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Colonic epithelial K+ secretion is a two-step transport process with initial K+ uptake over the basolateral membrane followed by K+ channel-dependent exit into the lumen. In this process the large-conductance, Ca2+-activated KCa1.1 (BK) channel has been identified as the only apparent secretory K+ channel in the apical membrane of the murine distal colon. The BK channel is responsible for both resting and Ca2+-activated colonic K+ secretion and is up-regulated by aldosterone. Agonists (e.g. adrenaline) that elevate cAMP are potent activators of distal colonic K+ secretion. However, the secretory K+ channel responsible for cAMP-induced K+ secretion remains to be defined. In this study we used the Ussing chamber to identify adrenaline-induced electrogenic K+ secretion. We found that the adrenaline-induced electrogenic ion secretion is a compound effect dominated by anion secretion and a smaller electrically opposing K+ secretion. Using tissue from (i) BK wildtype (BK+/+) and knockout (BK−/−) and (ii) cystic fibrosis transmembrane regulator (CFTR) wildtype (CFTR+/+) and knockout (CFTR−/−) mice we were able to isolate the adrenaline-induced K+ secretion. We found that adrenaline-induced K+ secretion: (1) is absent in colonic epithelia from BK−/− mice, (2) is greatly up-regulated in mice on a high K+ diet and (3) is present as sustained positive current in colonic epithelia from CFTR−/− mice. We identified two known C-terminal BK α-subunit splice variants in colonic enterocytes (STREX and ZERO). Importantly, the ZERO variant known to be activated by cAMP is differentially up-regulated in enterocytes from animals on a high K+ diet. In summary, these results strongly suggest that the adrenaline-induced distal colonic K+ secretion is mediated by the BK channel and probably involves aldosterone-induced ZERO splice variant up-regulation.

Mammalian K+ homeostasis results from highly regulated renal and intestinal absorption and secretion, which balances the unregulated K+ intake. Aldosterone is known to enhance both renal and colonic K+ secretion. In mouse distal colon K+ secretion occurs exclusively via luminal KCa1.1 (BK) channels. Here we investigate if aldosterone stimulates colonic K+ secretion via BK channels. Luminal Ba2+ and iberiotoxin (IBTX)-sensitive electrogenic K+ secretion was measured in Ussing chambers. In vivo aldosterone was augmented via a high K+ diet. High K+ diet led to a 2-fold increase of luminal Ba2+ and IBTX-sensitive short-circuit current in distal mouse colonic mucosa. This effect was absent in BK α-subunit-deficient (BK−/−) mice. The resting and diet-induced K+ secretion was stimulated by luminal ionomycin. In BK−/− mice luminal ionomycin did not stimulate K+ secretion. In vitro addition of aldosterone likewise triggered a 2-fold increase in K+ secretion, which was inhibited by the mineralocorticoid receptor antagonist spironolactone and the BK channel blocker IBTX. Semi-quantification of mRNA from colonic crypts showed up-regulation of BK α- and β2-subunits in high K+ diet mice. The BK channel could be detected luminally in colonic crypt cells by immunohistochemistry. The expression level of the channel in the luminal membrane was strongly up-regulated in K+-loaded animals. Taken together, these data strongly suggest that aldosterone-induced K+ secretion occurs via increased expression of luminal BK channels.

Non-technical summary Our ability to respond to stress is critically dependent upon the release of the stress hormone adrenocorticotrophic hormone (ACTH) from corticotroph cells of the anterior pituitary gland. ACTH release is controlled by the electrical properties of corticotrophs that are determined by the movement of ions through channel pores in the plasma membrane. We show that a calcium-activated potassium ion channel called SK4 is expressed in corticotrophs and regulates ACTH release. We provide evidence of how SK4 channels control corticotroph function, which is essential for understanding homeostasis and for treating stress-related disorders. Abstract The anterior pituitary corticotroph is a major control point for the regulation of the hypothalamic–pituitary–adrenal (HPA) axis and the neuroendocrine response to stress. Although corticotrophs are known to be electrically excitable, ion channels controlling the electrical properties of corticotrophs are poorly understood. Here, we exploited a lentiviral transduction system to allow the unequivocal identification of live murine corticotrophs in culture. We demonstrate that corticotrophs display highly heterogeneous spontaneous action-potential firing patterns and their resting membrane potential is modulated by a background sodium conductance. Physiological concentrations of corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) cause a depolarization of corticotrophs, leading to a sustained increase in action potential firing. A major component of the outward potassium conductance was mediated via intermediate conductance calcium-activated (SK4) potassium channels. Inhibition of SK4 channels with TRAM-34 resulted in an increase in corticotroph excitability and exaggerated CRH/AVP-stimulated ACTH secretion in vitro. In accordance with a physiological role for SK4 channels in vivo, restraint stress-induced plasma ACTH and corticosterone concentrations were significantly enhanced in gene-targeted mice lacking SK4 channels (Kcnn4−/−). In addition, Kcnn4−/− mutant mice displayed enhanced hypothalamic c-fos and nur77 mRNA expression following restraint, suggesting increased neuronal activation. Thus, stress hyperresponsiveness observed in Kcnn4−/− mice results from enhanced secretagogue-induced ACTH output from anterior pituitary corticotrophs and may also involve increased hypothalamic drive, thereby suggesting an important role for SK4 channels in HPA axis function.

Mammalian K(+) homeostasis results from highly regulated renal and intestinal absorption and secretion, which balances the unregulated K(+) intake. Aldosterone is known to enhance both renal and colonic K(+) secretion. In mouse distal colon K(+) secretion occurs exclusively via luminal K(Ca)1.1 (BK) channels. Here we investigate if aldosterone stimulates colonic K(+) secretion via BK channels. Luminal Ba(2+) and iberiotoxin (IBTX)-sensitive electrogenic K(+) secretion was measured in Ussing chambers. In vivo aldosterone was augmented via a high K(+) diet. High K(+) diet led to a 2-fold increase of luminal Ba(2+) and IBTX-sensitive short-circuit current in distal mouse colonic mucosa. This effect was absent in BK alpha-subunit-deficient (BK(-/-)) mice. The resting and diet-induced K(+) secretion was stimulated by luminal ionomycin. In BK(-/-) mice luminal ionomycin did not stimulate K(+) secretion. In vitro addition of aldosterone likewise triggered a 2-fold increase in K(+) secretion, which was inhibited by the mineralocorticoid receptor antagonist spironolactone and the BK channel blocker IBTX. Semi-quantification of mRNA from colonic crypts showed up-regulation of BK alpha- and beta(2)-subunits in high K(+) diet mice. The BK channel could be detected luminally in colonic crypt cells by immunohistochemistry. The expression level of the channel in the luminal membrane was strongly up-regulated in K(+)-loaded animals. Taken together, these data strongly suggest that aldosterone-induced K(+) secretion occurs via increased expression of luminal BK channels.