The degenerin channels, epithelial sodium channels, and acid-sensing ion channels (DEG/ENaC/ASICs) play important roles in sensing mechanical stimuli, regulating salt homeostasis, or responding to acidification in the nervous system. They share a common topology with two transmembrane domains separated by a large extracellular domain and are believed to assemble as homomeric or heteromeric trimers into non-voltage gated, sodiumselective, and amiloride-sensitive ion channels. Amiloride is not the only drug that targets DEG/ENaC/ASICs, however, they are also emerging as a target of nonsteroidal antiinflammatory drugs (NSAIDs) as well as other classes of small molecules. C. elegans has about 30 genes encoding DEG/ENaC/ASIC subunits and thus offers an excellent opportunity to examine variations in sensitivity to small molecules and biophysical properties. Here, we analyzed a subset of the C. elegans DEG/ENaC/ASIC proteins in order to test the hypothesis that individual family members have distinct properties. Toward this goal, we expressed five C. elegans isoforms in Xenopus laevis oocytes (DEGT-1d, DEL-1d, UNC-8d, MEC-10d and MEC-4d) and measured current amplitude, selectivity among monovalent cations, sensitivity to amiloride and its analogs, and sensitivity to NSAIDs. Of these five proteins, only DEGT-1d, UNC-8d, and MEC-4d form homomeric channels. Unlike MEC-4d and UNC-8d, DEGT-1d channels were insensitive to amiloride and its analogs and more permeable to K + than to Na + . As reported for rat ASIC1a, NSAIDs inhibit DEGT-1d and UNC-8d. Unexpectedly, MEC-4d was strongly potentiated by NSAIDs, an effect that was decreased by mutations in the extracellular domain that affect inhibition of rat ASIC1a. Collectively, these findings reveal that not all DEG/ENaC/ASIC channels are amiloride-sensitive and sodium-selective and that NSAIDs can both inhibit and potentiate these channels.The degenerin channels, epithelial sodium channels, and acid-sensing ion channels (DEG/ENaC/ASICs) are present in most, if not all metazoans and expressed in diverse tissues, including the epithelia of several organs and in the central and peripheral nervous systems (Eastwood and Goodman, 2012; Kellenberger and Schild, 2002). These channels vary in how they are activated in vivo, although the activation mechanisms are not yet known for all family members. At least two DEG proteins are known to be mechanosensitive, ENaCs are constitutively active and can be regulated by shear stress, and ASICs are activated by proton binding (Eastwood and Goodman, 2012). The DEG and ENaC proteins were the initial members of this superfamily. The DEGs were identified in C. elegans by virtue of their role in mechanosensation and neuronal degeneration (Chalfie and Wolinsky, 1990; Driscoll and Chalfie, 1991; Huang and Chalfie, 1994). The ENaCs were identified via expression of rodent cRNAs in Xenopus oocytes followed by functional screening (Canessa et al., 1995). The proteins that form acid-sensing ion channels (ASICs) were also identified in the 1990s (Waldman...

The degenerin channels, epithelial sodium channels, and acid-sensing ion channels (DEG/ENaC/ASICs) play important roles in sensing mechanical stimuli, regulating salt homeostasis, and responding to acidification in the nervous system. They have two transmembrane domains separated by a large extracellular domain and are believed to assemble as homomeric or heteromeric trimers. Based on studies of selected family members, these channels are assumed to form nonvoltage-gated and sodium-selective channels sensitive to the anti-hypertensive drug amiloride. They are also emerging as a target of nonsteroidal anti-inflammatory drugs (NSAIDs). Caenorhabditis elegans has more than two dozen genes encoding DEG/ENaC/ASIC subunits, providing an excellent opportunity to examine variations in drug sensitivity. Here, we analyze a subset of the C. elegans DEG/ENaC/ASIC proteins to test the hypothesis that individual family members vary not only in their ability to form homomeric channels but also in their drug sensitivity. We selected a panel of C. elegans DEG/ENaC/ASICs that are coexpressed in mechanosensory neurons and expressed gain-of-function or d mutants in Xenopus laevis oocytes. We found that only DEGT‑1d, UNC‑8d, and MEC‑4d formed homomeric channels and that, unlike MEC‑4d and UNC‑8d, DEGT‑1d channels were insensitive to amiloride and its analogues. As reported for rat ASIC1a, NSAIDs inhibit DEGT‑1d and UNC‑8d channels. Unexpectedly, MEC‑4d was strongly potentiated by NSAIDs, an effect that was decreased by mutations in the putative NSAID-binding site in the extracellular domain. Collectively, these findings reveal that not all DEG/ENaC/ASIC channels are amiloride-sensitive and that NSAIDs can both inhibit and potentiate these channels.

Pregnenolone (PREG) is a neurosteroid that modulates several brain functions, neuronal firing, glial growth and differentiation, these effects being primarily attributed to PREG actions on the neurons and glial cells. Despite the vital role of the cerebral circulation for brain function and the fact that PREG is a vasoactive agent, PREG actions on cerebral artery function remain unknown. Thus, we first obtained a concentration response curve to PREG on the diameter of de-endothelialized, in vitropressurized middle cerebral arteries (MCA) from C57BL/6 mice. PREG (1nM-100mM) constricted MCA, with maximal constriction reaching 7.2% at $10mM. This constriction was abolished by 1nM paxilline indicating involvement of Ca 2þ -and voltage-gated K þ channels of large conductance (BK). Furthermore, injection of 10mM PREG to mouse carotid artery in vivo constricted MCA by $7.5%, which was equivalent to PREG-induced in vitro constriction. The magnitude of PREG-induced vasoconstriction would result in robust decrease in local blood flow. Patch-clamp recordings on mouse MCA smooth muscle (SM) cells showed that PREG reduced BK activity (NPo) at concentrations that constricted de-endothelialized MCA, resulting in an average decrease in NPo of 56.7%. MCA SM BK include channel-forming (cbv1) and regulatory b1 subunits. Following reconstitution of cbv1 channels into POPE: POPS (3:1) (w/w) bilayers, application of (33mol%) PREG reduced channel activity, evidenced by a right shift in the NPo/NPo max -V curve with a change in V 0.5 from À21.24mV to 0.547mV. This inhibition was blunted by the Tyr450-Phe substitution in the cbv1 cholesterolrecognition amino acid consensus 4 domain (CRAC4). In conclusion, PREG is able of constricting cerebral artery independently of circulating and endothelial factors. Rather, this action involves inhibition of SM BK. PREG-induced BK inhibition does not require b1 subunits but is mediated by CRAC4 located in the cbv1 cytosolic tail. R01-HL147315(AMD).