Functional and Pharmacological Characterization of C. elegans DEG/ENaC/ASIC Channels

Abstract

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).

Mechano-gated channels in C. elegans

“…In contrast, degenerins (DEG) are C. elegans specific ion channels characterized for the unusual gain-offunction mutations which induce degenerations like swelling, vacuolation and eventually cell death (Hall et al, 1997). So far, about 30 genes encoding DEG/ENaC superfamily have been discovered in the C. elegans genome (Fechner et al, 2020). Studies in mechanoreceptor currents of DEG/ENaC channels revealed similarities to mechanoelectrical transduction of gentle and nociceptive mechanical stimuli.…”