Feedback inhibition of ENaC: Acute and chronic mechanisms

Abstract

modulates the activity of the epithelial Na channel (ENaC) to help prevent cell swelling and regulate epithelial Na C transport, but the underlying mechanisms remain unclear. We show here that short-term (60-80 min) incubation of ENaC-expressing oocytes in high Na C results in a 75% decrease in channel activity. When the b subunit was truncated, corresponding to a gain-of-function mutation found in Liddle's syndrome, the same maneuver reduced activity by 45% despite a larger increase in [Na C ] i . In both cases the inhibition occurred with little to no change in cell-surface expression of gENaC. Long-term incubation (18 hours) in high Na C reduced activity by 92% and 75% in wild-type channels and Liddle's mutant, respectively, with concomitant 70% and 52% decreases in cell-surface gENaC. In the presence of Brefeldin A to inhibit forward protein trafficking, high-Na C incubation decreased wt ENaC activity by 52% and 88% after 4 and 8 hour incubations, respectively. Cleaved gENaC at the cell surface had lifetimes at the surface of 6 hrs in low Na C and 4 hrs in high Na C , suggesting that [Na C ] i increased the rate of retrieval of cleaved g ENaC by 50%. This implies that enhanced retrieval of ENaC channels at the cell surface accounts for part, but not all, of the downregulation of ENaC activity shown with chronic increases in [Na C ] i .

Effects of syntaxins 2, 3, and 4 on rat and human epithelial sodium channel (ENaC) in Xenopus laevis oocytes

“…To distinguish between these mechanisms, we incubated oocytes expressing rENaC alone or together with syntaxin 3 in the presence of brefeldin A, a fungal metabolite which blocks channel delivery from the Golgi apparatus to the plasma membrane [42]. This method has previously been used in several studies with Xenopus laevis oocytes [1,26,41,44,59,65]. In the present study, the inhibitory effect of brefeldin A on ΔI ami was preserved in the presence of syntaxin 3.…”

The N terminus of α-ENaC mediates ENaC cleavage and activation by furin

“…Inhibition of ENaC proteolytic processing has been implicated in Na + feedback inhibition (Anantharam et al, 2006; Knight et al, 2008; Patel et al, 2014). We asked whether the influence of the α-ENaC N terminus that we detected upon ENaC cleavage related to that mode of ENaC regulation.…”

“…BFA is a fungal metabolite that potently and reversibly blocks trafficking of proteins from the ER to the plasma membrane (Misumi et al, 1986; Lippincott-Schwartz et al, 1989). BFA is often used to block ENaC anterograde trafficking and recycling (Butterworth et al, 2005; Patel et al, 2014). As expected from those observations, the I Na of oocytes moved to fresh medium after overnight incubation in BFA increased over 6 h (Fig.…”

“…In the simplest case, this homeostatic regulation of intracellular Na + activity controls the magnitude of epithelial Na + absorption and potentially influences other cellular processes affected by intracellular Na + activity (Ruan et al, 2012; Awayda, 2016). Recent work links the potential physiologic role of Na + feedback inhibition to cleavage of ENaC during its processing to the cell surface (Patel et al, 2014; Heidrich et al, 2015). The Frindt and Palmer (2015) and Myerburg et al (2006) groups showed that increasing intracellular Na + decreases the ENaC cleavage that could be ascribed to furin-like convertases.…”

Brakes and gas-regulation of ENaC by sodium

“…In the 8(5) issue of Channels Patel et al 8 examined the mechanism of feedback inhibition of ENaC by [Na C ] i . They subdivide feedback inhibition into one with a 1-2 hour time course and one with an overnight (>8 h) time course.…”