Bovine adrenal zona fasciculata (AZF) cells express a background K؉ channel (I AC ) that sets the resting potential and acts pivotally in ACTH-stimulated cortisol secretion. We have cloned a bTREK-1 (KCNK2) tandempore K ؉ channel cDNA from AZF cells with properties that identify it as the native I AC . The bTREK-1 cDNA is expressed robustly in AZF cells and includes transcripts of 4.9, 3.6, and 2.8 kb. In patch clamp recordings made from transiently transfected cells, bTREK-1 displayed distinctive properties of I AC in AZF cells. Specifically, bTREK-1 currents were outwardly rectifying with a large instantaneous and smaller time-dependent component. Similar to I AC , bTREK-1 increased spontaneously in amplitude over many minutes of whole cell recording and was blocked potently by Ca 2؉ antagonists including penfluridol and mibefradil and by 8-(4-chlorophenylthio)-cAMP. Unitary TREK-1 and I AC currents were nearly identical in amplitude. The native I AC current, in turn, displayed properties that together are specific to TREK-1 K ؉ channels. These include activation by intracellular acidification, enhancement by the neuroprotective agent riluzole, and outward rectification. bTREK-1 current differed from native K ؉ current only in its lack of ATP dependence. In contrast to I AC , the current density of bTREK-1 in human embryonic kidney-293 cells was not increased by raising pipette ATP from 0.1 to 5 mM. Further, the enhancement of I AC current in AZF cells by low pH and riluzole was facilitated by, and dependent on, ATP at millimolar concentrations in the pipette solution. Overall, these results establish the identity of I AC K ؉ channels, demonstrate the expression of bTREK-1 in a specific endocrine cell, identify potent new TREK-1 antagonists, and assign a pivotal role for these tandem-pore channels in the physiology of cortisol secretion. The activation of I AC by ATP indicates that native bTREK-1 channels may function as sensors that couple the metabolic state of the cell to membrane potential, perhaps through an associated ATP-binding protein.

The United States Food and Drug Administration (FDA) uses alfuzosin as an example of a drug having QT risk in humans that was not detected in nonclinical studies. FDA approval required a thorough clinical QT study (TCQS) that was weakly positive at high doses. The FDA has used the clinical/nonclinical discordance as a basis for mandatory TCQS, and this requirement has serious consequences for drug development. For this reason, we re-examined whether nonclinical signals of QT risk for alfuzosin were truly absent. Alfuzosin significantly prolonged action potential duration (APD) 60 in rabbit Purkinje fibers (p Ͻ 0.05) and QT in isolated rabbit hearts (p Ͻ 0.05) at the clinically relevant concentration of 300 nM. In man, the QT interval corrected with Fridericia's formula increased 7.7 ms, which exceeds the 5.0-ms threshold for a positive TCQS. Effects on hK v 11.1, hK v 4.3, and hK v 7.1/hKCNE1 potassium currents and calcium current were not involved. At 300 nM, ϳ30ϫ C max , alfuzosin significantly increased whole-cell peak sodium (hNa v 1.5) current (p Ͻ 0.05), increased the probability of late hNa v 1.5 single-channel openings, and significantly shortened the slow time constant for recovery from inactivation. Alfuzosin also increased hNa v 1.5 burst duration and number of openings per burst between 2-and 3-fold. Alfuzosin is a rare example of a non-antiarrhythmic drug that delays cardiac repolarization not by blocking hK v 11.1 potassium current, but by increasing sodium current. Nonclinical studies clearly show that alfuzosin increases plateau potential and prolongs APD and QT, consistent with QT prolongation in man. The results challenge the FDA grounds for the absolute primacy of TCQS based on the claim of a false-negative, nonclinical study on alfuzosin.

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In whole-cell and single-channel patch-clamp recordings from bovine adrenal fasciculata cells,

Bovine adrenocortical cells express bTREK-1 K ؉ channels that set the resting membrane potential (V m ) and couple angiotensin II (AngII) and adrenocorticotropic hormone (ACTH) receptors to membrane depolarization and corticosteroid secretion. In this study, it was discovered that AngII inhibits bTREK-1 by separate Ca 2؉ -and ATP hydrolysis-dependent signaling pathways. When whole cell patch clamp recordings were made with pipette solutions that support activation of both Ca 2؉ -and ATP-dependent pathways, AngII was significantly more potent and effective at inhibiting bTREK-1 and depolarizing adrenal zona fasciculata cells, than when either pathway is activated separately. External ATP also inhibited bTREK-1 through these two pathways, but ACTH displayed no Ca 2؉ -dependent inhibition. AngII-mediated inhibition of bTREK-1 through the novel Ca 2؉ -dependent pathway was blocked by the AT 1 receptor antagonist losartan, or by including guanosine-5-O-(2-thiodiphosphate) in the pipette solution. The Ca 2؉ -dependent inhibition of bTREK-1 by AngII was blunted in the absence of external Ca 2؉ or by including the phospholipase C antagonist U73122, the inositol 1,4,5-trisphosphate receptor antagonist 2-amino-ethoxydiphenyl borate, or a calmodulin inhibitory peptide in the pipette solution. The activity of unitary bTREK-1 channels in inside-out patches from adrenal zona fasciculata cells was inhibited by application of Ca 2؉ (5 or 10 M) to the cytoplasmic membrane surface. The Ca 2؉ ionophore ionomycin also inhibited bTREK-1 currents through channels expressed in CHO-K1 cells. These results demonstrate that AngII and selected paracrine factors that act through phospholipase C inhibit bTREK-1 in adrenocortical cells through simultaneous activation of separate Ca 2؉ -and ATP hydrolysis-dependent signaling pathways, providing for efficient membrane depolarization. The novel Ca 2؉ -dependent pathway is distinctive in its lack of ATP dependence, and is clearly different from the calmodulin kinase-dependent mechanism by which AngII modulates T-type Ca 2؉ channels in these cells.