo palmitoylation (a lipid modification that targets PSD-95 to lipid rafts) prevented its inhibition of ASIC3 current and cell surface expression. In addition, we found that cell surface ASIC3 is enriched in the lipid raft fraction. These data suggest that PSD-95 and ASIC3 interact within lipid rafts and that this raft interaction is required for PSD-95 to modulate ASIC3. protein trafficking; H ϩ -gated channel; PDZ protein THE ACID-SENSING ION CHANNEL 3 (ASIC3) is a member of the degenerin/epithelial sodium channel (DEG/ENaC) family of ion channels that is expressed primarily in mammalian sensory neurons (49). It localizes to nerve terminals, where it may function as a transducer of various sensory stimuli (35). Modest drops in pH activate ASIC3, and several lines of evidence support its role as a metabolic or pain sensor during acidic conditions: 1) ASIC3 is highly expressed in sensory nerves of cardiac (2, 41) and skeletal muscle (31, 32), tissues that have high metabolic activity and are thus susceptible to ischemia leading to production and accumulation of lactic acid; 2) lactate anion potentiates the pH sensitivity of ASIC3, providing a molecular explanation for why lactate is a more potent activator of sensory neurons compared with other acids (23); 3) pharmacological blockade of ASICs attenuate acid-induced pain in human skin (24, 48); and 4) mice lacking ASIC3 demonstrate diminished pain hypersensitivity in models of chronic hyperalgesia (25,40). In addition to its function as a pH sensor, ASIC3 has been implicated in playing a role in mechanosensation;ASIC3 null mice display altered responses to mechanical stimuli in specific populations of mechanosensory neurons (7,25,33,35). To begin to understand the regulation of ASIC3, it is essential to understand the anatomic and molecular environment in which the chann...