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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.

Although most L-type calcium channel alpha(1C) subunits isolated from heart or brain are approximately 190-kDa proteins that lack approximately 50 kDa of the C terminus, the C-terminal domain is present in intact cells. To test the hypothesis that the C terminus is processed but remains functionally associated with the channels, expressed, full-length alpha(1C) subunits were cleaved in vitro by chymotrypsin to generate a 190-kDa C-terminal truncated protein and C-terminal fragments of 30-56 kDa. These hydrophilic C-terminal fragments remained membrane-associated. A C-terminal proline-rich domain (PRD) was identified as the mediator of membrane association. The alpha(1C) PRD bound to SH3 domains in Src, Lyn, Hck, and the channel beta(2) subunit. Mutant alpha(1C) subunits lacking either approximately 50 kDa of the C terminus or the PRD produced increased barium currents through the channels, demonstrating that these domains participate in the previously described (Wei, X., Neely, a., Lacerda, A. E. Olcese, r., Stefani, E., Perez-Reyes, E., and Birnbaumer, L. (1994) J. Biol. Chem. 269, 1635-1640) inhibition of channel function by the C terminus.

Activation of protein kinase A (PKA) through the beta-adrenergic receptor pathway is crucial for the positive regulation of cardiac L-type currents; however it is still unclear which phosphorylation events cause the robust regulation of channel function. In order to study whether or not the recently identified PKA phosphorylation sites on the beta(2) subunit are of functional significance, we coexpressed wild-type (WT) or mutant beta(2) subunits in tsA-201 cells together with an alpha(1C) subunit, alpha(1C)Delta1905, that lacked the C-terminal 265 amino acids, including the only identified PKA site at Ser-1928. This truncated alpha(1C) subunit was similar to the truncated alpha(1C) subunit isolated from cardiac tissue not only in size ( approximately 190 kDa), but also with respect to its failure to serve as a PKA substrate. In cells transfected with the WT beta(2) subunit, voltage-activated Ba(2+) currents were significantly increased when purified PKA was included in the patch pipette. Furthermore, mutations of Ser-478 and Ser-479 to Ala, but not Ser-459 to Ala, on the beta(2) subunit, completely abolished the PKA-induced increase of currents. The data indicate that the PKA-mediated stimulation of cardiac L-type Ca(2+) currents may be at least partially caused by phosphorylation of the beta(2) subunit at Ser-478 and Ser-479.

G proteins play a critical role in transducing a large variety of signals into intracellular responses. Increasingly, there is evidence that G proteins may play other roles as well. Dominant-negative constructs of the alpha subunit of G proteins would be useful in studying the roles of G proteins in a variety of processes, but the currently available dominant-negative constructs, which target Mg2+-binding sites, are rather leaky. A variety of studies have implicated the carboxyl terminus of G protein alpha subunits in both mediating receptor-G protein interaction and in receptor selectivity. Thus we have made minigene plasmid constructs that encode oligonucleotide sequences corresponding to the carboxyl-terminal undecapeptide of Galphai, Galphaq, or Galphas. To determine whether overexpression of the carboxyl-terminal peptide would block cellular responses, we used as a test system the activation of the M2 muscarinic receptor activated K+ channels in HEK 293 cells. The minigenes were transiently transfected along with G protein-regulated inwardly rectifying K+ channels (GIRK) into HEK 293 cells that stably express the M2 muscarinic receptor. The presence of the Galphai carboxyl-terminal peptide results in specific inhibition of GIRK activity in response to agonist stimulation of the M2 muscarinic receptor. The Galphai minigene construct completely blocks agonist-mediated M2 mAChR K+ channel response whereas the control minigene constructs (empty vector, pcDNA3.1, and the Galpha carboxyl peptide in random order, pcDNA-GalphaiR) had no effect on agonist-mediated M2 muscarinic receptor GIRK response. The inhibitory effects of the Galphai minigene construct were specific because overexpression of peptides corresponding to the carboxyl terminus of Galphaq or Galphas had no effect on M2 muscarinic receptor stimulation of the K+ channel.

Activation of IK(ACh) is the major effect of the vagal neutrotransmitter acetylcholine in the heart. We report that both lysosphingomyelin (D‐erythro‐sphingosyl‐phosphorylcholine; SPC) and sphingosine 1‐phosphate (SPP) activate IK(ACh) in guinea pig atrial myocytes through the same receptor with an EC50 of 1.5 and 1.2 nM, respectively. Pertussis toxin abolished the activation of IK(ACh) by either lipid. The putative receptor showed an exquisite stereoselectivity for the naturally occurring D‐erythro‐(2S,3R)‐SPC stereoisomer, the structure of which was confirmed by mass spectroscopy and NMR. These lipids caused complete homologous and heterologous desensitization with each other but not with ACh, indicating that both act on the same receptor. This receptor displays a distinct structure‐activity relationship: it requires an unsubstituted amino group because N‐acetyl‐SPC, lysophosphatidic acid and lysophosphatidylcholine were inactive. Because SPP and SPC are naturally occurring products of membrane lipid metabolism, it appears that these compounds might be important extracellular mediators acting on a family of bona fide G protein‐coupled receptors. Expression of these receptors in the heart raises the possibility that sphingolipids may be a part of the physiological and/or pathophysiological regulation of the heart. Based on their ligand selectivity we propose a classification of the sphingolipid receptors.

1. Activation of muscarinic K+ current (IK(ACh)) by sphingosine‐1‐phosphate (Sph‐1‐P) was studied in isolated cultured guinea‐pig atrial myocytes using whole‐cell voltage clamp. 2. Sph‐1‐P caused activation of IK(ACh) with an EC50 of 1.2 nM. The maximal current that could be activated by Sph‐1‐P amounted to about 90% of the IK(ACh) caused by a saturating concentration of acetylcholine (ACh, 10 microM). Sphingosine (1 microM), which can mimic the signalling effects of Sph‐1‐P in other cells, failed to cause measurable activation of IK(ACh). 3. IK(ACh) activation by Sph‐1‐P was completely suppressed in cells treated with pertussis toxin. 4. Desensitization of muscarinic receptors by pre‐incubation of the cells with carbachol did not affect the response to Sph‐1‐P; likewise, pre‐incubation of the cells with Sph‐1‐P resulted in a reduced sensitivity to the phospholipid but not to ACh. In contrast, pre‐incubation with either Sph‐1‐P or a serum phospholipid previously described as activating atrial IK(ACh) resulted in reduced sensitivity to both phospholipids. 5. It is concluded that activation of IK(ACh) by Sph‐1‐P in atrial myocytes is induced by binding to a novel G protein‐coupled phospholipid receptor.

PD 81,723 has been shown to enhance binding of adenosine to A1receptors by stabilizing G protein‐receptor coupling (‘allosteric enhancement’). Evidence has been provided that in the perfused hearts and isolated atria PD 81,723 causes a sensitization to adenosine via this mechanism.We have studied the effect of PD 81,723 in guinea‐pig isolated atrial myocytes by use of whole‐cell measurement of the muscarinic K+current (IK(ACh)) activated by different Gi‐coupled receptors (A1, M2, sphingolipid). PD 81,273 caused inhibition of IK(ACh)(IC50≃5μm) activated by either of the three receptors. Receptor‐independent IK(ACh)in cells loaded with GTP‐γ‐S and background IK(ACh), which contributes to the resting conductance of atrial myocytes, were equally sensitive to PD 81,723. At no combination of concentrations of adenosine and PD 81,723 could an enhancing effect be detected.The compound was active from the outside only. Loading of the cells with PD 81,723 (50μm) via the patch pipette did not affect either IK(ACh)or its sensitivity to adenosine. We suggest that PD 81,723 acts as an inhibitor of inward rectifying K+channels; this is supported by the finding that ventricular IK1, which shares a large degree of homology with the proteins (GIRK1/GIRK4) forming IK(ACh)but is not G protein‐gated, was also blocked by this compound.It is concluded that the functional effects of PD 81,723 described in the literature are not mediated by the A1adenosine receptor‐Gi‐IK(ACh)pathway.

Activation of IK(ACh) is the major effect of the vagal neutrotransmitter acetylcholine in the heart. We report that both lysosphingomyelin (D‐erythro‐sphingosyl‐phosphorylcholine; SPC) and sphingosine 1‐phosphate (SPP) activate IK(ACh) in guinea pig atrial myocytes through the same receptor with an EC50 of 1.5 and 1.2 nM, respectively. Pertussis toxin abolished the activation of IK(ACh) by either lipid. The putative receptor showed an exquisite stereoselectivity for the naturally occurring D‐erythro‐(2S,3R)‐SPC stereoisomer, the structure of which was confirmed by mass spectroscopy and NMR. These lipids caused complete homologous and heterologous desensitization with each other but not with ACh, indicating that both act on the same receptor. This receptor displays a distinct structure‐activity relationship: it requires an unsubstituted amino group because N‐acetyl‐SPC, lysophosphatidic acid and lysophosphatidylcholine were inactive. Because SPP and SPC are naturally occurring products of membrane lipid metabolism, it appears that these compounds might be important extracellular mediators acting on a family of bona fide G protein‐coupled receptors. Expression of these receptors in the heart raises the possibility that sphingolipids may be a part of the physiological and/or pathophysiological regulation of the heart. Based on their ligand selectivity we propose a classification of the sphingolipid receptors.

Sphingosine-1-phosphate (SPP) has attracted much attention as a possible second messenger controlling cell proliferation and motility and as an intracellular Ca(2+)-releasing agent. Here, we present evidence that SPP activates a G protein-coupled receptor in the plasma membrane of various cells, leading to increase in cytoplasmic Ca2+ concentration ([Ca2+]i), inhibition of adenylyl cyclase, and opening of G protein-regulated potassium channels. In human enbryonic kidney (HEK) cells, SPP potently (EC50, 2 nM) and rapidly increased [Ca2+]i in a pertussis toxin-sensitive manner. Pertussis toxin-sensitive increase in [Ca2+]i was also observed with sphingosylphosphorylcholine (EC50, 460 nM), whereas other sphingolipids, including ceramide-1-phosphate, N-palmitoyl-sphingosine, psychosine, and D-erythro-sphingosine at micromolar concentrations did not or only marginally increased [Ca2+]i. Furthermore, SPP inhibited forskolin-stimulated cAMP accumulation in HEK cells and increased binding of guanosine 5'3-O-(thio) triphosphate to HEK cell membranes. Rapid [Ca2+]i responses were also observed in human transitional bladder carcinoma (J82) cells, monkey COS-1 cells, mouse NIH 3T3 cells, Chinese hamster ovary (CHO-K1) cells, and rat C6 glioma cells, whereas human HL-60 leukemia cells and human erythroleukemia cells failed to respond to SPP. In guinea pig atrial myocytes, SPP activated Gi protein-regulated inwardly rectifying potassium channels. Activation of these channels occurred strictly when SPP was applied at the extracellular face of atrial myocyte plasma membrane as measured in cell-attached and inside-out patch clamp current recordings. We conclude that SPP, in addition to its proposed direct action on intracellular Ca2+ stores, interacts with a high affinity Gi protein-coupled receptor in the plasma membrane of apparently many different cell types.

1. Muscarinic K+ current (IK(ACh)) was measured in cultured atrial myocytes from hearts of adult guinea‐pigs using whole‐cell voltage clamp. IK(ACh) was activated by superfusion with solutions containing either acetylcholine (ACh) or adenosine (Ado), in saturating concentrations of 2 microM (ACh) and 1 mM (Ado), respectively. 2. In freshly isolated cells the amplitude of the current activated by Ado (IK(Ado)) was 58% (mean) of the current that was induced by ACh. In serum‐free culture this relation, but also the absolute density of IK(ACh), remained fairly constant for up to 8 days. 3. If the culture medium was supplemented with fetal calf serum (FCS, 5%) the relation IK(Ado)/IK(ACh) gradually decayed, reaching a value of less than 0.1 on days 7‐8, whereas the response to ACh remained stable over this period of time. 4. After treatment of cells with FCS‐containing medium, no recovery was observed upon FCS withdrawal for up to 4 days. 5. The effect of FCS on responsiveness to Ado was half‐maximal at about 1% (v/v). The active principle can be dialysed (mol. mass exclusion: 10 kDa). It is not identical with an albumin‐associated factor that has been shown to be a potent activator of atrial IK(ACh) upon acute superfusion. Loss of responsiveness to Ado was paralleled by a reduction of binding sites to the A1 adenosine receptor‐specific radioligand 8‐cyclopentyl‐1,3‐dipropylxanthine ([3H]CPX). 6. It is concluded that FCS contains a factor that causes down‐regulation of A1 Ado receptors. The signalling pathway that leads to an increased opening activity of IK(ACh) channels and other receptors, such as the M2 muscarinic receptor, linked to this signalling pathway are not affected by this factor.

1. Desensitization of muscarinic K+ current (IK(ACh)) was studied in cultured atrial myocytes from guinea‐pig hearts using whole‐cell voltage clamp. 2. Three different types of desensitization could be identified. A fast component which upon rapid superfusion with ACh resulted in a partial relaxation of IK(ACh) within a few seconds to a plateau which was maintained in the presence of ACh. Recovery from this type of desensitization paralleled the decay of IK(ACh) after washout of the agonist. A second type of desensitization was observed within minutes. This was reversed around 10 min after washout of ACh. Both types were heterologous with regard to the A1 receptor and the novel phospholipid (Pl) receptor, both of which activate IK(ACh) via the same signalling pathway. 3. A third type of desensitization (downregulation) occurred upon exposure of the cultures for 24‐48 h to the muscarinic agonist carbachol (CCh). The level of downregulation depended on the concentration of CCh (0.1 microM < or = [CCh] < or = 10 microM). No recovery was observed within 5 h after washout of CCh. Thereafter sensitivity to ACh slowly returned (half‐time (t1/2), approximately 20 h). 4. Downregulation by CCh (0.1‐5 microM) was characterized by an increase in EC50 for ACh with no reduction in maximum IK(ACh). With 5 microM CCh, EC50 was increased from 0.1 to 3.7 microM. At 10 microM CCh EC50 was increased to 15 microM and maximal current that could be evoked by ACh was reduced to 15%. 5. Downregulation by CCh was homologous with regard to A1 and Pl receptors. Maximum IK(ACh), assayed by a saturating concentration of Pl, was not reduced in downregulated cells, suggesting a mechanism localized at the M2 receptor. 6. The changes in the concentration‐response curves can be accounted for by assuming an excess of M2 receptors relative to the subsequent component of the signalling pathway. 7. As the intact heart is under tonic vagal control, downregulation is likely to contribute to controlling the sensitivity of the heart to vagal activity in situ.

Here we report novel effects of regulators of G protein signaling (RGS) on G protein-regulated ion channels. RGS3 and RGS4 induced a substantial increase in currents through the Gbeta gamma-regulated inwardly rectifying K+ channels, IK(ACh), in the absence of receptor activation. Concomitantly, the amount of current that could be activated by agonist was reduced. Pretreatment with pertussis toxin or a muscarinic receptor antagonist abolished agonist-induced currents but did not modify RGS effects. Cotransfection of cells with a Gbetagamma-binding protein significantly reduced the RGS4-induced basal IK(ACh) currents. The RGS proteins also modified the properties of another Gbeta gamma effector, the N-type Ca2+ channels. These observations strongly suggest that RGS proteins increase the availability of Gbeta gamma in addition to their previously described GTPase-activating function.

The kinetics of G-protein-coupled receptor activation and deactivation has, so far, been measured only indirectly, most frequently by assessing the production of various second messengers. We have developed methods based on fluorescence resonance energy transfer to quantify the kinetics of receptor activation by agonist (measured as conformational change in the receptor), the kinetics of G-protein activation (measured as G-protein subunit rearrangement) and the kinetics of receptor inactivation by arrestins (measured as receptor–arrestin interaction). Using these methods, we show that receptor activation by agonists and signalling to G-proteins occur on the subsecond time scale, whereas receptor desensitization is limited by receptor phosphorylation and proceeds more slowly.