The focus of this presentation will be on emerging evidence for site-specific regulation of the epithelial sodium channel (ENaC) and its role in Na⁺ and K⁺ homeostasis [1]. In most textbooks ENaC function is discussed in the context of the cortical collecting duct (CCD). ENaC is localised in the apical membrane of CCD principal cells and is the rate-limiting step for Na⁺ absorption. It is inhibited by the K⁺ sparing diuretic amiloride and regulated by aldosterone. Aldosterone binds to a cytosolic mineralocortioid receptor (MR) and by complex transcriptional regulation and signaling pathways stimulates ENaC. This is evident in primary hyperaldosteronism or Conn syndrome. In simplified terms, inappropriately increased ENaC activity due to hyperaldosteronism results in enhanced NaCl absorption, expansion of extracellular fluid volume and hypertension. Conn syndrome is mimicked by gain-of-function mutations of ENaC causing Liddle syndrome, a rare genetic form of salt-sensitive hypertension. This highlights the importance of ENaC in Na⁺ homeostasis and long-term control of blood pressure. Hyperaldosteronism and Liddle syndrome are usually associated with hypokalemia due to a tight functional link between ENaC activity and renal K⁺ secretion via the renal outer medullary K⁺ channel ROMK. The classical concept that ENaC is strictly aldosterone-dependent was challenged by our patch-clamp studies exploring the site-specific regulation of ENaC in microdissected mouse tubules with a focus on two clearly distinguishable regions: The transition zone from the late convoluted tubule (CNT) to the CCD referred to as CNT/CCD, and the transition zone from the late distal convoluted tubule (DCT2) to the early connecting tubule referred to as DCT2/CNT. In an aldosterone synthase deficient mouse model we confirmed the aldosterone dependence of ENaC in CNT/CCD, but made the surprising observation that ENaC activity is aldosterone-independent in DCT2/CNT [2]. Using a mouse model with inducible nephron-specific MR deficiency, we demonstrated that aldosterone-dependent ENaC activity in CNT/CCD was completely MR dependent as expected. Interestingly, MR deficiency also largely reduced aldosterone-independent ENaC activity in DCT2/CNT [3], where MR can probably be stimulated by glucocorticoids due to low expression of 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2). Aldosterone-independent but MR-dependent ENaC activity in DCT2/CNT probably explains, why MR antagonists are efficient blood pressure lowering drugs even when aldosterone levels are not elevated. Moreover, in DCT2/CNT baseline apical ROMK activity is high and a low-K⁺ diet reduces ENaC activity, which highlights the important role of this nephron segment in regulating K⁺ secretion in an aldosterone-independent manner [4]. Future studies are needed to explore whether other hormones or local factors also regulate ENaC activity in a site-specific manner. In this context proteolytic channel activation, a unique feature of ENaC, may be of particular interest. To address this, the physiologically relevant endogenous proteases need to be elucidated. Recently, we identified TMPRSS2 as a candidate protease involved in proteolytic ENaC activation [5], but its role in the kidney remains to be determined. Finally, high resolution cryo-EM structures of ENaC are now available. These structural data open up exciting new horizons to study ENaC function and site-specific regulation at the molecular level.