Intracellular signalling involved in activation of the volume-sensitive K+ current in Ehrlich ascites tumour cells

The cell swelling-activated K+ channel in Ehrlich ascites tumour cells has a conductance of 5 pS estimated from noise analysis of the volume-sensitive whole-cell K+ current (I(K,vol)). I(K,vol) exhibits Goldman-Hodgkin-Katz type behaviour and is insensitive to clotrimazole, apamin and charybdotoxin (ChTX), but inhibited by clofilium. Its small conductance, lack of intrinsic voltage-dependence and peculiar pharmacological profile are similar to properties described for the two-pore domain background K+ TASK channels. Neither Ca2+ nor ATP work as initiators in the activation of I(K,vol). In contrast, several investigations in Ehrlich cells suggest an important role for leukotriene D4 (LTD4) in the activation of I(K,vol). Under isotonic conditions, LTD4 activates Ca2+-dependent, ChTX-sensitive K+ channels as well as Ca2+-independent. ChTX-insensitive K+ channels. The LTD4-activated, ChTX-insensitive K+ current exhibits a current-voltage relation, pharmacological profile and single channel conductance similar to that of I(K,vol), indicating that LTD4 is the signalling molecule responsible for activation of the volume-sensitive K+ channels in Ehrlich cells. Hypotonic swelling of Ehrlich cells results in translocation of the 85-kDa cytosolic (c) PLA2alpha to the nucleus where it is activated. This activation leads to an increase in arachidonic acid release followed by an increased release of leukotrienes, and is essential in cell swelling-induced activation of I(K,vol) and of the organic osmolyte channels.     

Novel signalling events mediated by muscarinic receptor subtypes

The M2 muscarinic acetylcholine receptor (mAChR) activates Gi protein coupled pathways, such as stimulation of G-protein activated inwardly rectifying K channels (GIRKs). Here we report a novel heterologous desensitization of these GIRK currents, which appeared to be specifically induced by M2/M4 mAChR stimulation, but not via adenosine (Ado) and alpha2-adrenergic receptors (AR). This heterologous desensitization reflected an inhibition of the GIRK signalling pathway downstream of G-protein activation. It was mediated in a membrane-delimited fashion via a PTX insensitive GTP dependent pathway and could be competed with exogenous Gbetagamma. The activation of M3 mAChR/Gq coupled receptors potently inhibited GIRK currents similar as M2 mAChR. By monitoring simultaneously the response of A1 adenosine receptor (AdoR) activation on N-type Ca2+ channels and GIRK channels, the stimulation of M3 mAChR was found to cause an inhibition of the Ado response in both effector systems, suggesting that the inhibition occurred at the level of the G-protein common to both effectors. These results indicated that Gq proteins inhibit pathways that are commonly regulated by Gbetagamma proteins.     

Reconstitution of somatostatin and muscarinic receptor mediated stimulation of K+ channels by isolated GK protein in clonal rat anterior pituitary cell membranes

Somatostatin (SS) inhibits secretion from many cells, including clonal GH3 pituitary cells, by a complex mechanism that involves a pertussis toxin (PTX)-sensitive step and is not limited to its cAMP lowering effect, since secretion induced by cAMP analogs and K+ depolarization are also inhibited. SS also causes membrane hyperpolarization which may lead to decreases in intracellular Ca2+ need for secretion. Using patch clamp techniques we now demonstrate: 1) that both (SS) and acetylcholine applied through the patch pipette to the extracellular face of a patch activate a 55-picosiemens K+ channel without using a soluble second messenger; 2) that, after patch excision, the active state of the ligand-stimulated channel is dependent on GTP in the bath, is abolished by treatment of the cytoplasmic face of the patch with activated PTX and NAD+, and after inactivation by PTX, is restored in a GTP-dependent manner by addition of a nonactivated human erythrocyte PTX-sensitive G protein, and 3) that the 55-picosiemens K+ channel can also be activated in a ligand-independent manner with guanosine [gamma-thio] triphosphate (GTP gamma S) or with Mg2+/GTP gamma S-activated erythrocyte G protein. We call this protein GK. It is an alpha-beta-gamma trimer of which we have previously shown that the alpha-subunit is the substrate for PTX and that it dissociates on activation with Mg2+/GTP gamma S into alpha-GTP gamma S plus beta-gamma. A similarly activated and dissociated preparation of GS, the stimulatory regulatory component of adenylyl cyclase, having a different alpha-subunit but the same beta-gamma-dimer, was unable to cause K+ opening.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms for picrotoxin block of alpha2 homomeric glycine receptors

It is well known that the convulsant alkaloid picrotoxin (PTX) can inhibit neuronal gamma-aminobutyric acid (GABA) and homomeric glycine receptors (GlyR). However, the mechanism for PTX block of alpha(2) homomeric GlyR is still unclear compared with that of alpha(1) homomeric GlyR, GABA(A), and GABA(C) receptors. Furthermore, PTX effects on GlyR kinetics have been poorly explored at the single-channel level. Hence, we used the patch-clamp technique in the outside-out configuration to investigate the mechanism of PTX suppression of currents carried by alpha(2) homomeric GlyRs stably transfected into Chinese hamster ovary cells. PTX inhibited the alpha(2) homomeric GlyR current elicited by glycine in a concentration-dependent and voltage-independent manner. Both competitive and noncompetitive mechanisms were observed. PTX decreased the mean open time of the GlyR channel in a concentration-dependent manner, suggesting that PTX can block channel openings and bind to the receptor in the open channel conformation. When PTX and glycine were co-applied, a small rebound current was observed during drug washout. Application of PTX during the deactivation phase of glycine-induced currents eliminated the rebound current and accelerated the deactivation time course in a concentration-dependent manner. PTX could not bind to the unbound conformation of GlyR, but could be trapped at its binding site when the channel closed during glycine dissociation. Based on these observations, we propose a kinetic Markov model in which PTX binds to the alpha(2) homomeric GlyR in both the open channel state and the fully liganded closed state. Our data suggest a new allosteric mechanism for PTX inhibition of wild-type homomeric alpha(2) GlyR.     

AMP-activated protein kinase mediates carotid body excitation by hypoxia

Early detection of an O2 deficit in the bloodstream is essential to initiate corrective changes in the breathing pattern of mammals. Carotid bodies serve an essential role in this respect; their type I cells depolarize when O2 levels fall, causing voltage-gated Ca2+ entry. Subsequent neurosecretion elicits increased afferent chemosensory fiber discharge to induce appropriate changes in respiratory function (1). Although depolarization of type I cells by hypoxia is known to arise from K+ channel inhibition, the identity of the signaling pathway has been contested, and the coupling mechanism is unknown (2). We tested the hypothesis that AMP-activated protein kinase (AMPK) is the effector of hypoxic chemotransduction. AMPK is co-localized at the plasma membrane of type I cells with O2-sensitive K+ channels. In isolated type I cells, activation of AMPK using 5-aminoimidazole-4-carboxamide riboside (AICAR) inhibited O2-sensitive K+ currents (carried by large conductance Ca2+-activated (BKCa) channels and TASK (tandem pore, acid-sensing potassium channel)-like channels, leading to plasma membrane depolarization, Ca2+ influx, and increased chemosensory fiber discharge. Conversely, the AMPK antagonist compound C reversed the effects of hypoxia and AICAR on type I cell and carotid body activation. These results suggest that AMPK activation is both sufficient and necessary for the effects of hypoxia. Furthermore, AMPK activation inhibited currents carried by recombinant BKCa channels, whereas purified AMPK phosphorylated thealpha subunit of the channel in immunoprecipitates, an effect that was stimulated by AMP and inhibited by compound C. Our findings demonstrate a central role for AMPK in stimulus-response coupling by hypoxia and identify for the first time a link between metabolic stress and ion channel regulation in an O2-sensing system.     

Regulators of G-protein signaling form a quaternary complex with the agonist, receptor, and G-protein. A novel explanation for the acceleration of signaling activation kinetics

Regulators of G-protein signaling (RGS) proteins modulate signaling through heterotrimeric G-proteins. They act to enhance the intrinsic GTPase activity of the Galpha subunit but paradoxically have also been shown to enhance receptor-stimulated activation. To study this paradox, we used a G-protein gated K+ channel to report the dynamics of the G-protein cycle and fluorescence resonance energy transfer techniques with cyan and yellow fluorescent protein-tagged proteins to report physical interaction. Our data show that the acceleration of the activation kinetics is dissociated from deactivation kinetics and dependent on receptor and RGS type, G-protein isoform, and RGS expression levels. By using fluorescently tagged proteins, fluorescence resonance energy transfer microscopy showed a stable physical interaction between the G-protein alpha subunit and RGS (RGS8 and RGS7) that is independent of the functional state of the G-protein. RGS8 does not directly interact with G-protein-coupled receptors. Our data show participation of the RGS in the ternary complex between agonist-receptor and G-protein to form a "quaternary complex." Thus we propose a novel model for the action of RGS proteins in the G-protein cycle in which the RGS protein appears to enhance the "kinetic efficacy" of the ternary complex, by direct association with the G-protein alpha subunit.     

Effect of pertussis toxin on insulin-induced signal transduction in rat adipocytes and soleus muscles

It has been reported that pertussis toxin (PTX) suppresses the function of trimeric guanine nucleotide binding protein (G-protein). We examined the effect of PTX on insulin-induced glucose uptake, diacylglycerol (DG)-protein kinase C (PKC) signalling, phosphatidylinositol (PI) 3-kinase and PKC zeta activation and insulin-induced tyrosine phosphorylation of Gialpha to clarify the role of G-protein for insulin-mediated signal transduction mechanism in rat adipocytes and soleus muscles. Isolated adipocytes and soleus muscles were preincubated with 0.01 approximately 1 ng/ml PTX for 2 hours, followed by stimulation with 10-100 nM insulin or 1 microM tetradecanoyl phorbol-13-acetate (TPA). Pretreatment with PTX resulted in dose-responsive decreases in insulin-stimulated [3H]2-deoxyglucose (DOG) uptake, and unchanged TPA-stimulated [3H]2-DOG uptake, without affecting basal [3H]2-DOG uptake. In adipocytes, insulin-induced DG-PKC signalling, PI 3-kinase activation and PKC zeta translocation from cytosol to the membrane were suppressed when treated with PTX, despite no changes in [125I]insulin-specific binding and insulin receptor tyrosine kinase activity. Moreover, to elucidate insulin-stimulated tyrosine phosphorylation of 40 kDa alpha-subunit of G-protein (Gialpha-2), adipocytes were stimulated with 10 nM insulin for 10 minutes, homogenized, immunoprecipitated with anti-phosphotyrosine antibody, and immunoblotted with anti-Gialpha-2 antibody. Insulin-induced tyrosine phosphorylation of Gialpha-2 was found by immunoblot analysis with anti-Gialpha-2 antibody. These results suggest that G-protein regulates DG-PKC signalling by binding of Gialpha-2 with GTP and PI 3-kinase-PKC zeta signalling by releasing of Gbetagamma via dissociation of trimeric G-protein after insulin receptor tyrosine phosphorylation in insulin-sensitive tissues.     

Trp-Lys-Tyr-Met-Val-Met activates mitogen-activated protein kinase via a PI-3 kinase-mediated pathway independent of PKC

Trp-Lys-Tyr-Met-Val-Met (WKYMVM) is a novel potent peptide which can stimulate phosphoinositide hydrolysis in U937 as well as U266 and HL-60 cells (Baek et al., J. Biol. Chem. 271, 8170 (1996)). The peptide also induces superoxide generation in human neutrophils (Seo et al., J. Immunol. 158, 1896 (1997)). However, the signaling pathway down-stream of PLC set in motion by the peptide is not yet completely understood. We studied the signaling pathway of the peptide with the goal of elucidating the mechanism of the peptide's action. WKYMVM induced a rapid and transient activation of the ERKs in human histiocytic lymphoma cells, U937. The ERK1 activation peaked at 5 min and returned to the basal level after 30 min. The ERK1 stimulation by the peptide was partially inhibited by pretreatment of the cells with pertussis toxin (PTX), implicating G-protein involvement in the peptide's action. Pretreatment of staurosporine, protein kinase C (PKC) inhibitor, or PKC down-regulating PMA had no impact on the ERK1 activation by the peptide, indicating that the signaling pathway is independent of PKC activation. Pretreatment of the cells with neomycin and intracellular Ca2+ mobilizing reagents had also no effect on the ERK1 activation by the peptide. However, pretreatment with wortmannin or LY294002, the inhibitor of phosphatidylinositol 3 kinase (PI-3K), strongly inhibited peptide-stimulated ERK1 activation. Our results suggest that PI-3K may be an important participant in the ERK cascade induced by the peptide. Furthermore, the treatment of U937 cells with the peptide activated p74Raf-1, an upstream kinase of ERK. Taken together, our results suggest that the peptide activate ERK via a G-protein/PI-3K/Ras/Raf-1 mediated signaling pathway in U937 cells.     

Activation of an ATP-dependent K(+) conductance in Xenopus oocytes by expression of adenylate kinase cloned from renal proximal tubules

In rabbit proximal convoluted tubules, an ATP-sensitive K(+) (K(ATP)) channel has been shown to be involved in membrane cross-talk, i.e. the coupling (most likely mediated through intracellular ATP) between transepithelial Na(+) transport and basolateral K(+) conductance. This K(+) conductance is inhibited by taurine. We sought to isolate this K(+) channel by expression cloning in Xenopus oocytes. Injection of renal cortex mRNA into oocytes induced a K(+) conductance, largely inhibited by extracellular Ba(2+) and intracellular taurine. Using this functional test, we isolated from our proximal tubule cDNA library a unique clone, which induced a large K(+) current which was Ba(2+)-, taurine- and glibenclamide-sensitive. Surprisingly, this clone is not a K(+) channel but an adenylate kinase protein (AK3), known to convert NTP+AMP into NDP+ADP (N could be G, I or A). AK3 expression resulted in a large ATP decrease and activation of the whole-cell currents including a previously unknown, endogenous K(+) current. To verify whether ATP decrease was responsible for the current activation, we demonstrated that inhibition of glycolysis greatly reduces oocyte ATP levels and increases an inwardly rectifying K(+) current. The possible involvement of AK in the K(ATP) channel's regulation provides a means of explaining their observed activity in cytosolic environments characterized by high ATP concentrations.     

Effects of palytoxin on contractile response and calcium movement in guinea-pig taenia coli

PTX (10(-8)M) induced a rapid increase followed by a gradual decrease in muscle tension in normal physiological salt solution (PSS), while it induced a slow increase in muscle tension in low-Na+ solution. These contractions were inhibited by Ca2+ channel blockers, verapamil and nicardipine. PTX rapidly increased tissue Na+ and decreased tissue K+ contents in normal PSS. In low-Na+ solution, PTX decreased tissue K+ content with a slower rate than that in normal PSS. PTX increased uptake of 45Ca2+ in normal as well as low-Na+ solutions with similar time course as the increase in muscle tension. However, 45Ca2+ uptake still remained high when the PTX-induced transient contraction ceased. These results suggest that PTX increases Ca2+ influx through voltage-dependent Ca2+ channels to cause contraction. After a prolonged exposure to PTX, however, muscle tension is uncoupled from Ca2+ influx.     

Modulation of potassium channel gating by external divalent cations

We have examined the actions of Zn2+ ions on Shaker K channels. We found that low (100 microM) concentrations of Zn2+ produced a substantial (approximately three-fold) slowing of the kinetics of macroscopic activation and inactivation. Channel deactivation was much less affected. These results were obtained in the presence of 5 mM Mg2+ and 4 mM Ca2+ in the external solution and so are unlikely to be due to modification of membrane surface charges. Furthermore, the action of 100 microM Zn2+ on activation was equivalent to a 70-mV reduction of a negative surface potential whereas the effects on deactivation would require a 15-mV increase in surface potential. External H+ ions reduced the Zn-induced slowing of macroscopic activation with an apparent pK of 7.3. Treatment of Shaker K channels with the amino group reagent, trinitrobenzene sulfonic acid (TNBS), substantially reduced the effects of Zn2+. All these results are qualitatively similar to the actions of Zn2+ on squid K channels, indicating that the binding site may be a common motif in potassium channels. Studies of single Shaker channel properties showed that Zn2+ ions had little or no effect on the open channel current level or on the open channel lifetime. Rather, Zn2+ substantially delayed the time to first channel opening. Thus, K channels appear to contain a site to which divalent cations bind and in so doing act to slow one or more of the rate constants controlling transitions among closed conformational states of the channel.     

Effect of palytoxin on the sodium-potassium pump: model and simulation

We propose a reaction model for the palytoxin-sodium-potassium (PTX-Na(+)/K(+)) pump complex. The model, which is similar to the Albers-Post model for Na(+)/K(+)-ATPase, is used to elucidate the effect of PTX on Na(+)/K(+)-ATPase during the enzyme interactions with Na(+) and/or K(+) ions. Conformational substates and reactions for the pump are incorporated into the Albers-Post model to represent enzymes with or without bound PTX. A mathematical model based on the reaction scheme is used in simulations modeling experimental studies of PTX-induced ionic currents. Our simulations suggest that (i) extracellular Na(+) as well as K(+) promotes PTX-induced channel blockage; (ii) extracellular K(+) accelerates PTX unbinding; and (iii) K(+) occlusion in the PTX-pump complex is essential for describing the PTX-induced current dynamics.     

Mutations in the yeast two pore K+ channel YKC1 identify functional differences between the pore domains

The K+ channel of Saccharomyces cerevisiae encoded by the YKC1 gene includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore. Gating of the channel is promoted by membrane depolarisation and is regulated by the extracellular K+ concentration ([K+]o) both in the yeast and when expressed in Xenopus oocytes. Our previous work showed that substitutions of equivalent residues L293 and A428 within the pore-loops had qualitatively similar effects on both the [K+]o-sensitivity of channel gating and its voltage-dependence. Here, we report that mutations of equivalent residues N275 and N410, N-terminal from the K+ channel signature sequences of the two pores, have very different actions on channel gating and, in this case, are without effect on its voltage-sensitivity. The mutation N410D slowed current activation in a [K+]o-dependent manner and it accelerated deactivation, but without significant effect on the apparent affinity for K+. The N275D mutant, by contrast, had little effect on the [K+]o-sensitivity for activation and it greatly altered the. [K+]o-dependence of current deactivation. Neither mutant affected the voltage-dependence of the steady-state current nor the ability for other alkali cations to substitute for K+ in regulating gating. The double mutant N410D-N275D showed characteristics of N410D in the [K+]o-sensitivity of current activation and of N275D in the [K+]o-sensitivity of deactivation, suggesting that little interaction occurs between pore domains with mutations at these sites. The results indicate that the two pore domains are not functionally equivalent and they suggest that the regulation of gating by external K+ is mediated by K+ binding at two physically distinct sites with different actions.     

Perinatal PTX-sensitive G-protein expression and regulation of conductive 22Na+ transport in lung apical membrane vesicles.

Using apical membrane vesicles (AMV) prepared from mature foetal and early neonatal guinea pig lung we show that pertussis toxin (PTX)-sensitive G-protein regulation of conductive 22Na+ uptake undergoes rapid changes following birth. Thus, G-protein activation by intravesicular incorporation of 100 microM GTPgammaS into vesicles resuspended in NaCl, which in late gestation stimulated uptake, consistently induced inhibition of conductive Na+ uptake into AMV prepared from neonatal lung at 4 days of age (N4) (52+/-9%, n=8, P<0.05). This response was not significantly different in the presence of the relatively impermeant anion isethionate (Ise-) (69+/-9%, n=7, P<0.05). Changes in the regulation of uptake were already detectable on the day of birth (N0) in AMV resuspended in NaCl, with GTPgammaS inducing both stimulatory and inhibitory responses. These data indicate that the processes by which 22Na+ uptake into AMV is regulated by G-proteins undergoes a change at birth and by 4 days of age, G-protein regulation of uptake occurs predominantly via modulation of co-localised Na+ channels. Intravesicular incorporation of GDPbetaS or pre-treatment with PTX did not significantly alter conductive 22Na+ uptake in the presence of NaCl or NaIse suggesting that constitutively active G-proteins are not involved in this process. Pre-treatment of AMV with PTX prevented the inhibition of conductive 22Na+ uptake by GTPgammaS (105+/-16% n=7) indicating that a PTX-sensitive G-protein mediates the inhibition of channels in neonatal AMV. Western blotting demonstrated enrichment of Gialpha1, Gialpha2, Gialpha3 and Goalpha in the apical membrane preparations. We also show that there is a significant rise in the levels of Gialpha3 during the early neonatal period providing a potential candidate for the G-protein mediated changes in regulation of conductive 22Na+ uptake in neonatal AMV.     

A mutation in the pore of the sodium channel alters gating.

Ion permeation and channel gating are classically considered independent processes, but site-specific mutagenesis studies in K channels suggest that residues in or near the ion-selective pore of the channel can influence activation and inactivation. We describe a mutation in the pore of the skeletal muscle Na channel that alters gating. This mutation, I-W53C (residue 402 in the mu 1 sequence), decreases the sensitivity to block by tetrodotoxin and increases the sensitivity to block by externally applied Cd2+ relative to the wild-type channel, placing this residue within the pore near the external mouth. Based on contemporary models of the structure of the channel, this residue is remote from the regions of the channel known to be involved in gating, yet this mutation abbreviates the time to peak and accelerates the decay of the macroscopic Na current. At the single-channel level we observe a shortening of the latency to first opening and a reduction in the mean open time compared with the wild-type channel. The acceleration of macroscopic current kinetics in the mutant channels can be simulated by changing only the activation and deactivation rate constants while constraining the microscopic inactivation rate constants to the values used to fit the wild-type currents. We conclude that the tryptophan at position 53 in the domain IP-loop may act as a linchpin in the pore that limits the opening transition rate. This effect could reflect an interaction of I-W53 with the activation voltage sensors or a more global gating-induced change in pore structure.     

Lysophosphatidylglycerol stimulates chemotactic migration in human natural killer cells

We observed that lysophosphatidylglycerol (LPG) stimulates chemotactic migration in human natural killer (NK) cells. The LPG-induced chemotactic migration of NK cells was completely inhibited by pertussis toxin (PTX). LPG also stimulated the extracellular signal-regulated kinase (ERK) and Akt activities in NK cells. LPG-stimulated ERK activity was inhibited by PTX, indicating the involvement of PTX-sensitive G-proteins. The preincubation of NK cells with an ERK inhibitor (PD98059) or phosphoinositide-3-kinase (PI3K) inhibitors (wortmannin and LY294002) completely inhibited LPG-induced chemotactic migration, suggesting the essential role of ERK and PI3K in the process. Moreover, LPG-induced chemotactic migration in NK cell was inhibited by Ki16425, an LPA_1/3 receptor-selective antagonist, suggesting the involvement of the Ki16425-sensitive G-protein coupled receptor (GPCR) in the process. Taken together, the results indicate that LPG stimulates chemotactic migration in NK cells through GPCR, suggesting a new function of LPG as a modulator of NK cell functioning.     

Signal pathway of mitogen-induced Ca2+-activated K+ currents in young and aged T-cell clones of C57BL/6 mice.

Signal transduction pathways of mitogenic plant lectin, concanavalin A (Con A)- and ionomycin (INM)-induced (Ca2+-dependent K+ currents (I(Con A) and I(INM)) have been compared in young and aged T-cell clones by using the nystatin perforated patch-clamp whole-cell recording technique. In young T-cell clones, Con A evoked a long-lasting outward current which is mediated by the activation of the Ca2+-dependent K+ channels. The Ca2+ ionophore, INM, evoked a short-lasting Ca2+-dependent outward K+ current (I(INM)). The protein tyrosine kinase (PTK) inhibitor, herbimycin A (3 x 10(-6) M), but not the G protein blocker, pertussis toxin (PTX, 500 ng ml(-1)), completely prevented the I(Con A), but did not affect the I(INM). In aged T-cell clones, Con A fails to evoke any current response, while INM evokes an outward current which is comparable to that in a young T-cell clone. It is concluded that PTK, but not PTX-sensitive G proteins, plays a critical role in mediation of the signal transduction from Con A stimulation to activation of the Ca2+-dependent K+ channels, and that an impairment of the early signal pathway, perhaps the PTK, might be involved in the mechanism of the age-related decline of the proliferative response of T-lymphocytes to mitogenic stimulation.     

Rapid signaling responses in Sertoli cell membranes induced by follicle stimulating hormone and testosterone: calcium inflow and electrophysiological changes

This minireview describes the rapid signaling actions of follicle stimulating hormone (FSH) and testosterone in immature Sertoli cells mainly related to Ca(2+) inflow and the electrophysiological changes produced by hormones. The rapid membrane actions of FSH occur in a time frame of seconds to minutes, which include membrane depolarization and the stimulation of (45)Ca(2+) uptake. These effects can be prevented by pertussis toxin (PTX), suggesting that they are likely mediated by Gi-protein coupled receptor activation. Furthermore, these effects were inhibited by verapamil, a blocker of the L-type voltage-dependent Ca(2+) channel (VDCC). Finally, FSH stimulation of (45)Ca(2+) uptake was inhibited by the (phosphoinositide 3-kinase) PI3K inhibitor wortmannin. These results suggest that the rapid action of FSH on L-type Ca(2+) channel activity in Sertoli cells from pre-pubertal rats is mediated by the Gi/Gβγ/PI3Kγ pathway, independent of its effects on insulin-like growth factor type I (IGF-I). Testosterone depolarizes the membrane potential and increases the resistance and the (45)Ca(2+) uptake in Sertoli cells of the seminiferous tubules of immature rats. These actions were nullified by diazoxide (K(+)(ATP) channel opener). Testosterone actions were blocked by both PTX and the phospholipase C (PLC) inhibitor U73122, suggesting the involvement of PLC - phosphatidylinositol 4-5 bisphosphate (PIP2) hydrolysis via the Gq protein in the testosterone-mediated pathway. These results indicate that testosterone acts on the Sertoli cell membrane through the K(+)(ATP) channels and PLC-PIP2 hydrolysis, which closes the channel, depolarizes the membrane and stimulates (45)Ca(2+) uptake. These results demonstrate the existence of rapid non-classical pathways in immature Sertoli cells regulated by FSH and testosterone.     

Concerted All-or-none Subunit Interactions Mediate Slow Deactivation of Human ether-à-go-go-related Gene K+ Channels

During the repolarization phase of a cardiac action potential, hERG1 K⁺ channels rapidly recover from an inactivated state then slowly deactivate to a closed state. The resulting resurgence of outward current terminates the plateau phase and is thus a key regulator of action potential duration of cardiomyocytes. The intracellular N-terminal domain of the hERG1 subunit is required for slow deactivation of the channel as its removal accelerates deactivation 10-fold. Here we investigate the stoichiometry of hERG1 channel deactivation by characterizing the kinetic properties of concatenated tetramers containing a variable number of wild-type and mutant subunits. Three mutations known to accelerate deactivation were investigated, including R56Q and R4A/R5A in the N terminus and F656I in the S6 transmembrane segment. In all cases, a single mutant subunit induced the same rapid deactivation of a concatenated channel as that observed for homotetrameric mutant channels. We conclude that slow deactivation gating of hERG1 channels involves a concerted, fully cooperative interaction between all four wild-type channel subunits.     

Tyrosine kinase-dependent modulation by interferon-alpha of the ATP-sensitive K+ current in rabbit ventricular myocytes

We examined the effects of interferon-alpha on the ATP-sensitive K+ current (IK,ATP) in rabbit ventricular cells using the patch-clamp technique. IK,ATP was induced by NaCN. Whole-cell experiments indicated that interferon-alpha (5 x 10(2) - 2.4 x 10(4) U/ml) inhibited IK,ATP in a concentration-dependent manner (60.7+/-7.5% with 2.4 x 10(4) U/ml). In cell-attached configuration, interferon-alpha (2.4 x 10(4) U/ml) applied to the external solution also inhibited the activity of the single ATP-sensitive K+ (KATP) channel by 56.0+/-5.8% without affecting the single channel conductance. The inhibitory effect of IK,ATP by interferon-alpha was blocked by genistein and herbimycin A, tyrosine kinase inhibitors, but was not affected by N-(2-metylpiperazyl)-5-isoquinolinesulfoamide (H-7), an inhibitor of protein kinase C and cAMP-dependent protein kinase. These findings suggest that interferon-alpha inhibits the cardiac KATP channel through the activation of tyrosine kinase. The tyrosine kinase-mediated inhibition of IK,ATP by cytokines may aggravate cell damage during myocardial ischemia.