HERG potassium channel regulation by the N-terminal eag domain

Human ether-á-go-go related gene (hERG, K(v)11.1) potassium channels play a significant role in cardiac excitability. Like other K(v) channels, hERG is activated by membrane voltage; however, distinct from other K(v) channels, hERG channels have unusually slow kinetics of closing (deactivation). The mechanism for slow deactivation involves an N-terminal "eag domain" which comprises a PAS (Per-Arnt-Sim) domain and a short Cap domain. Here we review recent advances in understanding how the eag domain regulates deactivation, including several new Nuclear Magnetic Resonance (NMR) solution structures of the eag domain, and evidence showing that the eag domain makes a direct interaction with the C-terminal C-linker and Cyclic Nucleotide-Binding Homology Domain.     

Comparison of effects of a potassium channel opener BRL34915, a specific potassium ionophore valinomycin and calcium channel blockers on endothelin-induced vascular contraction

The effects of a potassium (K+) channel opener BRL34915 and a specific K+ ionophore valinomycin on vasoconstriction induced by endothelin (ET) were compared with those of calcium (Ca2+) channel blockers, nicardipine and verapamil, using helical strips from rat thoracic aorta. ET induced potent and persistent contraction in control solution and similar but smaller contraction in Ca2+-free solution. BRL34915 and valinomycin inhibited the ET-induced contraction dose-dependently in control solution, but not in Ca2+-free solution. The ET-induced contraction was also inhibited by nicardipine and verapamil, though less strongly. On the other hand, high K+ (35 mM)-induced vasoconstriction was strongly inhibited by nicardipine and verapamil, but not by BRL34915 or valinomycin. These results support the idea that the extracellular Ca2+-dependent component of the ET-induced contraction may be mediated by Ca2+ influx by a route other than voltage-dependent Ca2+-channels.     

hKv4.3 channel characterization and regulation by calcium channel antagonists

Relative expression pattern of short and long isoforms of hKv4.3 channels was evaluated by RT-PCR and RPA. Electrophysiological studies were performed in HEK293 cells transfected with short or long hKv4.3 cDNA. The long variant L-hKv4.3 was the only form present in lung, pancreas, and small intestine. The short variant S-hKv4.3 was predominant in brain whereas expression levels of the two isoforms were similar in cardiac and skeletal muscles. Properties of the ionic channels encoded by L-hKv4.3 and S-hKv4.3 cDNAs were essentially similar. Cadmium chloride and verapamil inhibited hKv4.3 current (with EC50s of 0.110 +/- 0.004 mM and 492.9 +/- 15.1 microM, respectively). Verapamil also accelerated current inactivation. Another calcium channel antagonist nicardipine was found inactive. In conclusion, this study confirms that both isoforms underlie the transient outward potassium current. Moreover, calcium channel inhibitors markedly affect hKv4.3 current, an effect which must be considered when evaluating transient outward potassium channel properties in native tissues.     

Cell-specific regulation of Fas exon 6 splicing mediated by Hu antigen R

The differential expression levels of T-cell intracellular antigens (TIA) and Hu antigen R (HuR) are concomitant with a splicing switch in apoptosis receptor Fas in HCT-116 cells. Thus, overexpression and knockdown of HuR led to Fas exon 6 skipping and inclusion, respectively. These results suggest that the TIA and HuR cellular ratio influences cell-type specific Fas exon 6 splicing pattern.     

Effects of calcium channel blockers on potassium homeostasis.

The known effects of calcium channel blockers on various aspects of potassium homeostasis are reviewed. Regulation of potassium homeostasis requires both renal and external handling mechanisms. Signaling by calcium appears to mediate both of these. Calcium channels have been identified in adrenal glomerulosa cells, and cellular calcium entry has been demonstrated in vitro to be necessary for the synthesis and secretion of aldosterone. Calcium channel antagonists such as verapamil and nifedipine, at pharmacologic doses, can block aldosterone production. In vivo, however, only chronic administration of verapamil appears to attenuate aldosterone responsiveness to angiotensin II. Chronic administration of nifedipine does not have a dramatic effect on aldosterone production following potassium loading. Other studies have demonstrated improved extrarenal potassium disposal following treatment with calcium channel blocking agents. Clinically, there are no reports of either hyperkalemia or hypokalemia with the routine therapeutic use of these agents given alone. This review was prompted by the development of hyperkalemia in a patient with chronic renal failure following the initiation of therapy with the calcium channel blocker diltiazem: however, numerous other etiologies may also have contributed to the development of hyperkalemia in this case. Review of the data indicates that current evidence implicating this class of drugs in the pathogenesis of disordered potassium regulation remains equivocal.     

Single channel studies of inward rectifier potassium channel regulation by muscarinic acetylcholine receptors

Negative regulation of the heartbeat rate involves the activation of an inwardly rectifying potassium current (I(KACh)) by G protein-coupled receptors such as the m2 muscarinic acetylcholine receptor. Recent studies have shown that this process involves the direct binding of G(betagamma) subunits to the NH(2)- and COOH-terminal cytoplasmic domains of the proteins termed GIRK1 and GIRK4 (Kir3.1 and Kir3.4/CIR), which mediate I(KACh). Because of the very low basal activity of native I(KACh), it has been difficult to determine the single channel effect of G(betagamma) subunit binding on I(KACh) activity. Through analysis of a novel G protein-activated chimeric inward rectifier channel that displays increased basal activity relative to I(KACh), we find that single channel activation can be explained by a G protein-dependent shift in the equilibrium of open channel transitions in favor of a bursting state of channel activity over a long-lived closed state.     

Structure and regulation of the MinK potassium channel

MinK is a novel protein which induces an extremely slowly activating potassium channel when expressed in Xenopus oocytes. We discuss the properties and regulation of the current and localization and possible physiological roles of the MinK protein.Special issue dedicated to Dr. Alan N. Davison.     

Regulation of Fas alternative splicing by antagonistic effects of TIA-1 and PTB on exon definition

Fas exon 6 can be included or skipped to generate mRNAs encoding, respectively, a membrane bound form of the receptor that promotes apoptosis or a soluble isoform that prevents programmed cell death. We report that the apoptosis-inducing protein TIA-1 promotes U1 snRNP binding to the 5' splice site of intron 6, which in turn facilitates exon definition by enhancing U2AF binding to the 3' splice site of intron 5. The polypyrimidine tract binding protein (PTB) promotes exon skipping by binding to an exonic splicing silencer and inhibiting the association of U2AF and U2 snRNP with the upstream 3' splice site, without affecting recognition of the downstream 5' splice site by U1. Remarkably, U1 snRNP-mediated recognition of the 5' splice site is required both for efficient U2AF binding and for U2AF inhibition by PTB. We propose that TIA-1 and PTB regulate Fas splicing and possibly Fas-mediated apoptosis by targeting molecular events that lead to exon definition.     

Reciprocal regulation of calcium dependent and calcium independent cyclic AMP hydrolysis by protein phosphorylation

The hydrolysis of cyclic nucleotide second messengers takes place through multiple cyclic nucleotide phosphodiesterases (PDEs). The significance of this diversification is not fully understood. Here we report the differential regulation of low K(m) Ca2+-activated (PDE1C) and Ca2+-independent, rolipram-sensitive (PDE4) PDEs by protein phosphorylation in the neuroendocrine cell line AtT20. Incubation of cells with 8-(4-chlorophenylthio)-cyclic AMP (CPT-cAMP) enhanced PDE4 and reduced PDE1C activity. These effects were blocked by H89 indicating mediation by cAMP-dependent protein kinase (PKA), furthermore in broken cell preparations PKA produced the same reciprocal changes of PDE activities. Calyculin A, an inhibitor of protein phosphatases 1 and 2 A, stimulated PDE4 and enhanced the inhibitory effect of CPT-cAMP on PDE1C. The reduction of PDE1C activity was characterized by a marked attenuation of the activation by Ca2+/calmodulin. Stimulation of PDE4 activity by CPT-cAMP or calyculin A was attributable to PDE4D3 and these effects could also be reproduced in human embryonic kidney cells expressing epitope-tagged PDE4D3. Together, these data show reciprocal regulation of PDE1C and PDE4D by PKA, which represents a novel scheme for plasticity in intracellular signalling.     

ATP-sensitive potassium channel traffic regulation by adenosine and protein kinase C

ATP-sensitive potassium (K(ATP)) channels activate under metabolic stress to protect neurons and cardiac myocytes. However, excessive channel activation may cause arrhythmia in the heart and silence neurons in the brain. Here, we report that PKC-mediated downregulation of K(ATP) channel number, via dynamin-dependent channel internalization, can act as a brake mechanism to control K(ATP) activation. A dileucine motif in the pore-lining Kir6.2 subunit of K(ATP), but not the site of PKC phosphorylation for channel activation, is essential for PKC downregulation. Whereas K(ATP) activation results in a rapid shortening of the action potential duration (APD) in metabolically inhibited ventricular myocytes, adenosine receptor stimulation and consequent PKC-mediated K(ATP) channel internalization can act as a brake to lessen this APD shortening. Likewise, in hippocampal CA1 neurons under metabolic stress, PKC-mediated, dynamin-dependent K(ATP) channel internalization can also act as a brake to dampen the rapid decline of excitability due to K(ATP) activation.     

NO调控下丘脑钙激活钾通道的机制研究

目的 :研究NO对下丘脑神经元钙激活钾通道 (KCa)的作用及其机制。方法 :采用膜片钳技术内面向外式及细胞贴附式。结果 :NO可直接或通过升高cGMP来提高KCa通道的开放概率 (Po) ,这种增强作用是因为通道开放时间延长及开放频率增加。结论 :下丘脑神经元中NO可通过不同机制激活KCa。     

Homeostatic regulation of Kv1.2 potassium channel trafficking by cyclic AMP

The Shaker family potassium channel, Kv1.2, is a key determinant of membrane excitability in neurons and cardiovascular tissue. Kv1.2 is subject to multiple forms of regulation and therefore integrates cellular signals involved in the homeostasis of excitability. The cyclic AMP/protein kinase A (PKA) pathway enhances Kv1.2 ionic current; however, the mechanisms for this are not fully known. Here we show that cAMP maintains Kv1.2 homeostasis through opposing effects on channel trafficking. We found that Kv1.2 is regulated by two distinct cAMP pathways, one PKA-dependent and the other PKA-independent. PKA inhibitors elevate Kv1.2 surface levels, suggesting that basal levels of cAMP control steady-state turnover of the channel. Elevation of cAMP above basal levels also increases the amount of Kv1.2 at the cell surface. This effect is not blocked by PKA inhibitors, but is blocked by inhibition of Kv1.2 endocytosis. We conclude that Kv1.2 levels at the cell surface are kept in dynamic balance by opposing effects of cAMP.     

Differential regulation of calcium channel coding genes by prolonged depolarization

Calcium channels must be subjected to a very precise regulation in order to preserve cell function and viability. Voltage gated calcium channels (VGCC) represent the main pathway for calcium entry in excitable cells. This explains why depolarization induces a rapid-onset and short-term inactivation of calcium currents. Contrarily to this well-documented mechanism to maintain calcium below toxic levels, the regulatory pathways inducing longer-lasting changes and cell surface expression of functional calcium channels are largely unknown. Since calcium is a main player in the activity-dependent regulation of many genes, we hypothesize that calcium channel coding genes could be also subjected to activity-dependent regulation. We have used prolonged depolarization to analyze the effects of sustained intracellular calcium elevation on the mRNAs coding for the different alpha(1) pore-forming subunits of the calcium channels expressed in chromaffin cells. Our findings reveal that persistent depolarization is accompanied by a prolonged intracellular calcium elevation and reduction of calcium current. This calcium current inhibition could be mediated, at least partially, by the downregulation of the mRNAs coding for several alpha(1) subunits. Thus, we show here that depolarization inhibits the expression of Ca(V)1.1, Ca(V)1.2, Ca(V)1.3, Ca(V)2.2 and Ca(V)2.3 mRNAs, while the Ca(V)2.1 mRNA remains unmodified. Moreover, such downregulation of channels depends on calcium entry through the L-type calcium channel, as both mRNA and calcium current changes induced by depolarization are abrogated by L-type channel specific blockers.     

Effects of calcium channel blocker, mibefradil, and potassium channel opener, pinacidil, on the contractile response of mid-pregnant goat myometrium

The present study was undertaken to investigate the in vitro influence of mibefradil, a calcium channel blocker, and pinacidil, a potassium channel opener, on pregnant goat myometrial spontaneous rhythmic contractility and contractions induced with the agonist, oxytocin. Longitudinal strips from the distal region of uterus, collected from goats at midgestation, were mounted in an organ bath for recording isometric contractions. Mibefradil (10(-8)-10(-4) M) or pinacidil (10(-10)-10(-4) M), added cumulatively to the bath at an increment of 1 log unit, caused concentration-dependent inhibition of the spontaneous rhythmic contractions of isolated uterine strips. The rhythmic contraction was, respectively, abolished at 100 and 10 microM concentrations of mibefradil and pinacidil. In a concentration-dependent manner, mibefradil (1 and 10 microM) antagonized the contractions elicited with oxytocin (10(-5)-10(-2) IU). Pretreatment of uterine strips with glibenclamide (10 microM), a selective KATP channel blocker, caused a rightward shift of the concentration-response curve of pinacidil with a concomitant decrease in its pD2 value. Pinacidil (0.3, 1 and 3 microM), in a concentration-related manner, antagonized the oxytocin (10(-5)-10(-2) IU)-induced contractile response. The inhibition of spontaneous rhythmic contractions and antagonism of oxytocin-induced contraction by mibefradil in the pregnant goat myometrium may be related to the antagonism of voltage-dependent Ca2+ channels, while by pinacidil suggests that KATP channel could be a therapeutic target for tocolysis.     

Kinase-dependent regulation of the intermediate conductance, calcium-dependent potassium channel, hIK1

We determined the effect of nucleotides and protein kinase A (PKA) on the Ca(2+)-dependent gating of the cloned intermediate conductance, Ca(2+)-dependent K(+) channel, hIK1. In Xenopus oocytes, during two-electrode voltage-clamp, forskolin plus isobutylmethylxanthine induced a Ca(2+)-dependent increase in hIK1 activity. In excised inside-out patches, addition of ATP induced a Ca(2+)-dependent increase in hIK1 activity (NP(o)). In contrast, neither nonhydrolyzable (AMP-PNP, AMP-PCP) nor hydrolyzable ATP analogs (GTP, CTP, UTP, and ITP) activated hIK1. The ATP-dependent activation of hIK1 required Mg(2+) and was reversed by either exogenous alkaline phosphatase or the PKA inhibitor PKI(5-24). The Ca(2+) dependence of hIK1 activation was best fit with a stimulatory constant (K(s)) of 350 nM and a Hill coefficient (n) of 2.3. ATP increased NP(o) at [Ca(2+)] >100 nM while having no effect on K(s) or n. Mutation of the single PKA consensus phosphorylation site at serine 334 to alanine (S334A) had no effect on the PKA-dependent activation during either two-electrode voltage-clamp or in excised inside-out patches. When expressed in HEK293 cells, ATP activated hIK1 in a Mg(2+)-dependent fashion, being reversed by alkaline phosphatase. Neither PKI(5-24) nor CaMKII(281-309) or PKC(19-31) affected the ATP-dependent activation. Northern blot analysis revealed hIK1 expression in the T84 colonic cell line. Endogenous hIK1 was activated by ATP in a Mg(2+)- and PKI(5-24)-dependent fashion and was reversed by alkaline phosphatase, whereas CaMKII(281-309) and PKC(19-31) had no effect on the ATP-dependent activation. The Ca(2+)-dependent activation (K(s) and n) was unaffected by ATP. In conclusion, hIK1 is activated by a membrane delimited PKA when endogenously expressed. Although the oocyte expression system recapitulates this regulation, expression in HEK293 cells does not. The effect of PKA on hIK1 gating is Ca(2+)-dependent and occurs via an increase in NP(o) without an effect on either Ca(2+) affinity or apparent cooperativity.     

Differential regulation of basic protein phosphorylation by calcium phospholipid and cyclic-AMP-dependent protein kinases

Myelin basic protein, an 80-kilodalton (kDa) protein in rat oligodendrocytes, and an 80-kDa basic protein in neuroblastoma x neonatal Chinese hamster brain explant hybrids were phosphorylated extensively when the cells were treated with either phorbol esters (TPA) or diacylglycerols (e.g., oleyoyl-acetylglycerol). TPA-stimulated phosphorylation was inhibited by pre-incubation with 50 microM psychosine (galactosyl-sphingosine), confirming that it is mediated through the phospholipid-dependent protein kinase C (PK-C). Surprisingly, phosphorylation of these proteins was inhibited by incubation of cells with agents which result in activation of cyclic-AMP-dependent protein kinase (dibutyryl cyclic AMP or forskolin). In contrast, phosphorylation of other nonbasic proteins, for example, the oligodendrocyte-specific 2',3'-cyclic nucleotide phosphohydrolase, was stimulated under these conditions (Vartanian et al.: Proceedings of the National Academy of Sciences of the United States of America 85:939, 1988). The possible role of cyclic AMP in activating specific phosphatases or restricting the availability of diacylglycerol for PK-C activation is discussed.     

Activation process of calcium-dependent potassium channel in Euhadra neurons: involvement of calcium/calmodulin and subsequent protein phosphorylation.

1. The activation process of Ca(2+)-dependent potassium channel was studied electrophysiologically and pharmacologically using identified neurons of the land snail, Euhadra peliomphala. 2. Ca(2+)-mediated delayed outward K current (IKD) was dose-dependently reduced by the calmodulin inhibitors, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5, week) and N-(6-aminohexyl)-5-chloro-naphthalenesulfonamide (W-7, potent). These antagonists also caused a slight membrane depolarization and increase in impulse discharge frequency with decrease in the amplitude of both action potential and after hyperpolarization. 3. The cAMP-dependent protein kinase inhibitor N-[2-(methylamino) ethyl]-5-isoquinoline-sulfonamide (H-8) did not produce any significant effect on IKD and membrane potential. 4. Calmodulin, when injected into the neuron which had been treated with either W-5 or W-7, transiently restored the suppressed IKD nearly to the pretreatment level, and caused hyperpolarization of the cell. In contrast, calcium chloride, intracellularly injected in the same way, had little effect on both the IKD and the membrane potential shifted by these antagonists. 5. Intracellular injection of kinase II, a Ca2+/calmodulin-dependent protein kinase, caused an increase in the IKD and membrane hyperpolarization. Similar but weak effects were produced when a catalytic subunit (CS) of cAMP-dependent protein kinase was intracellularly injected. However, the neurons pretreated with W-7 no longer had any detectable increase in the IKD and hyperpolarization of the membrane. 6. These results suggest the possibility that Ca2+/camodulin-dependent protein phosphorylation may finally mediate the activation of a certain number of potassium channels.     

Differential regulation of a CLC anion channel by SPAK kinase ortholog-mediated multisite phosphorylation

Shrinkage-induced inhibition of the Caenorhabditis elegans cell volume and cell cycle-dependent CLC anion channel CLH-3b occurs by concomitant phosphorylation of S742 and S747, which are located on a 175 amino acid linker domain between cystathionine-β-synthase 1 (CBS1) and CBS2. Phosphorylation is mediated by the SPAK kinase homolog GCK-3 and is mimicked by substituting serine residues with glutamate. Type 1 serine/threonine protein phosphatases mediate swelling-induced channel dephosphorylation. S742E/S747E double mutant channels are constitutively inactive and cannot be activated by cell swelling. S742E and S747E mutant channels were fully active in the absence of GCK-3 and were inactive when coexpressed with the kinase. Both channels responded to cell volume changes. However, the S747E mutant channel activated and inactivated in response to cell swelling and shrinkage, respectively, much more slowly than either wild-type or S742E mutant channels. Slower activation and inactivation of S747E was not due to altered rates of dephosphorylation or dephosphorylation-dependent conformational changes. GCK-3 binds to the 175 amino acid inter-CBS linker domain. Coexpression of wild-type CLH-3b and GCK-3 with either wild-type or S742E linkers gave rise to similar channel activity and regulation. In contrast, coexpression with the S747E linker greatly enhanced basal channel activity and increased the rate of shrinkage-induced channel inactivation. Our findings suggest the intriguing possibility that the phosphorylation state of S742 in S747E mutant channels modulates GCK-3/channel interaction and hence channel phosphorylation. These results provide a foundation for further detailed studies of the role of multisite phosphorylation in regulating CLH-3b and GCK-3 activity.