TASK-3 dominates the background potassium conductance in rat adrenal glomerulosa cells

In a preceding study we showed that the highly negative resting membrane potential of rat adrenal glomerulosa cells is related to background potassium channel(s), which belong to the two-pore domain channel family. TWIK-related acid-sensitive K+ channel (TASK-1) expression was found in glomerulosa tissue, and the currents elicited by injection of glomerulosa mRNA (I(glom)) or TASK-1 cRNA (I(TASK-1)) showed remarkable similarity in Xenopus laevis oocytes. However, based on the different sensitivity of these currents to acidification, we concluded that TASK-1 may be responsible for a maximum of 25% of the weakly pH-dependent glomerulosa background K+ current. Here we demonstrate that TASK-3, a close relative of TASK-1, is expressed abundantly in glomerulosa cells. Northern blot detected TASK-3 message in adrenal glomerulosa, but not in other tissues. Quantitative RT-PCR experiments indicated even higher mRNA expression of TASK-3 than TASK-1 in glomerulosa tissue. Similarly to the glomerulosa background current, the current expressed by injection of TASK-3 cRNA (I(TASK-3)) was less acid-sensitive than I(TASK-1). Ruthenium red in the micromolar range inhibited I(glom) and I(TASK-3), but not I(TASK-1). Like I(TASK-1), I(TASK-3) was inhibited by stimulation of AT1a angiotensin II receptor coexpressed with the potassium channel. The high level of expression and its pharmacological properties suggest that TASK-3 dominates the resting potassium conductance of glomerulosa cells.     

Intracellular mediators of potassium-induced aldosterone secretion

We have investigated the intracellular messengers of potassium in eliciting aldosterone secretion in calf adrenal glomerulosa cells since there were unresolved issues relating to the role of phosphoinositides, cAMP and protein kinases. We observed no evidence of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in 3H-inositol labeled alf adrenal cells or increase of cAMP in response to potassium. Addition of calcium channel blocker, nitrendipine after stimulating adrenal glomerulosa cells with potassium, markedly inhibited aldosterone secretion. A calmodulin inhibitor (W-7) produced greater reduction of aldosterone secretion than an inhibitor of protein kinase C (H-7). These results suggest that a rise in cytosolic free calcium concentration through voltage-dependent calcium channel and calmodulin are the critical determinants of aldosterone secretion stimulated by potassium.     

TREK-1 K+ channels couple angiotensin II receptors to membrane depolarization and aldosterone secretion in bovine adrenal glomerulosa cells

Bovine adrenal glomerulosa (AZG) cells were shown to express bTREK-1 background K(+) channels that set the resting membrane potential and couple angiotensin II (ANG II) receptor activation to membrane depolarization and aldosterone secretion. Northern blot and in situ hybridization studies demonstrated that bTREK-1 mRNA is uniformly distributed in the bovine adrenal cortex, including zona fasciculata and zona glomerulosa, but is absent from the medulla. TASK-3 mRNA, which codes for the predominant background K(+) channel in rat AZG cells, is undetectable in the bovine adrenal cortex. In whole cell voltage clamp recordings, bovine AZG cells express a rapidly inactivating voltage-gated K(+) current and a noninactivating background K(+) current with properties that collectively identify it as bTREK-1. The outwardly rectifying K(+) current was activated by intracellular acidification, ATP, and superfusion of bTREK-1 openers, including arachidonic acid (AA) and cinnamyl 1-3,4-dihydroxy-alpha-cyanocinnamate (CDC). Bovine chromaffin cells did not express this current. In voltage and current clamp recordings, ANG II (10 nM) selectively inhibited the noninactivating K(+) current by 82.1 +/- 6.1% and depolarized AZG cells by 31.6 +/- 2.3 mV. CDC and AA overwhelmed ANG II-mediated inhibition of bTREK-1 and restored the resting membrane potential to its control value even in the continued presence of ANG II. Vasopressin (50 nM), which also physiologically stimulates aldosterone secretion, inhibited the background K(+) current by 73.8 +/- 9.4%. In contrast to its potent inhibition of bTREK-1, ANG II failed to alter the T-type Ca(2+) current measured over a wide range of test potentials by using pipette solutions of identical nucleotide and Ca(2+)-buffering compositions. ANG II also failed to alter the voltage dependence of T channel activation under these same conditions. Overall, these results identify bTREK-1 K(+) channels as a pivotal control point where ANG II receptor activation is transduced to depolarization-dependent Ca(2+) entry and aldosterone secretion.     

A Ba2+-resistant, acid-sensitive K+ conductance in Na+-absorbing H441 human airway epithelial cells

By analysis of whole cell membrane currents in Na(+)-absorbing H441 human airway epithelial cells, we have identified a K(+) conductance (G(K)) resistant to Ba(2+) but sensitive to bupivacaine or extracellular acidification. In polarized H441 monolayers, we have demonstrated that bupivacaine, lidocaine, and quinidine inhibit basolateral membrane K(+) current (I(Bl)) whereas Ba(2+) has only a weak inhibitory effect. I(Bl) was also inhibited by basolateral acidification, and, although subsequent addition of bupivacaine caused a further fall in I(Bl), acidification had no effect after bupivacaine, demonstrating that cells grown under these conditions express at least two different bupivacaine-sensitive K(+) channels, only one of which is acid sensitive. Basolateral acidification also inhibited short-circuit current (I(SC)), and basolateral bupivacaine, lidocaine, quinidine, and Ba(2+) inhibited I(SC) at concentrations similar to those needed to inhibit I(Bl), suggesting that the K(+) channels underlying I(Bl) are part of the absorptive mechanism. Analyses using RT-PCR showed that mRNA encoding several two-pore domain K(+) (K2P) channels was detected in cells grown under standard conditions (TWIK-1, TREK-1, TASK-2, TWIK-2, KCNK-7, TASK-3, TREK-2, THIK-1, and TALK-2). We therefore suggest that K2P channels underlie G(K) in unstimulated cells and so maintain the driving force for Na(+) absorption. Since this ion transport process is vital to lung function, K2P channels thus play an important but previously undocumented role in pulmonary physiology.     

TASK-5, a new member of the tandem-pore K(+) channel family.

TASKs are members of the recently identified K(+) channel family (KCNKx). Four TASKs (TASK1-4) identified so far form functional K(+) channels and encode background K(+) channels in various cell types. Recently, another member (TASK-5) was identified in the human genome. We cloned it and studied its tissue expression and functional properties. TASK-5 shares 51% amino acid identity with TASK-1 and TASK-3. Northern blot analysis showed that TASK-4 mRNA was expressed primarily in the adrenal gland and pancreas. Single nucleotide polymorphism (SNP) was found at amino acid position 95 that normally forms part of the K(+) channel selectivity filter. Neither form of TASK-5 showed channel activity when transfected in COS-7 cells. Exchange of C-termini of TASK-3 and TASK-5 failed to generate whole-cell currents. Thus, TASK-5 is a new member of the tandem-pore K(+) channel family but does not produce a functional plasma membrane K(+) current by itself.     

Sequential phosphorylation mediates receptor- and kinase-induced inhibition of TREK-1 background potassium channels

Background potassium channels determine membrane potential and input resistance and serve as prominent effectors for modulatory regulation of cellular excitability. TREK-1 is a two-pore domain background K+ channel (KCNK2, K2P2.1) that is sensitive to a variety of physicochemical and humoral factors. In this work, we used a recombinant expression system to show that activation of G alpha(q)-coupled receptors leads to inhibition of TREK-1 channels via protein kinase C (PKC), and we identified a critical phosphorylation site in a key regulatory domain that mediates inhibition of the channel. In HEK 293 cells co-expressing TREK-1 and either the thyrotropin-releasing hormone receptor (TRHR1) or the Orexin receptor (Orx1R), agonist stimulation induced robust channel inhibition that was suppressed by a bisindolylmaleimide PKC inhibitor but not by a protein kinase A blocker ((R(p))-cAMP-S). Channel inhibition by agonists or by direct activators of PKC (phorbol dibutyrate) and PKA (forskolin) was disrupted not only by alanine or aspartate mutations at an identified PKA site (Ser-333) in the C terminus, but also at a more proximal regulatory site in the cytoplasmic C terminus (Ser-300); S333A and S300A mutations enhanced basal TREK-1 current, whereas S333D and S300D substitutions mimicked phosphorylation and strongly diminished currents. When studied in combination, TREK-1 current density was enhanced in S300A/S333D but reduced in S300D/S333A mutant channels. Channel mutants were expressed and appropriately targeted to cell membranes. Together, these data support a sequential phosphorylation model in which receptor-induced kinase activation drives modification at Ser-333 that enables subsequent phosphorylation at Ser-300 to inhibit TREK-1 channel activity.     

Evidence for two distinct voltage-gated calcium channel currents in bovine adrenal glomerulosa cells

We analyzed inward Ca2+ currents in single bovine adrenal glomerulosa cell using whole-cell patch clamp techniques. Two types of voltage-gated Ca2+ channel currents were identified. One was a transient (T) type which decayed within 100 ms, characterized by a low threshold voltage (about -70 mv) similar to that seen in rat adrenal glomerulosa cells (Matsunaga, H. et al. (1987) Pflügers Arch. 408, 351-355.) Another was a long-lasting (L) type which shows a more positive threshold potential. The present results suggest that while T type Ca2+ channels may explain initial calcium influx in response to an elevation in extracellular K+, L type Ca2+ channels may allow sustained calcium influx which is necessary for sustained aldosterone secretion.     

Invalidation of TASK1 potassium channels disrupts adrenal gland zonation and mineralocorticoid homeostasis

TASK1 (KCNK3) and TASK3 (KCNK9) are two-pore domain potassium channels highly expressed in adrenal glands. TASK1/TASK3 heterodimers are believed to contribute to the background conductance whose inhibition by angiotensin II stimulates aldosterone secretion. We used task1-/- mice to analyze the role of this channel in adrenal gland function. Task1-/- exhibited severe hyperaldosteronism independent of salt intake, hypokalemia, and arterial 'low-renin' hypertension. The hyperaldosteronism was fully remediable by glucocorticoids. The aldosterone phenotype was caused by an adrenocortical zonation defect. Aldosterone synthase was absent in the outer cortex normally corresponding to the zona glomerulosa, but abundant in the reticulo-fasciculata zone. The impaired mineralocorticoid homeostasis and zonation were independent of the sex in young mice, but were restricted to females in adults. Patch-clamp experiments on adrenal cells suggest that task3 and other K+ channels compensate for the task1 absence. Adrenal zonation appears as a dynamic process that even can take place in adulthood. The striking changes in the adrenocortical architecture in task1-/- mice are the first demonstration of the causative role of a potassium channel in development/differentiation.     

双孔钾通道TASK-1的研究进展

双孔钾离子通道是一种背景钾离子通道,广泛分布于各种兴奋和非兴奋细胞中,并具有许多重要的生理功能。TASK-1是双孔钾离子通道家族的重要一员,它对缺氧和细胞外酸化敏感,参与形成心肌动作电住平台期,调节呼吸、肺动脉平滑肌收缩和醛固酮的分泌,并且是麻醉剂的作用靶点,人们不断对其进行研究并取得了很多重要结果,本文将概述双孔钾通道TASK-1的研究进展。     

Diazepam inhibits potassium-induced aldosterone secretion in adrenal glomerulosa cell

In an attempt to elucidate a possible role of peripheral benzodiazepine receptor in adrenal glomerulosa cell, effect of diazepam on potassium-induced aldosterone secretion was studied using isolated bovine adrenal glomerulosa cell. Diazepam inhibited aldosterone secretion stimulated by 8mM potassium in a dose dependent manner. The ID50 was approximately 14 nM. Although diazepam inhibited potassium action effectively, forskolin-induced aldosterone secretion was not affected by diazepam. These results indicate that peripheral benzodiazepine receptor may have an active role in regulating aldosterone secretion. The voltage dependent calcium channel may be a possible site of benzodiazepine action in this tissue.     

Differential effects of volatile and intravenous anesthetics on the activity of human TASK-1

Volatile anesthetics have been shown to activate various two-pore (2P) domain K⁺ (K_2P) channels such as TASK-1 and TREK-1 (TWIK-related acid-sensitive K⁺ channel), and mice deficient in these channels are resistant to halothane-induced anesthesia. Here, we investigated whether K_2P channels were also potentially important targets of intravenous anesthetics. Whole cell patch-clamp techniques were used to determine the effects of the commonly used intravenous anesthetics etomidate and propofol on the acid-sensitive K⁺ current in rat ventricular myocytes (which strongly express TASK-1) and selected human K_2P channels expressed in Xenopus laevis oocytes. In myocytes, etomidate decreased both inward rectifier K⁺ (K_ir) current (I_K1) and acid-sensitive outward K⁺ current at positive potentials, suggesting that this drug may inhibit TASK channels. Indeed, in addition to inhibiting guinea pig Kir2.1 expressed in oocytes, etomidate inhibited human TASK-1 (and TASK-3) in a concentration-dependent fashion. Propofol had no effect on human TASK-1 (or TASK-3) expressed in oocytes. Moreover, we showed that, similar to the known effect of halothane, sevoflurane and the purified R-(–)- and S-(+)-enantiomers of isoflurane, without stereoselectivity, activated human TASK-1. We conclude that intravenous and volatile anesthetics have dissimilar effects on K_2P channels. Human TASK-1 (and TASK-3) are insensitive to propofol but are inhibited by supraclinical concentrations of etomidate. In contrast, stimulatory effects of sevoflurane and enantiomeric isoflurane on human TASK-1 can be observed at clinically relevant concentrations. volatile anesthetics; etomidate; propofol; ion channels     

3H]nitrendipine binding to adrenal capsular membranes

The physiologic regulation of aldosterone secretion is dependent on extracellular calcium and appears to be mediated by increases in cytosolic free calcium concentration in the zona glomerulosa cell. A specific role for voltage-dependent calcium channels was suggested by previous studies with the calcium channel antagonist verapamil. We therefore studied the [3H]nitrendipine calcium channel binding site in adrenal capsules. These studies revealed a single class of saturable, high affinity sites with KD = .26 +/- .04 nM and Bmax = 105 +/- 5.7 fmol/mg protein. Specific binding of [3H]nitrendipine was inhibited by calcium channel antagonists with potencies nitrendipine = nifedipine much greater than verapamil, while diltiazem had no inhibitory effect. In the rat, binding sites for [3H]nitrendipine were located in the adrenal capsule and medulla and were undetectable in the zona fasciculata. Physiologic studies with collagenase-dispersed adrenal glomerulosa cells demonstrated that nifedipine selectively inhibited angiotensin-II and potassium-stimulated steroidogenesis. These observations suggest both a pharmacologic and physiologic role for the nitrendipine binding site in aldosterone production.     

Expression of Stretch-Activated Two-Pore Potassium Channels in Human Myometrium in Pregnancy and Labor

Background

We tested the hypothesis that the stretch-activated, four-transmembrane domain, two pore potassium channels (K2P), TREK-1 and TRAAK are gestationally-regulated in human myometrium and contribute to uterine relaxation during pregnancy until labor.

Methodology

We determined the gene and protein expression of K2P channels in non-pregnant, pregnant term and preterm laboring myometrium. We employed both molecular biological and functional studies of K2P channels in myometrial samples taken from women undergoing cesarean delivery of a fetus.

Principal Findings

TREK-1, but not TREK-2, channels are expressed in human myometrium and significantly up-regulated during pregnancy. Down-regulation of TREK-1 message was seen by Q-PCR in laboring tissues consistent with a role for TREK-1 in maintaining uterine quiescence prior to labor. The TRAAK channel was unregulated in the same women. Blockade of stretch-activated channels with a channel non-specific tarantula toxin (GsMTx-4) or the more specific TREK-1 antagonist L-methionine ethyl ester altered contractile frequency in a dose-dependent manner in pregnant myometrium. Arachidonic acid treatment lowered contractile tension an effect blocked by fluphenazine. Functional studies are consistent with a role for TREK-1 in uterine quiescence.

Conclusions

We provide evidence supporting a role for TREK-1 in contributing to uterine quiescence during gestation and hypothesize that dysregulation of this mechanism may underlie certain cases of spontaneous pre-term birth.     

Modulation of TASK-1 (Kcnk3) and TASK-3 (Kcnk9) potassium channels: volatile anesthetics and neurotransmitters share a molecular site of action

TASK-1 and TASK-3, members of the two-pore-domain channel family, are widely expressed leak potassium channels responsible for maintenance of cell membrane potential and input resistance. They are sites of action for a variety of modulatory agents, including volatile anesthetics and neurotransmitters/hormones, the latter acting via mechanisms that have remained elusive. To clarify these mechanisms, we generated mutant channels and found that alterations disrupting anesthetic (halothane) activation of these channels also disrupted transmitter (thyrotropin-releasing hormone, TRH) inhibition and did so to a similar degree. For both TASK-1 and TASK-3, mutations (substitutions with corresponding residues from TREK-1) in a six-residue sequence at the beginning of the cytoplasmic C terminus virtually abolished both anesthetic activation and transmitter inhibition. The only sequence motif identified with a classical signaling mechanism in this region is a potential phosphorylation site; however, mutation of this site failed to disrupt modulation. TASK-1 and TASK-3 differed insofar as a large portion of the C terminus was necessary for the full effects of halothane and TRH on TASK-3 but not on TASK-1. Finally, tandem-linked TASK-1/TASK-3 heterodimeric channels were fully modulated by anesthetic and transmitter, and introduction of the identified mutations either into the TASK-1 or the TASK-3 portion of the channel was sufficient to disrupt both effects. Thus, both anesthetic activation and transmitter inhibition of these channels require a region at the interface between the final transmembrane domain and the cytoplasmic C terminus that has not been associated previously with receptor signal transduction. Our results also indicate a close molecular relationship between these two forms of modulation, one endogenous and the other clinically applied.     

Angiotensin II negatively modulates L-type calcium channels through a pertussis toxin-sensitive G protein in adrenal glomerulosa cells.

In bovine adrenal glomerulosa cells, angiotensin II and extracellular K+ stimulate aldosterone secretion in a calcium-dependent manner. In these cells, physiological concentrations of extracellular potassium activate both T-type (low threshold) and L-type (high threshold) voltage-operated calcium channels. Paradoxically, the cytosolic calcium response to 9 mM K+ is inhibited by angiotensin II. Because K+-induced calcium changes observed in the cytosol are almost exclusively due to L-type channel activity, we therefore studied the mechanisms of L-type channel regulation by angiotensin II. Using the patch-clamp method in its perforated patch configuration, we observed a marked inhibition (by 63%) of L-type barium currents in response to angiotensin II. This effect of the hormone was completely prevented by losartan, a specific antagonist of the AT1 receptor subtype. Moreover, this inhibition was strongly reduced when the cells were previously treated for 1 night with pertussis toxin. An effect of pertussis toxin was also observed on the modulation by angiotensin II of the K+ (9 mM)-induced cytosolic calcium response in fura-2-loaded cells, as well as on the angiotensin II-induced aldosterone secretion, at both low (3 mM) and high (9 mM) K+ concentrations. Finally, the expression of both Go and Gi proteins in bovine glomerulosa cells was detected by immunoblotting. Altogether, these results strongly suggest that in bovine glomerulosa cells, a pertussis toxin-sensitive G protein is involved in the inhibition of L-type channel activity induced by angiotensin II.     

Role of voltage-gated calcium channels in potassium-stimulated aldosterone secretion from rat adrenal zona glomerulosa cells

The mineralocorticoid aldosterone plays an important role in the regulation of plasma electrolyte homeostasis. Exposure of acutely isolated rat adrenal zona glomerulosa cells to elevated K(+) activates voltage-gated calcium channels and initiates a calcium-dependent increase in aldosterone synthesis. We developed a novel 96-well format aldosterone secretion assay to rapidly evaluate the effect of known T- and L-type calcium channel antagonists on K(+)-stimulated aldosterone secretion and better define the role of voltage-gated calcium channels in this process. Reported T-type antagonists, mibefradil and Ni(2+), and selected L-type antagonist dihydropyridines, inhibited K(+)-stimulated aldosterone synthesis. Dihydropyridine-mediated inhibition occurred at concentrations which had no effect on rat alpha1H T-type Ca(2+) currents. In contrast, below 10 microM, the L-type antagonists verapamil and diltiazem showed only minimal inhibitory effects. To examine the selectivity of the calcium channel antagonist-mediated inhibition, we established an aldosterone secretion assay in which 8Br-cAMP stimulates aldosterone secretion independent of extracellular calcium. Mibefradil remained inhibitory in this assay, while the dihydropyridines had only limited effects. Taken together, these data demonstrate a role for the L-type calcium channel in K(+)-stimulated aldosterone secretion. Further, they confirm the need for selective T-type calcium channel antagonists to better address the role of T-type channels in K(+)-stimulated aldosterone secretion.