Mechanism of fluoride activation of G protein-gated muscarinic atrial K+ channels.

Aluminum fluoride (AlF4-) activates the heterotrimeric G protein Gs (stimulatory G protein of adenylylcyclase) (Sternweis, P. C., and Gilman, A. G. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 4888-4891) and GT (transducin), and for GT, Bigay et al. (Bigay, J., Deterre, P., Pfister, C., and Chabre, M. (1985) FEBS Lett. 191, 181-185) have made the intriguing proposal that AlF4- acts by mimicking the gamma-phosphate of GTP. The endogenous G protein (probably G alpha i-2 or G alpha i-3 (Yatani, A., Mattera, R., Codina, J., Graf, R., Okabe, K., Padrell, E., Iyengar, R., Brown, A. M., and Birnbaumer, L. (1988) Nature 336, 680-682) that stimulates the muscarinic atrial K+ (K+[ACh]) channel is also thought to be activated by AlF4- (Kurachi, Y., Nakajima, T., and Ito, H. (1987) Circulation 76, 105P). To investigate the AlF4- mechanism, we applied potassium fluoride (KF) to the cytoplasmic face of inside-out membrane patches excised from guinea pig atria. We found that KF activated single K+[ACh] channel currents in both a concentration- and a Mg(2+)-dependent manner. Activation persisted following removal of KF, but unlike activation by guanosine 5'-(3-thiotriphosphate) (GTP gamma S), was fully reversed by removal of Mg2+. Evidence for Al3+ involvement was that the Al3+ chelator deferoxamine (500 microM) inhibited KF activation and that at low concentrations of KF (less than 1 mM), micromolar AlCl3 concentrations potentiated KF stimulation. The rate of activation produced by KF was far slower than the rate produced by GTP or GTP gamma S, and unlike these guanine nucleotides, the rate was unchanged in the presence of agonist. To test the gamma-phosphate-mimicking hypothesis, we evaluated the requirement for GDP; and to accomplish this, it was necessary to establish a condition that ensured exchange of guanine nucleotides. This condition was satisfied by using the muscarinic agonist carbachol because both the rate and the extent of activation of the K+[ACh] channels produced by GTP were much faster in carbachol, and both were greatly slowed when GDP was added along with GTP. By contrast, the effects of KF were unchanged by carbachol in the presence or absence of GDP. Further evidence that GDP is not essential for activation by AlF4- was provided by the observation that during carbachol activation and following extensive washing with GMP, guanosine 5'-O-(2-thiodiphosphate) at blocking concentrations had no effect on activation produced by KF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Beta gamma dimers of G proteins inhibit atrial muscarinic K+ channels

It has been proposed that beta gamma dimers of signal-transducing G proteins mediate muscarinic activation of atrial K+ channels. We examined this hypothesis by testing the effects of beta gamma dimers from four sources (human erythrocytes, human placenta, bovine brain, and bovine retina) on single channel muscarinic K+ (K+[acetylcholine (ACh)]) currents in inside-out membrane patches of adult guinea pig atria. None of the four beta gamma dimer preparations stimulated K+[ACh] currents; on the contrary, each inhibited the currents whether the currents were activated with GTP alone (agonist-independent activity) or with GTP plus a muscarinic agonist (agonist-dependent activity). Detergents at concentrations used to suspend erythrocyte, brain, and placental beta gamma dimers had no effect by themselves, and detergents were not used with the retinal beta gamma dimers. We conclude that beta gamma dimers do not mediate stimulatory effects of the endogenous G protein that regulates the K+ channels. In fact beta gamma dimers appear to inhibit activation by the endogenous G alpha subunits. Further insight into the role of beta gamma dimers came from the observation that agonist-independent GTP-activated K+[ACh] currents were inhibited by beta gamma dimers at about one-tenth the concentration required to inhibit agonist-dependent activation. One possibility is that dimeric beta gamma may have a higher affinity for free alpha subunits than for alpha subunits associated with agonist-occupied receptors. Thus, in addition to the known requirement of beta gamma dimers for the interaction of alpha subunits with receptors, beta gamma dimers may also improve the signal-to-noise ratio for agonists by reducing agonist-independent background activities.     

Yeast pheromone receptor endocytosis and hyperphosphorylation are independent of G protein-mediated signal transduction.

When alpha factor binds to the yeast alpha factor receptor a signal is transmitted via a tripartite G protein that prepares the cell for conjugation. As a result of alpha factor binding the receptor also undergoes a regulated internalization and hyperphosphorylation. Using cells that lack activity of this tripartite G protein, we show that G protein-mediated pheromone signal transduction is not necessary for regulation of receptor internalization or hyperphosphorylation. Therefore, the processes of signal transduction and down regulation can be uncoupled. We propose that binding of alpha factor to its receptor results in a receptor conformation change that permits receptor hyperphosphorylation and interaction with the endocytic machinery.     

Effects of anions on the G protein-mediated activation of the muscarinic K+ channel in the cardiac atrial cell membrane. Intracellular chloride inhibition of the GTPase activity of GK

The effects of various intracellular anions on the G protein (GK)-mediated activation of the muscarinic K+ (KACh) channel were examined in single atrial myocytes isolated from guinea pig hearts. The patch clamp technique was used in the inside-out patch configuration. With acetylcholine (ACh, 0.5 microM) in the pipette, 1 microM GTP caused different magnitudes of KACh channel activation in internal solutions containing different anions. The order of potency of anions to induce the KACh channel activity at 0.5 microM ACh and 1 microM GTP was Cl- greater than or equal to Br- greater than 1-. In the SO4(2-) or aspartic acid internal solution, no channel openings were induced by 1 microM GTP with 0.5 microM ACh. In both the Cl- and SO4(2-) internal solutions (with 0.5 microM ACh) the relationship between the concentration of GTP and the channel activity was fit by the Hill equation with a Hill coefficient of approximately 3-4. However, the concentration of GTP at the half-maximal activation (Kd) was 0.2 microM in the Cl- and 10 microM in the SO4(2-) solution. On the other hand, the quasi-steady-state relationship between the concentration of guanosine-5'-o-(3-thiotriphosphate) and the channel activity did not differ significantly between the Cl- and SO4(2-) solutions; i.e., the Hill coefficient was approximately 3-4 and the Kd was approximately 0.06-0.08 microM in both solutions. The decay of channel activity after washout of GTP in the Cl- solution was much slower than that in the SO4(2-) solution. These results suggest that intracellular Cl- does not affect the turn-on reaction but slows the turn-off reaction of GK, resulting in higher sensitivity of the KACh channel for GTP. In the Cl- solution, even in the absence of agonists, GTP (greater than 1 microM) or ATP (greater than 1 mM) alone caused activation of the KACh channel, while neither occurred in the SO4(2-) solution. These observations suggest that the activation of the KACh channel by the basal turn-on reaction of GK or by phosphate transfer to GK by nucleoside diphosphate-kinase may depend at least partly on the intracellular concentration of Cl-.     

Folic acid stimulation of the Gα4 G protein-mediated signal transduction pathway inhibits anterior prestalk cell development in Dictyostelium

In Dictyostelium discoideum, several G proteins are known to mediate the transduction of signals that direct chemotactic movement and regulate developmental morphogenesis. The G protein alpha subunit encoded by the Galpha4 gene has been previously shown to be required for chemotactic responses to folic acid, proper developmental morphogenesis, and spore production. In this study, cells overexpressing the wild type Galpha4 gene, due to high copy gene dosage (Galpha4HC), were found to be defective in the ability to form the anterior prestalk cell region, express prespore- and prestalk-cell specific genes, and undergo spore formation. In chimeric organisms, Galpha4HC prespore cell-specific gene expression and spore production were rescued by the presence of wild-type cells, indicating that prespore cell development in Galpha4HC cells is limited by the absence of an intercellular signal. Transplanted wild-type tips were sufficient to rescue Galpha4HC prespore cell development, suggesting that the rescuing signal originates from the anterior prestalk cells. However, the deficiencies in prestalk-specific gene expression were not rescued in the chimeric organisms. Furthermore, Galpha4HC cells were localized to the prespore region of these chimeric organisms and completely excluded from the anterior prestalk region, suggesting that the Galpha4 subunit functions cell-autonomously to prevent anterior prestalk cell development. The presence of exogenous folic acid during vegetative growth and development delayed anterior prestalk cell development in wild-type but not galpha4 null mutant aggregates, indicating that folic acid can inhibit cell-type-specific differentiation by stimulation of the Galpha4-mediated signal transduction pathway. The results of this study suggest that Galpha4-mediated signals can regulate cell-type-specific differentiation by promoting prespore cell development and inhibiting anterior prestalk-cell development.     

Application of surface plasmon resonance for analysis of protein-protein interactions in the G protein-mediated signal transduction pathway

Hundreds of extracellular stimuli are received by cells via the pathways consisting of three basic components: cell-surface receptors, heterotrimeric G proteins, and intracellular effector enzymes or ion channels. A number of additional molecules, including G protein-coupled receptor kinases (GRKs), phosducin and Ca(2+)-binding proteins modulate signal transduction through these cascades. Understanding how these universal pathways work requires a detailed analysis of the interactions between these proteins. The recently emerged technology of surface plasmon resonance (SPR) can study protein-protein interactions by measuring not only the equilibrium binding constants, but also the association and dissociation rates. This article reviews experimental design used by researchers to analyze different components of the G protein pathway by SPR and focuses on the insights this technique provides regarding the kinetics, structure-function aspects and regulation of specific molecular events in the cascade.     

Effects of abscisic acid on K+ channels in Vicia faba guard cell protoplasts

Potassium channels were resolved in Vicia faba guard cell protoplasts by patch voltage-clamp. Whole-cell currents and single K+ channels had linear instantaneous current-voltage relations, reversing at the calculated Nernst potential for K+. Whole cell K+ currents activated exponentially during step depolarizations, with half-activation times of 400-450 msec at +80 mV and 90-110 msec at +150 mV. Single K+ channel conductance was 65 +/- 5 pS with a mean open time of 1.25 +/- 0.30 msec at 150 mV. Potassium channels were blocked by internal Cs+ and by external TEA+, but they were insensitive to external 4-aminopyridine. Application of 10 microM abscisic acid increased mean open time and caused long-lasting bursts of channel openings. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology.     

G regulatory proteins and muscarinic receptor signal transduction in mucous acini of rat submandibular gland

The involvement of G regulatory proteins in muscarinic receptor signal transduction was examined in electrically permeabilized rat submandibular acinar cells. The guanine nucleotide analog, GTP gamma S, caused the dose dependent hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to release IP3. This response was insensitive to pertussis toxin treatment and was duplicated by NaF but not by GDP beta S. Enhanced IP3 synthesis was observed with a combination of GTP gamma S and carbachol. Exogenous IP3, as well as carbachol and GTP gamma S, provoked the release of sequestered 45Ca2+ from non-mitochondrial stores. In intact cells, carbachol significantly reduced the level of cyclic AMP induced by the beta-adrenergic agonist, isoproterenol, to 69% of its normal value. Pertussis toxin abolished this inhibitory action of carbachol on cyclic nucleotide levels. These results suggest that muscarinic receptors are coupled to two separate G regulatory proteins in submandibular mucous acini-the pertussis toxin-insensitive Gp of the phosphoinositide transduction pathway associated with elevated cytosolic calcium levels, and the pertussis toxin-sensitive Gi inhibitory protein of the adenylate cyclase complex.     

Essential fatty acids and their metabolites in signal transduction.

The above examples argue that the diversity of eicosanoid structures is reflected in distinct actions, that the membrane-permeable nature of these molecules provides a system for interactive cellular signalling that may be particularly important in the nervous system, and that convincing evidence exists for direct actions of eicosanoids on both ion channels and kinases, as well as G-protein-coupled receptors.     

Detecting and imaging protein-protein interactions during G protein-mediated signal transduction in vivo and In situ by using fluorescence-based techniques

An important goal in cell biology has been to observe dynamic interactions between protein molecules within a living cell as they execute the reactions of a particular biochemical pathway. An important step toward achieving this goal has been the development of noninvasive fluorescence-based detection and imaging techniques for determining whether and when specific biomolecules in a cell become associated with one another. Furthermore, these techniques, which take advantage of phenomena known as bioluminescence- and fluorescence resonance energy transfer (BRET and FRET, respectively) as well as biomolecular fluorescence complementation (BiFC), can provide information about where and when protein-protein interactions occur in the cell. Increasingly BRET, FRET, and BiFC are being used to probe interactions between components involved in G protein-mediated signal transduction. Heptahelical (7TM) receptors, heterotrimeric guanine nucleotide binding proteins (G proteins) and their proximal downstream effectors constitute the core components of these ubiquitous signaling pathways. Signal transduction is initiated by the binding of agonist to heptahelical (7TM) receptors that in turn activate their cognate G proteins. The activated G protein subsequently regulates the activity of specific effectors. 7TM receptors, G proteins, and effectors are all membrane-associated proteins, and for decades two opposing hypotheses have vied for acceptance. The predominant hypothesis has been that these proteins move about independently of one another in membranes and that signal trandduction occurs when they encounter each other as the result of random collisions. The contending hypothesis is that signaling is propagated by organized complexes of these proteins. Until recently, the data supporting these hypotheses came from studying signaling proteins in solution, in isolated membranes, or in fixed cells. Although the former hypothesis has been favored, recent studies using BRET and FRET have generally supported the latter hypothesis as being the most likely scenario operating in living cells. In addition to the core components, there are many other proteins involved in G protein signaling, and BRET and FRET studies have been used to investigate their interactions as well. This review describes various BRET, FRET, and BiFC techniques, how they have been or can be applied to the study of G protein signaling, what caveats are involved in interpreting the results, and what has been learned about G protein signaling from the published studies.     

G protein-activated K+ channels: a reporter for rapid activation of G proteins by lysophosphatidic acid in Xenopus oocytes

Threshold concentrations of lysophosphatidic acid (LPA) or acetylcholine (ACh) induce pertussis toxin (PTX)-sensitive rapid desensitization of responses to LPA in Xenopus oocytes. To demonstrate that threshold [LPA] rapidly activates Gi/o proteins, we used the G protein-activated K+ channel (GIRK) as a reporter. Low [LPA] induced IK+ in <3 s of the agonist addition with little or no activation of chloride current. Depletion of Galphao/Galphao1 each decreased the LPA-induced IK+ by approximately 40-50%, while PTX completely abolished it. This is the first direct evidence showing the activation of GIRK by LPA, and the involvement of G proteins of the Go family in rapid desensitization of LPA responses.     

Production of N-lauroylated G protein alpha-subunit in Sf9 insect cells: the type of N-acyl group of Galpha influences G protein-mediated signal transduction

The alpha-subunit of rod photoreceptor G protein transducin (G(t1)alpha) is heterogeneously modified at the N-terminus by a mixture of acyl groups, laurate (C12:0), myristate (C14:0), and two unsaturated fatty acids (C14:1 and C14:2). Although the N-fatty acylation of G(t1)alpha plays important roles in protein-protein and protein-membrane interactions in light signaling, the biological significance of the heterogeneous acylation remains unclear due to the difficulty in isolating each G(t1)alpha isoform from the retinal rod cells. Here we found that G(t1)alpha/G(i1)alpha chimera (G(t/i)alpha) expressed in Sf9 cells is also heterogeneously modified by myristate (approximately 90%) and laurate (approximately 10%), raising the possibility that the N-acyl group of recombinant G(t/i)alpha may be manipulated by modifying culture media. In fact, addition of myristic acid to the medium decreased the relative content of lauroylated G(t/i)alpha to an undetectable level, whereas exogenously added lauric acid significantly increased the relative content of lauroylated G(t/i)alpha in a concentration-dependent manner. By culturing the G(t/i)alpha-virus infected Sf9 cells with fatty acids, we obtained four different preparations of N-acylated G(t/i)alpha, in which the relative abundance of lauroylated isoform was 0%, 20%, 33% and approximately 70%, respectively. Functional analysis of these proteins showed that an increase in the relative content of the lauroylated isoform remarkably slowed down the steady-state GTP hydrolysis rate of G(t/i)alpha; the steady-state GTPase activity of the lauroylated isoform was estimated to be one order of magnitude lower than that of the myristoylated isoform. These results suggest that the retinal G(t1)alpha is composed of isoforms having functionally heterogeneous signaling properties.     

Differential G protein-mediated coupling of D2 dopamine receptors to K+ and Ca2+ currents in rat anterior pituitary cells.

In anterior pituitary cells, dopamine, acting on D2 dopamine receptors, concomitantly reduces calcium currents and increases potassium currents. These dopamine effects require the presence of intracellular GTP and are blocked by pretreatment of the cells with pertussis toxin, suggesting that one or more G protein is involved. To identify the G proteins involved in coupling D2 receptors to these currents, we performed patch-clamp recordings in the whole-cell configuration using pipettes containing affinity-purified polyclonal antibodies raised against either Go alpha, Gi3 alpha, or Gi1,2 alpha. Dialysis with Go alpha antiserum significantly reduced the inhibition of calcium currents induced by dopamine, while increase of potassium currents was markedly attenuated only by Gi3 alpha antiserum. We therefore conclude that in pituitary cells, two different G proteins are involved in the signal transduction mechanism that links D2 receptor activation to a specific modulation of the four types of ionic channels studied here.     

Hydrophobic substitution mutations in the S4 sequence alter voltage-dependent gating in Shaker K+ channels

Voltage-activated Na+, Ca2+, and K+ channels contain a common motif, the S4 sequence, characterized by a basic residue at every third position interspersed mainly with hydrophobic residues. The S4 sequence is proposed to function as the voltage sensor and to move in response to membrane depolarization, triggering conformational changes that open the channel. This hypothesis has been tested in previous studies which revealed that mutations of the S4 basic residues often shift the curve of voltage dependence of activation along the voltage axis. We find that comparable or larger shifts are caused by conservative substitutions of hydrophobic residues in the S4 sequence of the Shaker K+ channel. We suggest that the S4 structure plays an essential role in determining the relative stabilities of the closed and open states of the channel.     

Effects of quinine on K+ transport in heart mitochondria

Quinine inhibits the respiration-dependent extrusion of K⁺ from Mg²⁺-depleted heart mitochondria and the passive osmotic swelling of these mitochondria in K⁺ and Na⁺ acetate at alkaline pH. These observations concur with those of Nakashima and Garlid (J. Biol. Chem. 257, 9252, 1982) using rat liver mitochondria. Quinine also inhibits the respiration-dependent contraction of heart mitochondria swollen passively in Na⁺ or K⁺ nitrate and the increment of elevated respiration associated with the extrusion of ions from these mitochondria. Quinine, at concentrations up to 0.5 mM, inhibits the respiration-dependent⁴²K⁺/K⁺ exchange seen in the presence of mersalyl, but higher levels of the drug produce increased membrane permeability and net K⁺ loss from the matrix. These results are all consistent with an inhibition of the putative mitochondrial K⁺/H⁺ antiport by quinine. However, quinine has other effects on the mitochondrial membrane, and possible alternatives to this interpretation are discussed.     

植物体内Ca2+信号转导过程的研究进展

Ca2+是高等植物细胞内普遍存在的一种信使分子,在植物体内起着非常广泛的作用,参与了植物体内多种刺激-反应的藕联过程。本文介绍了植物体内Ca2+转移系统,Ca2+信号的产生、终止和传递途径,Ca2+信号编码的多样性的最近研究进展。     

Energy-dependent exchange of K+ in heart mitochondria. K+ influx.

The tuberculostatic and carcinogenic drug isonicotinic acid hydrazide (“isoniazid”) is oxidized to pyridine-4-carboxaldehyde by the horseradish peroxidase/Mn²⁺/O₂ system. Eosin and related sensitizers greatly accelerate the reaction and generate light detectable with the liquid scintillation counter or with the photon counter. If the isoniazid concentration is raised, the rate and extent of O₂ uptake are increased, but above a certain concentration of isoniazid, emission is reduced and even suppressed. The strong quencher of triplet eosin, potassium ferricyanide, abolished both effects of eosin, that is, catalysis and light emission. Superoxide dismutase at high concentrations partially suppressed the emission and almost totally removed the catalytic effect. It is tentatively proposed that the isoniazid/peroxidase/Mn²⁺/O₂ system efficiently produces the aldehyde in the triplet state, which in turn transfers energy to eosin. Because of the presence of oxygen, only a small yield of triplet eosin is obtained and only a small fraction of these triplet eosin molecules is able to react with isoniazid. Nevertheless, it will contribute efficiently to a cyclic process of oxidation of the isoniazid.     

Energy-dependence exchange of K+ in heart mitochondria. K+ efflux.

The efflux ⁴²K⁺ from isolated beef heart mitochondria under conditions of near steadystate K⁺ is increased by repiration and is sensitive to uncouplers and to exogenous Mg² The respiration-dependent efflux is strongly activated by inorganic phosphate in the presence of external K⁺, but not Na⁺, and is inhibited by oxidative phosphorylation. Low concentrations of mersalyl also activate respiration-dependent efflux of ⁴²K⁺ in the absence of net alteration in matrix K⁺. Acetate in the presence of mersalyl brings about net accumulation of K⁺ with retention of internal ⁴²K⁺. The results are consistent with a model in which nearly constant matrix K⁺ is maintained by the regulated interplay between a K⁺ uniport (which is responsive to membrane potential and which is the pathway for K⁺ influx) and a $\text{K}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchanger (which responds to the transmembrane pH differential and which is the pathway for net K⁺ efflux).