Blots were washed in 0.2 × SSC (1× SSC = 0.15 M sodium chloride/0.015 M sodium citrate, pH 7.0)/0.5% SDS for 40 min. 32P-labeled cDNA probes were hybridized with blots in 5 ml of ExpressHyb solution (CLONTECH) at 68☌ for 1 h. Gel electrophoresis and Northern blotting were performed by using a standard method. Total RNA (5–10 mg) was loaded in each lane in a formaldehyde agarose gel. RNA was isolated from tissues by using TRIzol (Invitrogen). elegans, mutations in DEG/ENaC proteins such as MEC-4 and MEC-10 lead to impaired touch responses ( 8, 9), and the targeted deletion of ASIC2 in mice resulted in a reduced sensitivity of low-threshold mechanoreceptors ( 7). However, there is evidence also that they are involved in mechanosensation many DEG/ENaC proteins are localized to mechanosensitive cells in Caenorhabditis elegans, Drosophila melanogaster, rat, and mouse ( 1, 6, 7). Because these receptors are gated by protons, it has been suggested that they might be involved in the perception of pain during tissue acidosis ( 5). ASIC proteins associate as homo- or heteromultimers to form functional receptors.
In vertebrates, the DEG/ENaC family includes several related subunits of Na +-selective (P NA/P K, 8–40) acid- sensing ion channels (ASIC1a, ASIC1b, ASIC2a, ASC2b, ASIC3, and ASIC4 previously named ASIC-α/BNC2, ASIC-β, MDEG1/BNC1, MDEG2, DRASIC, and SPASIC, respectively) ( 2– 4). The structural hallmarks of these proteins are two hydrophobic transmembrane domains, with short N and C termini and a large extracellular loop. Cation channels of the degenerin/epithelial sodium channel family (DEG/ENaC) have been proposed as transducers of somatosensory stimuli in several species ( 1).
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