Peripheral initiators of muscle pain are virtually unknown, but likely key to development of chronic pain after muscle insult. The current study tested the hypothesis that ASIC3 in muscle is necessary for development of cutaneous mechanical, but not heat hyperalgesia induced by muscle inflammation. Using mechanical and heat stimuli, we assessed behavioral responses in ASIC3−/− and ASIC3+/+ mice after induction of carrageenan muscle inflammation. ASIC3−/−mice did not develop cutaneous mechanical hyperalgesia after muscle inflammation when compared to ASIC3+/ + mice; heat hyperalgesia developed similarly between groups. We then tested if the phenotype could be rescued in ASIC3−/− mice by using a recombinant herpes virus vector to express ASIC3 in skin (where testing occurred) or muscle (where inflammation occurred). Infection of mouse DRG neurons with ASIC3-encoding virus resulted in functional expression of ASICs. Injection of ASIC3-encoding virus into muscle or skin of ASIC3−/− mice resulted in ASIC3 mRNA in DRG and protein expression in DRG and the peripheral injection site. Injection of ASIC3-encoding virus into muscle, but not skin, resulted in development of mechanical hyperalgesia similar to that observed in ASIC3+/+ mice. Thus, ASIC3 in primary afferent fibers innervating muscle is critical to development of hyperalgesia that results from muscle insult.

Acid-sensing ion channels (ASICs) are H؉ -gated members of the degenerin/epithelial Na ؉ channel (DEG/ ENaC) family in vertebrate neurons. Several ASICs are expressed in sensory neurons, where they play a role in responses to nociceptive, taste, and mechanical stimuli; others are expressed in central neurons, where they participate in synaptic plasticity and some forms of learning. Stomatin is an integral membrane protein found in lipid/protein-rich microdomains, and it is believed to regulate the function of ion channels and transporters. In Caenorhabditis elegans, stomatin homologs interact with DEG/ENaC channels, which together are necessary for normal mechanosensation in the worm. Therefore, we asked whether stomatin interacts with and modulates the function of ASICs. We found that stomatin co-immunoprecipitated and co-localized with ASIC proteins in heterologous cells. Moreover, stomatin altered the function of ASIC channels. Stomatin potently reduced acid-evoked currents generated by ASIC3 without changing steady state protein levels or the amount of ASIC3 expressed at the cell surface. In contrast, stomatin accelerated the desensitization rate of ASIC2 and heteromeric ASICs, whereas current amplitude was unaffected. These data suggest that stomatin binds to and alters the gating of ASICs. Our findings indicate that modulation of DEG/ENaC channels by stomatin-like proteins is evolutionarily conserved and may have important implications for mammalian nociception and mechanosensation.

Water-borne illness, primarily caused by fecal contamination of drinking water, is a major health burden in the state of Andhra Pradesh, India. Currently drinking water is treated at the reservoir level and supplied on alternate days, necessitating storage in households for up to 48 hrs. We hypothesized that fecal contamination occurs principally during storage due to poor water handling. In this study we tested for coliform bacteria in water samples collected at distribution points as household storage containers were filled, and then tested containers in the same households 24-36 hours after collection. We also conducted an observational survey to make an assessment of water handling and hygiene. Ninety-two percent (47/51) of samples tested at supply points were adequately chlorinated and bacterial contamination was found in two samples with no residual chlorine. Samples collected from household storage containers showed an increase in contamination in 18/50 houses (36%). Households with contaminated stored samples did not show significant differences in demographics, water handling, hygiene practices, or sanitation. Nevertheless, the dramatic increase in contamination after collection indicates that until an uninterrupted water supply is possible, the point at which the biggest health impact can be made is at the household level.

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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...