Abstract
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) tandem-pore 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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY148474
BTREK-1 in adrenal zona fasciculata (AZF) Cells—Previous studies using whole cell and single channel patch clamp recording showed that IAC channels resembled 2P/4TMS channels of the TREK-1 and TRAAK with regard to single channel conductance and rectification (5, 16)
Degenerate primers were designed using the P1 and M2 regions of hTREK-1 and hTRAAK as templates, and 3ЈRACE PCR was performed using AZF cell double-stranded cDNA generated from poly(A)ϩ mRNA
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY148474. IAC displays a number of properties typical of background or leak-type channels that function in setting the resting membrane potential of various cells. In whole cell patch clamp recordings, IAC appears as a noninactivating, outwardly rectifying Kϩ current that displays weak voltage dependence and grows spontaneously over many minutes (1, 3, 4). At the single channel level, the conductance and rectifying properties of these channels depend strongly on the presence of divalent cations (5). In whole cell patch clamp recordings, IAC is inhibited potently by ACTH and angiotensin II, the two peptide hormones that regulate cortisol secretion physiologically (1, 6). Second messengers synthesized or released upon activation of these receptors, including Ca2ϩ and cAMP, inhibit IAC channels. Inhibition appears to involve atypical signaling pathways, because responses to peptides and second messengers are insensitive to inhibition of cAMP and Ca2ϩ-activated protein kinases (3, 5, 6, 9)
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