Novel inhibition of gbetagamma-activated potassium currents induced by M(2) muscarinic receptors via a pertussis toxin-insensitive pathway

G(i) protein-coupled receptors such as the M(2) muscarinic acetylcholine receptor (mAChR) and A(1) adenosine receptor have been shown to activate G protein-activated inwardly rectifying K(+) channels (GIRKs) via pertussis toxin-sensitive G proteins in atrial myocytes and in many neuronal cells. Here we show that muscarinic M(2) receptors not only activate but also reversibly inhibit these K(+) currents when stimulated with agonist for up to 2 min. The M(2) mAChR-mediated inhibition of the channel was also observed when the channels were first activated by inclusion of guanosine 5'-O-(thiotriphosphate) in the pipette. Under these conditions the M(2) mAChR-induced inhibition was quasi-irreversible, suggesting a role for G proteins in the inhibitory process. In contrast, when GIRK currents were maximally activated by co-expressing exogenous Gbetagamma, the extent of acetylcholine (ACh)-induced inhibition was significantly reduced, suggesting competition between the receptor-mediated inhibition and the large pool of available Gbetagamma subunits. The signaling pathway that led to the ACh-induced inhibition of GIRK channels was unaffected by pertussis toxin pretreatment. Furthermore, the internalization and agonist-induced phosphorylation of M(2) mAChR was not required because a phosphorylation- and internalization-deficient mutant of the M(2) mAChR was as potent as the wild-type counterpart. Pharmacological agents modulating various protein kinases or phosphatidylinositol 3-kinase did not affect the inhibition of GIRK currents. Furthermore, the signaling pathway that mediates GIRK current inhibition was found to be membrane-delimited because bath application of ACh did not inhibit GIRK channel activity in cell-attached patches. Other G protein-coupled receptors including M(4) mAChR and alpha(1A) adrenergic receptors also caused the inhibition, whereas other G protein-coupled receptors including A(1) and A(3) adenosine receptors and alpha(2A) and alpha(2C) adrenergic receptors could not induce the inhibition. The presented results suggest the existence of a novel signaling pathway that can be activated selectively by M(2) and M(4) mAChR but not by adenosine receptors and that involves non-pertussis toxin-sensitive G proteins leading to an inhibition of Gbetagamma-activated GIRK currents in a membrane-delimited fashion.     

Signaling pathway underlying stimulation of L-type Ca2+ channels in rabbit portal vein myocytes by recombinant Gbetagamma subunits

In previous studies, we (Callaghan B, Koh SD, and Keef KD, Circ Res 94: 626-633, 2004) have shown that voltage-dependent L-type Ca(2+) channels (Cav) in portal vein myocytes are enhanced when muscarinic M2 receptors are activated with ACh. Current stimulation was coupled to the G protein subunit Gbetagamma along with the downstream mediators phosphatidylinositol-3-kinase (PI3K), protein kinase C (PKC), and c-Src. The present study was designed to determine whether the same second messenger pathway could be identified when exogenous recombinant Gbetagamma subunits are introduced into cells. Smooth muscle myocytes were freshly isolated from rabbit portal vein, and Cav currents were recorded by using the patch-clamp technique. Dialysis of cells with recombinant Gbetagamma (50 nM) significantly increased Cav currents (141%). Nifedipine (1 microM) reduced both control and stimulated currents by approximately 90%. The enhancement of current by Gbetagamma was equivalent to that produced by ACh (142%), whereas the PKC activator phorbol 12,13-dibutyrate (PdBu) gave rise to greater current stimulation (192%). Current stimulation with Gbetagamma, ACh, and PdBu were not associated with changes in the voltage dependence of activation or inactivation. The PI3K inhibitor LY-294002 (20 microM) reduced peak currents by 32% in cells dialyzed with Gbetagamma, whereas the inactive analog LY-303511 resulted in a small but significant reduction in current (12%). The c-Src inhibitor PP2 (1 microM) also significantly reduced currents (34%), whereas the inactive analog PP3 was without effect. These data provide further evidence for the hypothesis that Gbetagamma leads to stimulation of Cav currents in rabbit portal vein myocytes via a signaling pathway that includes PI3K, PKC, and c-Src.     

Overexpression of beta 1 and beta 2 adrenergic receptors in rat atrial myocytes. Differential coupling to G protein-gated inward rectifier K(+) channels via G(s) and G(i)/o

G protein-activated inwardly rectifying K(+) (GIRK) channels, expressed in atrial myocytes, various neurons, and endocrine cells, represent the paradigmatic target of beta gamma subunits released from activated heterotrimeric G proteins. These channels contribute to physiological slowing of cardiac frequency and synaptic inhibition. They are activated by beta gamma dimers released upon stimulation of receptors coupled to pertussis toxin-sensitive G proteins (G(i/o)), whereas beta gamma released from G(s) do not converge on the channel subunits. This is in conflict with the finding that dimeric combinations of various beta and gamma subunits can activate GIRK channels with little specificity. In the present study, we have overexpressed the major subtypes of cardiac beta-adrenergic receptors (beta(1)-AR and beta(2)-AR) in atrial myocytes by transient transfection. Whereas in native cells beta-adrenergic stimulation with isoproterenol failed to induce measurable GIRK current, robust currents were recorded from myocytes overexpressing either beta(1)-AR or beta(2)-AR. Whereas the beta(2)-AR-induced current showed the same sensitivity to pertussis toxin as the current evoked by the endogenous G(i/o)-coupled muscarinic M(2) receptor, isoproterenol-activated currents were insensitive to pertussis toxin treatment in beta(1)-AR-overexpressing myocytes. In contrast to a recent publication (Leaney, J. L., Milligan, G., and Tinker, A. (2000) J. Biol. Chem. 275, 921-929), sizable GIRK currents could also be activated by isoproterenol when the signaling pathway was reconstituted by transient transfection in two different standard cell lines (Chinese hamster ovary and HEK293). These results demonstrate that specificity of receptor-G protein signaling can be disrupted by overexpression of receptors. Moreover, the alpha subunit of heterotrimeric G proteins does not confer specificity to G beta gamma-mediated signaling.     

Specificity of Gbetagamma signaling to Kir3 channels depends on the helical domain of pertussis toxin-sensitive Galpha subunits

Acetylcholine signaling through muscarinic type 2 receptors activates atrial G protein-gated inwardly rectifying K(+) (Kir3) channels via the betagamma subunits of G proteins (Gbetagamma). Different combinations of recombinant Gbetagamma subunits have been shown to activate Kir3 channels in a similar manner. In native systems, however, only Gbetagamma subunits associated with the pertussis toxin-sensitive Galpha(i/o) subunits signal to K(+) channels. Additionally, in vitro binding experiments supported the notion that the C terminus of Kir3 channels interacts preferentially with Galpha(i) over Galpha(q). In this study we confirmed in two heterologous expression systems a preference of Galpha(i) over Galpha(q) in the activation of K(+) currents. To identify determinants of Gbetagamma signaling specificity, we first exchanged domains of Galpha(i) and Galpha(q) subunits responsible for receptor coupling selectivity and swapped their receptor coupling partners. Our results established that the G proteins, regardless of the receptor type to which they coupled, conferred specificity to Kir3 activation. We next tested signaling through chimeras between the Galpha(i) and Galpha(q) subunits in which the N terminus, the helical, or the GTPase domains of the Galpha subunits were exchanged. Our results revealed that the helical domain of Galpha(i) (residues 63-175) in the background of Galpha(q) could support Kir3 activation, whereas the reverse chimera could not. Moreover, the helical domain of the Galpha(i) subunit conferred "Galpha(i)-like" binding of the Kir3 C terminus to the Galpha(q) subunits that contained it. These results implicate the helical domain of Galpha(i) proteins as a critical determinant of Gbetagamma signaling specificity.     

Galphai1 and Galphai3 differentially interact with, and regulate, the G protein-activated K+ channel

G protein-activated K(+) channels (GIRKs; Kir3) are activated by direct binding of Gbetagamma subunits released from heterotrimeric G proteins. In native tissues, only pertussis toxin-sensitive G proteins of the G(i/o) family, preferably Galpha(i3) and Galpha(i2), are donors of Gbetagamma for GIRK. How this specificity is achieved is not known. Here, using a pull-down method, we confirmed the presence of Galpha(i3-GDP) binding site in the N terminus of GIRK1 and identified novel binding sites in the N terminus of GIRK2 and in the C termini of GIRK1 and GIRK2. The non-hydrolyzable GTP analog, guanosine 5'-3-O-(thio)triphosphate, reduced the binding of Galpha(i3) by a factor of 2-4. Galpha(i1-GDP) bound to GIRK1 and GIRK2 much weaker than Galpha(i3-GDP). Titrated expression of components of signaling pathway in Xenopus oocytes and their activation by m2 muscarinic receptors revealed that G(i3) activates GIRK more efficiently than G(i1), as indicated by larger and faster agonist-evoked currents. Activation of GIRK by purified Gbetagamma in excised membrane patches was strongly augmented by coexpression of Galpha(i3) and less by Galpha(i1). Differences in physical interactions of GIRK with GDP-bound Galpha subunits, or Galphabetagamma heterotrimers, may dictate different extents of Galphabetagamma anchoring, influence the efficiency of GIRK activation by Gbetagamma, and play a role in determining signaling specificity.     

M2 muscarinic receptors induce airway smooth muscle activation via a dual, Gbetagamma-mediated inhibition of large conductance Ca2+-activated K+ channel activity

Airway smooth muscle is richly endowed with muscarinic receptors of the M(2) and M(3) subtype. Stimulation of these receptors inhibits large conductance calcium-activated K(+) (BK) channels, a negative feed back regulator, in a pertussis toxin-sensitive manner and thus facilitates contraction. The underlying mechanism, however, is unknown. We therefore studied the activity of bovine trachea BK channels in HEK293 cells expressing the M(2) or M(3) receptor (M(2)R or M(3)R). In M(2)R- but not M(3)R-expressing cells, maximal effective concentrations of carbamoylcholine (CCh) inhibited whole cell BK currents by 53%. This M(2)R-induced inhibition was abolished by pertussis toxin treatment or overexpression of the Gbetagamma scavenger transducin-alpha. In inside-out patches, direct application of 300 nm purified Gbetagamma decreased channel open probability by 55%. The physical interaction of Gbetagamma with BK channels was confirmed by co-immunoprecipitation. Interestingly, inhibition of phospholipase C as well as protein kinase C activities also reversed the CCh effect but to a smaller (approximately 20%) extent. Mouse tracheal cells responded similarly to CCh, purified Gbetagamma and phospholipase C/protein kinase C inhibition as M(2)R-expressing HEK293 cells. Our results demonstrate that airway M(2)Rs inhibit BK channels by a dual, Gbetagamma-mediated mechanism, a direct membrane-delimited interaction, and the activation of the phospholipase C/protein kinase C pathway.     

Coupling of exogenous receptors to phospholipase C in Xenopus oocytes through pertussis toxin-sensitive and -insensitive pathways. Cross-talk through heterotrimeric G-proteins

Heterotrimeric guanine nucleotide-binding proteins (G-proteins) can be categorized into molecularly divergent groups by their differential sensitivity to pertussis toxin. Receptors specifically use either pertussis toxin-sensitive or-insensitive G-proteins to couple to specific effectors. Receptor stimulation of phospholipase C, however, is pertussis toxin sensitive in some systems and pertussis toxin insensitive in others. We studied the coupling of receptors to phospholipase C by expressing receptors from both systems into a single cell, the Xenopus oocyte. [Arg8]Vassopressin (AVP) receptors from liver and cholecystokinin-8(sulfated) (CCK) receptors from brain were expressed in oocytes by intracellular injection of RNA. Both receptors stimulated a Ca2+-dependent Cl- current which can also be evoked by intracellular injection of inositol 1,4,5-tris-phosphate. Hence, receptor stimulation of phospholipase C was measured as the evoked Ca2+-dependent Cl- current. The liver AVP receptor, which is known to stimulate phospholipase C in a pertussis toxin-insensitive manner (Lynch, C. J., Prpic, V., Blackmore, P. F., and Exton, J. H. (1986) Mol. Pharmacol. 29, 196-203), was found to stimulate phospholipase C through a pertussis toxin-sensitive pathway in the Xenopus oocyte. The CCK receptor from brain stimulated phospholipase C through a pertussis toxin-insensitive pathway. Both AVP and CCK stimulation of phospholipase C were attenuated by the intracellular injection of excess G-protein beta gamma subunits. Neither pertussis toxin treatment nor intracellular injection of beta gamma subunits affected any steps subsequent to inositol 1,4,5-tris-phosphate production. From these data we conclude that both the pertussis toxin-sensitive and -insensitive pathways for receptor coupling to phospholipase C are transduced by heterotrimeric G-proteins. We also find that there is a lack of coupling fidelity of receptors to G-proteins in stimulation of phospholipase C which can be influenced by the membrane environment.     

Neuronal Platelet-Activating Factor Receptor Signal Transduction Involves a Pertussis Toxin-Sensitive G-Protein

In most nonneural systems, platelet-activating factor (PAF) receptor effects are mediated by G-proteins that are often pertussis toxin-sensitive. The activation of pertussis toxin-sensitive G-proteins linked to PAF receptors results in the mobilization of intracellular calcium, at least in part, through the second messenger inositol triphosphate. We have sought to determine if a pertussis toxin-sensitive G-protein is involved in the PAF receptor-mediated phenomena of growth cone collapse and of synaptic enhancement in primary neuronal culture. Using infrared differential interference contrast microscopy and patch-clamp recording techniques, pertussis toxin, but not the inactive B oligomer of the toxin, was found to block both the growth cone collapse and the enhanced synaptic release of excitatory transmitter induced by a nonhydrolyzable PAF receptor agonist, making it likely that G_o, G_q, or G_i is the G-protein transducer of PAF receptors in primary neurons. We believe that PAF acts directly on neuronal receptors, which are linked to pertussis toxin-sensitive G-proteins, on the tips of developing neurites, and on presynaptic nerve terminals, leading to growth cone collapse and enhanced synaptic release of transmitter.     

The G protein alpha subunit has a key role in determining the specificity of coupling to, but not the activation of, G protein-gated inwardly rectifying K(+) channels

In neuronal and atrial tissue, G protein-gated inwardly rectifying K(+) channels (Kir3.x family) are responsible for mediating inhibitory postsynaptic potentials and slowing the heart rate. They are activated by Gbetagamma dimers released in response to the stimulation of receptors coupled to inhibitory G proteins of the G(i/o) family but not receptors coupled to the stimulatory G protein G(s). We have used biochemical, electrophysiological, and molecular biology techniques to examine this specificity of channel activation. In this study we have succeeded in reconstituting such specificity in an heterologous expression system stably expressing a cloned counterpart of the neuronal channel (Kir3.1 and Kir3.2A heteromultimers). The use of pertussis toxin-resistant G protein alpha subunits and chimeras between G(i1) and G(s) indicate a central role for the G protein alpha subunits in determining receptor specificity of coupling to, but not activation of, G protein-gated inwardly rectifying K(+) channels.     

The M2 muscarinic G-protein-coupled receptor is voltage-sensitive

G-protein coupled receptors are not considered to exhibit voltage sensitivity. Here, using Xenopus oocytes, we show that the M2 muscarinic receptor (m2R) is voltage-sensitive. The m2R-mediated potassium channel (GIRK) currents were used to assay the activity of m2R. We found that the apparent affinity of m2R toward acetylcholine (ACh) was reduced upon depolarization. Binding experiments of [3H]ACh to individual oocytes expressing m2R confirmed the electrophysiological findings. When the GIRK channels were activated either by overexpression of Gbetagamma subunits or by injection of GTPgammaS, the ratio between the currents measured at -60 mV and +40 mV was the same as for the basal activity of the GIRK channel. Thus, the steps downstream to agonist activation of m2R are not voltage-sensitive. We further found that, in contrast to m2R, the apparent affinity of m1R was increased upon depolarization. We also found that the voltage sensitivity of binding of [3H]ACh to oocytes expressing m2R was greatly diminished following pretreatment with pertussis toxin. The cumulative results suggest that m2R is, by itself, voltage-sensitive. Furthermore, the voltage sensitivity does not reside in the ACh binding site, rather, it most likely resides in the receptor region that couples to the G-protein.     

Overexpression of monomeric and multimeric GIRK4 subunits in rat atrial myocytes removes fast desensitization and reduces inward rectification of muscarinic K(+) current (I(K(ACh))). Evidence for functional homomeric GIRK4 channels

K(+) channels composed of G-protein-coupled inwardly rectifying K(+) channel (GIRK) (Kir3.0) subunits are expressed in cardiac, neuronal, and various endocrine tissues. They are involved in inhibiting excitability and contribute to regulating important physiological functions such as cardiac frequency and secretion of hormones. The functional cardiac (K((ACh))) channel activated by G(i)/G(o)-coupled receptors such as muscarinic M(2) or purinergic A(1) receptors is supposed to be composed of the subunits GIRK1 and GIRK4 in a heterotetrameric (2:2) fashion. In the present study, we have manipulated the subunit composition of the K((ACh)) channels in cultured atrial myocytes from hearts of adult rats by transient transfection of vectors encoding for GIRK1 or GIRK4 subunits or GIRK4 concatemeric constructs and investigated the effects on properties of macroscopic I(K(ACh)). Transfection with a GIRK1 vector did not cause any measurable effect on properties of I(K(ACh)), whereas transfection with a GIRK4 vector resulted in a complete loss in desensitization, a reduction of inward rectification, and a slowing of activation. Transfection of myocytes with a construct encoding for a concatemeric GIRK4(2) subunit had similar effects on desensitization and inward rectification. Following transfection of a tetrameric construct (GIRK4(4)), these changes in properties of I(K(ACh)) were still observed but were less pronounced. Heterologous expression in Chinese hamster ovary cells and human embryonic kidney 293 cells of monomeric, dimeric, and tetrameric GIRK4 resulted in robust currents activated by co-expressed A(1) and M(2) receptors, respectively. These data provide strong evidence that homomeric GIRK4 complexes form functional G(beta)gamma gated ion channels and that kinetic properties of GIRK channels, such as activation rate, desensitization, and inward rectification, depend on subunit composition.     

Coupling to Gs and G(q/11) of histamine H2 receptors heterologously expressed in adult rat atrial myocytes

The predominant histamine receptor subtype in the supraventricular and ventricular tissue of various mammalian species is the H2 receptor (H2-R) subtype, which is known to couple to stimulatory G proteins (Gs), i.e. the major effects of this autacoid are an increase in sinus rate and in force of contraction. To investigate histamine effects in H2-R-transfected rat atrial myocytes, endogenous GIRK currents and L-type Ca2+ currents were used as functional assays. In H2-R-transfected myocytes, exposure to His resulted in a reversible augmentation of L-type Ca2+ currents, consistent with the established coupling of this receptor to the Gs-cAMP-PKA signalling pathway. Mammalian K+ channels composed of GIRK (Kir3.x) subunits are directly controlled by interaction with betagamma subunits released from G proteins, which couple to seven-helix receptors. In mock-transfected atrial cardiomyocytes, activation of muscarinic K+ channels (IK(ACh)) was limited to Gi-coupled receptors (M2R, A1R). In H2-R-overexpressing cells, histamine activated IK(ACh) via Gs-derived betagamma subunits since the histamine-induced current was insensitive to pertussis toxin. These data indicate that overexpression of Gs-coupled H2-R results in a loss of target specificity due to an increased agonist-induced release of Gs-derived betagamma subunits. When IK(ACh) was maximally activated by GTP-gamma-S, histamine induced an irreversible inhibition of the inward current in a fraction of H2-R-transfected cells. This inhibition is supposed to be mediated via a G(q/11)-PLC-mediated depletion of PIP2, suggesting a partial coupling of overexpressed H2-R to G(q/11). Dual coupling of H2-Rs to Gs and Gq is demonstrated for the first time in cardiac myocytes. It represents a novel mechanism to augment positive inotropic effects by activating two different signalling pathways via one type of histamine receptor. Activation of the Gs-cAMP-PKA pathway promotes Ca2+ influx through phosphorylation of L-type Ca2+ channels. Simultaneous activation of Gq-signalling pathways might result in phosphoinositide turnover and Ca2+ release from intracellular stores, thereby augmenting H2-induced increases in [Ca2+]i.     

Molecular events associated with the regulation of signaling by M2 muscarinic receptors

Multiple events are associated with the regulation of signaling by the M2 muscarinic cholinergic receptors (mAChRs). Desensitization of the attenuation of adenylyl cyclase by the M2 mAChRs appears to involve agonist-dependent phosphorylation of M2 mAChRs by G-protein coupled receptor kinases (GRKs) that phosphorylate the receptors in a serine/threonine rich motif in the 3rd intracellular domain of the receptors. Mutation of residues 307-311 from TVSTS to AVAAA in this domain of the human M2 mAChR results in a loss of receptor/G-protein uncoupling and a loss of arrestin binding. Agonist-induced sequestration of receptors away from their normal membrane environment is also regulated by agonist-induced phosphorylation of the M2 mAChRs on the 3rd intracellular domain, but in HEK cells, the predominant pathway of internalization is not regulated by GRKs or arrestins. This pathway of internalization is not inhibited by a dominant negative dynamin, and does not appear to involve either clathrin coated pits or caveolae. The signaling of the M2 mAChR to G-protein regulated inwardly rectifying K channels (GIRKs) can be modified by RGS proteins. In HEK cells, expression of RGS proteins leads to a constitutive activation of the channels through a mechanism that depends on Gbetagamma. RGS proteins appear to increase the concentration of free Gbetagamma in addition to acting as GAPs. Thus multiple mechanisms acting at either the level of the M2 mAChRs or the G-proteins can contribute to the regulation of signaling via the M2 mAChRs.     

Regulation of melatonin 1a receptor signaling and trafficking by asparagine-124.

Melatonin is a pineal hormone that regulates seasonal reproduction and has been used to treat circadian rhythm disorders. The melatonin 1a receptor is a seven- transmembrane domain receptor that signals predominately via pertussis toxin-sensitive G-proteins. Point mutations were created at residue N124 in cytoplasmic domain II of the receptor and the mutant receptors were expressed in a neurohormonal cell line. The acidic N124D- and E-substituted receptors had high-affinity (125)I-melatonin binding and a subcellular localization similar to the neutral N124N wild-type receptor. Melatonin efficacy for the inhibition of cAMP by N124D and E mutations was significantly decreased. N124D and E mutations strongly compromised melatonin efficacy and potency for inhibition of K(+)-induced intracellular Ca(++) fluxes and eliminated control of spontaneous calcium fluxes. However, these substitutions did not appear to affect activation of Kir3 potassium channels. The hydrophobic N124L and N124A or basic N124K mutations failed to bind (125)I-melatonin and appeared to aggregate or traffic improperly. N124A and N124K receptors were retained in the Golgi. Therefore, mutants at N124 separated into two sets: the first bound (125)I-melatonin with high affinity and trafficked normally, but with reduced inhibitory coupling to adenylyl cyclase and Ca(++) channels. The second set lacked melatonin binding and exhibited severe trafficking defects. In summary, asparagine-124 controls melatonin receptor function as evidenced by changes in melatonin binding, control of cAMP levels, and regulation of ion channel activity. Asparagine-124 also has a unique structural effect controlling receptor distribution within the cell.     

The Stoichiometry of Gbeta gamma binding to G-protein-regulated inwardly rectifying K+ channels (GIRKs)

G-protein-coupled inwardly rectifying K(+) (GIRK; Kir3.x) channels are the primary effectors of numerous G-protein-coupled receptors. GIRK channels decrease cellular excitability by hyperpolarizing the membrane potential in cardiac cells, neurons, and secretory cells. Although direct regulation of GIRKs by the heterotrimeric G-protein subunit Gbetagamma has been extensively studied, little is known about the number of Gbetagamma binding sites per channel. Here we demonstrate that purified GIRK (Kir 3.x) tetramers can be chemically cross-linked to exogenously purified Gbetagamma subunits. The observed laddering pattern of Gbetagamma attachment to GIRK4 homotetramers was consistent with the binding of one, two, three, or four Gbetagamma molecules per channel tetramer. The fraction of channels chemically cross-linked to four Gbetagamma molecules increased with increasing Gbetagamma concentrations and approached saturation. These results suggest that GIRK tetrameric channels have four Gbetagamma binding sites. Thus, GIRK (Kir 3.x) channels, like the distantly related cyclic nucleotide-gated channels, are tetramers and exhibit a 1:1 subunit/ligand binding stoichiometry.     

Kappa opiate agonists inhibit Ca2+ influx in rat spinal cord-dorsal root ganglion cocultures. Involvement of a GTP-binding protein

The aim of the present study has been to characterize the regulation by opiates of 45Ca2+ influx in rat spinal cord-dorsal root ganglion cocultures. We have demonstrated that K+-induced depolarization, in the presence of the Ca2+ channel agonist Bay K8644, stimulated Ca2+ influx (3-4-fold) via the dihydropyridine class of voltage-dependent Ca2+ channels. While mu and delta opiates had no effect, kappa opiate agonists (e.g. U50488, dynorphin) profoundly depressed the stimulated Ca2+ influx (86% inhibition at 100 microM U50488). The kappa agonist action was stereospecific and could be reversed by the opiate antagonist naloxone. The inhibition produced by kappa agonists was greatly diminished following pertussis toxin treatment, and this effect was accompanied by toxin-induced ADP-ribosylation of a 40-41-kDa protein. This suggests that kappa opiate receptors are negatively coupled to voltage-dependent Ca2+ channels, via a pertussis toxin-sensitive GTP-binding protein. Basal 45Ca2+ uptake, stimulated by adenylate cyclase activators (forskolin and cholera toxin), was potently inhibited by kappa opiates suggesting that, under conditions of neurohormonal stimulation of adenylate cyclase, kappa receptors are coupled to Ca2+ channels indirectly via the adenylate cyclase complex. In addition, cAMP-independent coupling pathways may also be involved.     

Gbetagamma-dependent and Gbetagamma-independent basal activity of G protein-activated K+ channels

Cardiac and neuronal G protein-activated K+ channels (GIRK; Kir3) open following the binding of Gbetagamma subunits, released from Gi/o proteins activated by neurotransmitters. GIRKs also possess basal activity contributing to the resting potential in neurons. It appears to depend largely on free Gbetagamma, but a Gbetagamma-independent component has also been envisaged. We investigated Gbetagamma dependence of the basal GIRK activity (A(GIRK,basal)) quantitatively, by titrated expression of Gbetagamma scavengers, in Xenopus oocytes expressing GIRK1/2 channels and muscarinic m2 receptors. The widely used Gbetagamma scavenger, myristoylated C terminus of beta-adrenergic kinase (m-cbetaARK), reduced A(GIRK,basal) by 70-80% and eliminated the acetylcholine-evoked current (I(ACh)). However, we found that m-cbetaARK directly binds to GIRK, complicating the interpretation of physiological data. Among several newly constructed Gbetagamma scavengers, phosducin with an added myristoylation signal (m-phosducin) was most efficient in reducing GIRK currents. m-phosducin relocated to the membrane fraction and did not bind GIRK. Titrated expression of m-phosducin caused a reduction of A(GIRK,basal) by up to 90%. Expression of GIRK was accompanied by an increase in the level of Gbetagamma and Galpha in the plasma membrane, supporting the existence of preformed complexes of GIRK with G protein subunits. Increased expression of Gbetagamma and its constitutive association with GIRK may underlie the excessively high A(GIRK,basal) observed at high expression levels of GIRK. Only 10-15% of A(GIRK,basal) persisted upon expression of both m-phosducin and cbetaARK. These results demonstrate that a major part of Ibasal is Gbetagamma-dependent at all levels of channel expression, and only a small fraction (<10%) may be Gbetagamma-independent.     

The sensitivity of G protein-activated K+ channels toward halothane is essentially determined by the C terminus

G protein-activated K(+) channels (GIRKs or Kir3.x) are targets for the volatile anesthetic, halothane. When coexpressed with the m(2) acetylcholine (ACh) receptor in Xenopus oocytes, agonist-activated GIRK1(F137S)- and GIRK2-mediated currents are inhibited by halothane, whereas in the absence of ACh, high concentrations of halothane induce GIRK1(F137S)-mediated currents. To elucidate the molecular mechanism of halothane action on GIRK currents of different subunit compositions, we constructed deletion mutants of GIRK1(F137S) (GIRK1(Delta363*)) and GIRK2 (GIRK2(Delta356)) lacking the C-terminal ends, as well as chimeric GIRK channels. Mutated GIRK channels showed normal currents when activated by ACh but exhibited different pharmacological properties toward halothane. GIRK2(Delta356) showed no sensitivity against the inhibitory action of halothane but was activated by halothane in the absence of an agonist. GIRK1(Delta363*) was activated by halothane more efficiently. Currents mediated by chimeric channels were inhibited by anesthetic concentrations that were at least 30-fold lower than those necessary to decrease GIRK2 wild type currents. Glutathione S-transferase pulldown experiments did not show displacement of bound Gbetagamma by halothane, indicating that halothane does not interfere with Gbetagamma binding. Single channel experiments revealed an influence of halothane on the gating of the channels: The agonist-induced currents of GIRK1 and GIRK2, carried mainly by brief openings, were inhibited, whereas higher concentrations of the anesthetic promoted long openings of GIRK1 channels. Because the C terminus is crucial for these effects, an interaction of halothane with the channel seems to be involved in the mechanism of current modulation.     

The C terminus of the metabotropic glutamate receptor subtypes 2 and 7 specifies the receptor signaling pathways

There is accumulating evidence that the specificity of the transduction cascades activated by G protein-coupled receptors cannot solely depend on the nature of the coupled G protein. To identify additional structural determinants, we studied two metabotropic glutamate (mGlu) receptors, the mGlu2 and mGlu7 receptors, that are both coupled to G(o) proteins but are known to affect different effectors in neurons. Thus, the mGlu2 receptor selectively blocks N- and L-type Ca(2+) channels via a protein kinase C-independent pathway, whereas the mGlu7 receptor selectively blocks P/Q-type Ca(2+) channels via a protein kinase C-dependent pathway, and both effects are pertussis toxin-sensitive. We examined the role of the C-terminal domain of these receptors in this coupling. Chimeras were constructed by exchanging the C terminus of these receptors and transfected into neurons. Different chimeric receptors bearing the C terminus of mGlu7 receptor blocked selectively P/Q-type Ca(2+) channels, whereas chimeras bearing the C terminus of mGlu2 receptor selectively blocked N- and L-type Ca(2+) channels. These results show that the C terminus of mGlu2 and mGlu7 receptors is a key structural determinant that allows these receptors to select a specific signaling pathway in neurons.     

Pertussis toxin-dependent and -independent hormonal effects on cultured renal epithelioid cells

The present study has been performed to test for the involvement of pertussis toxin-sensitive GTP-binding proteins (G-proteins) in the cellular transduction of hormone-induced activation of potassium channels. In Madin Darby canine kidney (MDCK) cells, a permanent cell line from dog kidney, epinephrine, acetylcholine, bradykinin, serotonin and ATP hyperpolarize the cell membrane by activation of potassium channels. In cells pretreated with pertussis toxin the hyperpolarizations elicited by either acetylcholine or serotonin are completely abolished; that following epinephrine is blunted and only transient. The hyperpolarizing effects of ATP or bradykinin are not affected by pertussis toxin. Thus, in MDCK cells both pertussis toxin-dependent and -independent mechanisms operate in parallel to enhance the potassium conductance of the cell membrane.