The epithelial Na ؉ channel (ENaC) regulates epithelial salt and water reabsorption, processes that require significant expenditure of cellular energy. To test whether the ubiquitous metabolic sensor AMP-activated kinase (AMPK) regulates ENaC, we examined the effects of AMPK activation on amiloride-sensitive currents in Xenopus oocytes and polarized mouse collecting duct mpkCCD c14 cells. Microinjection of oocytes expressing mouse ENaC (mENaC) with either active AMPK protein or an AMPK activator inhibited mENaC currents relative to controls as measured by two-electrode voltageclamp studies. Similarly, pharmacological AMPK activation or overexpression of an activating AMPK mutant in mpkCCD c14 cells inhibited amiloride-sensitive short circuit currents. Expression of a degenerin mutant -mENaC subunit (S518K) along with wild type ␣ and ␥ increased the channel open probability (P o ) to ϳ1. However, AMPK activation inhibited currents similarly with expression of either degenerin mutant or wild type mENaC. Single channel recordings under these conditions demonstrated that neither P o nor channel conductance was affected by AMPK activation. Moreover, expression of a Liddle's syndrome-type -mENaC mutant (Y618A) greatly enhanced ENaC whole cell currents relative to wild type ENaC controls and prevented AMPKdependentinhibition.ThesefindingsindicatethatAMPKdependent ENaC inhibition is mediated through a decrease in the number of active channels at the plasma membrane (N), presumably through enhanced Nedd4-2-dependent ENaC endocytosis. The AMPK-ENaC interaction appears to be indirect; AMPK did not bind ENaC in cells, as assessed by in vivo pull-down assays, nor did it phosphorylate ENaC in vitro. In summary, these results suggest a novel mechanism for coupling ENaC activity and renal Na ؉ handling to cellular metabolic status through AMPK, which may help prevent cellular Na ؉ loading under hypoxic or ischemic conditions.The maintenance of transmembrane ion and solute gradients is a crucial process that permits normal cellular functioning, viability, and coordinated transepithelial transport but also consumes a substantial portion of total cellular energy (1). Under conditions of metabolic stress, the expression and activity of many membrane transport proteins including ENaC 1 are inhibited, thereby limiting the dissipation of ionic gradients and preserving the cellular energy required to maintain them (2). However, the cellular mechanisms that link membrane transport to energy production and metabolic status have remained elusive.AMPK is a ubiquitous metabolic-sensing Ser/Thr kinase that exists as a heterotrimer composed of a catalytic ␣ subunit and regulatory  and ␥ subunits. Its activity increases during conditions of metabolic stress, in response to elevated intracellular AMP:ATP ratios (3). A parallel activation pathway involves phosphorylation of the ␣ subunit by a recently identified upstream LKB1 kinase complex at a Thr residue in its activation loop (4). The earliest discovered substrates of AMPK were important ...