Distinct specificities of inwardly rectifying K(+) channels for phosphoinositides

Activation of several inwardly rectifying K(+) channels (Kir) requires the presence of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). The constitutively active Kir2.1 (IRK1) channels interact with PtdIns(4,5)P(2) strongly, whereas the G-protein activated Kir3.1/3.4 channels (GIRK1/GIRK4), show only weak interactions with PtdIns(4,5)P(2). We investigated whether these inwardly rectifying K(+) channels displayed distinct specificities for different phosphoinositides. IRK1, but not GIRK1/GIRK4 channels, showed a marked specificity toward phosphates in the 4,5 head group positions. GIRK1/GIRK4 channels were activated with a similar efficacy by PtdIns(3,4)P(2), PtdIns(3,5)P(2), PtdIns(4,5)P(2), and PtdIns(3,4,5)P(3). In contrast, IRK1 channels were not activated by PtdIns(3,4)P(2) and only marginally by high concentrations of PtdIns(3,5)P(2). Similarly, high concentrations of PtdIns(3,4,5)P(3) were required to activate IRK1 channels. For either channel, PtdIns(4)P was much less effective than PtdIns(4,5)P(2), whereas PtdIns was inactive. In contrast to the dependence on the position of phosphates of the phospholipid head group, GIRK1/GIRK4, but not IRK1 channel activation, showed a remarkable dependence on the phospholipid acyl chains. GIRK1/GIRK4 channels were activated most effectively by the natural arachidonyl stearyl PtdIns(4,5)P(2) and much less by the synthetic dipalmitoyl analog, whereas IRK1 channels were activated equally by dipalmitoyl and arachidonyl stearyl PtdIns(4,5)P(2). Incorporation of PtdInsP(2) into the membrane is necessary for activation, as the short chain water soluble diC(4) PtdIns(4,5)P(2) did not activate either channel, whereas activation by diC(8) PtdIns(4, 5)P(2) required high concentrations.     

Coupling Gbetagamma-dependent activation to channel opening via pore elements in inwardly rectifying potassium channels

G protein-coupled inwardly rectifying potassium channels, GIRK/Kir3.x, are gated by the Gbetagamma subunits of the G protein. The molecular mechanism of gating was investigated by employing a novel yeast-based random mutagenesis approach that selected for channel mutants that are active in the absence of Gbetagamma. Mutations in TM2 were found that mimicked the Gbetagamma-activated state. The activity of these channel mutants was independent of receptor stimulation and of the availability of heterologously expressed Gbetagamma subunits but depended on PtdIns(4,5)P(2). The results suggest that the TM2 region plays a key role in channel gating following Gbetagamma binding in a phospholipid-dependent manner. This mechanism of gating in inwardly rectifying K+ channels may be similar to the involvement of the homologous region in prokaryotic KcsA potassium channel and, thus, suggests evolutionary conservation of the gating structure.     

A sodium-mediated structural switch that controls the sensitivity of Kir channels to PtdIns(4,5)P(2)

Inwardly rectifying potassium (Kir) channels are gated by the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)). Among them, Kir3 requires additional molecules, such as the betagamma subunits of G proteins or intracellular sodium, for channel gating. Using an interactive computational-experimental approach, we show that sodium sensitivity of Kir channels involves the side chains of an aspartate and a histidine located across from each other in a crucial loop in the cytosolic domain, as well as the backbone carbonyls of two more residues and a water molecule. The location of the coordination site in the vicinity of a conserved arginine shown to affect channel-PtdIns(4,5)P(2) interactions suggests that sodium triggers a structural switch that frees the crucial arginine. Mutations of the aspartate and the histidine that affect sodium sensitivity also enhance the channel's sensitivity to PtdIns(4,5)P(2). Furthermore, on the basis of the molecular characteristics of the coordination site, we identify and confirm experimentally a sodium-sensitive phenotype in Kir5.1.     

Mechanosensitivity of GIRK channels is mediated by protein kinase C-dependent channel-phosphatidylinositol 4,5-bisphosphate interaction

Gprotein-activated inwardly rectifying K+ channel (GIRK or Kir3) currents are inhibited by mechanical stretch of the cell membrane, but the underlying mechanisms are not understood. In Xenopus oocytes heterologously expressing GIRK channels, membrane stretch induced by 50% reduction of osmotic pressure caused a prompt reduction of GIRK1/4, GIRK1, and GIRK4 currents by 16.6-42.6%. Comparable GIRK current reduction was produced by protein kinase C (PKC) activation (phorbol 12-myristate 13-acetate). The mechanosensitivity of the GIRK4 current was abolished by pretreatment with PKC inhibitors (staurosporine or calphostin C). Neither hypo-osmotic challenge nor PKC activation affected IRK1 currents. GIRK4 chimera (GIRK4-IRK1-(Lys207-Leu245)) and single point mutant (GIRK4(I229L)), in which the phosphatidylinositol 4,5-bisphosphate (PIP2) binding domain or residue was replaced by the corresponding region of IRK1 to strengthen the channel-PIP2 interaction, showed no mechanosensitivity and minimal PKC sensitivity. IRK1 gained mechanosensitivity and PKC sensitivity by reverse double point mutation of the PIP2 binding domain (L222I/R213Q). Overexpression of Gbetagamma, which is known to strengthen the channel-PIP2 interaction, attenuated the mechanosensitivity of GIRK4 channels. In oocytes expressing a pleckstrin homology domain of PLC-delta tagged with green fluorescent protein, hypo-osmotic challenge or PKC activation caused a translocation of the fluorescence signal from the cell membrane to the cytosol, reflecting PIP2 hydrolysis. The translocation was prevented by pretreatment with PKC inhibitors. Involvement of PKC activation in the mechanosensitivity of muscarinic K+ channels was confirmed in native rabbit atrial myocytes. These results suggest that the mechanosensitivity of GIRK channels is mediated primarily by channel-PIP2 interaction, with PKC playing an important role in modulating the interaction probably through PIP2 hydrolysis.     

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.     

Molecular determinants for sodium-dependent activation of G protein-gated K+ channels

G protein-gated inwardly rectifying K+ channels (GIRKs) are activated by a direct interaction with Gbetagamma subunits and also by raised internal [Na+]. Both processes require the presence of phosphatidylinositol bisphosphate (PIP2). Here we show that the proximal C-terminal region of GIRK2 mediates the Na+-dependent activation of both the GIRK2 homomeric channels and the GIRK1/GIRK2 heteromeric channels. Within this region, GIRK2 has an aspartate at position 226, whereas GIRK1 has an asparagine at the equivalent position (217). A single point mutation, D226N, in GIRK2, abolished the Na+-dependent activation of both the homomeric and heteromeric channels. Neutralizing a nearby negative charge, E234S had no effect. The reverse mutation in GIRK1, N217D, was sufficient to restore Na+-dependent activation to the GIRK1N217D/GIRK2D226N heteromeric channels. The D226N mutation did not alter either the single channel properties or the ability of these channels to be activated via the m2-muscarinic receptor. PIP2 dramatically increased the open probability of GIRK1/GIRK2 channels in the absence of Na+ or Gbetagamma but did not preclude further activation by Na+, suggesting that Na+ is not acting simply to promote PIP2 binding to GIRKs. We conclude that aspartate 226 in GIRK2 plays a crucial role in Na+-dependent gating of GIRK1/GIRK2 channels.     

A switch mechanism for G beta gamma activation of I(KACh)

G protein-gated inwardly rectifying potassium (GIRK) channels are a family of K(+)-selective ion channels that slow the firing rate of neurons and cardiac myocytes. GIRK channels are directly bound and activated by the G protein G beta gamma subunit. As heterotetramers, they comprise the GIRK1 and the GIRK2, -3, or -4 subunits. Here we show that GIRK1 but not the GIRK4 subunit is phosphorylated when heterologously expressed. We found also that phosphatase PP2A dephosphorylation of a protein in the excised patch abrogates channel activation by G beta gamma. Experiments with the truncated molecule demonstrated that the GIRK1 C-terminal is critical for both channel phosphorylation and channel regulation by protein phosphorylation, but the critical phosphorylation sites were not located on the C terminus. These data provide evidence for a novel switch mechanism in which protein phosphorylation enables G beta gamma gating of the channel complex.     

Stabilization of the activity of ATP-sensitive potassium channels by ion pairs formed between adjacent Kir6.2 subunits

ATP-sensitive potassium (KATP) channels are formed by the coassembly of four Kir6.2 subunits and four sulfonylurea receptor subunits (SUR). The cytoplasmic domains of Kir6.2 mediate channel gating by ATP, which closes the channel, and membrane phosphoinositides, which stabilize the open channel. Little is known, however, about the tertiary or quaternary structures of the domains that are responsible for these interactions. Here, we report that an ion pair between glutamate 229 and arginine 314 in the intracellular COOH terminus of Kir6.2 is critical for maintaining channel activity. Mutation of either residue to alanine induces inactivation, whereas charge reversal at positions 229 and 314 (E229R/R314E) abolishes inactivation and restores the wild-type channel phenotype. The close proximity of these two residues is demonstrated by disulfide bond formation between cysteine residues introduced at the two positions (E229C/R314C); disulfide bond formation abolishes inactivation and stabilizes the current. Using Kir6.2 tandem dimer constructs, we provide evidence that the ion pair likely forms by residues from two adjacent Kir6.2 subunits. We propose that the E229/R314 intersubunit ion pairs may contribute to a structural framework that facilitates the ability of other positively charged residues to interact with membrane phosphoinositides. Glutamate and arginine residues are found at homologous positions in many inward rectifier subunits, including the G-protein-activated inwardly rectifying potassium channel (GIRK), whose cytoplasmic domain structure has recently been solved. In the GIRK structure, the E229- and R314-corresponding residues are oriented in opposite directions in a single subunit such that in the tetramer model, the E229 equivalent residue from one subunit is in close proximity of the R314 equivalent residue from the adjacent subunit. The structure lends support to our findings in Kir6.2, and raises the possibility that a homologous ion pair may be involved in the gating of GIRKs.     

Phosphorylation of a tyrosine at the N-terminus regulates the surface expression of GIRK5 homomultimers

The G protein-coupled inwardly rectifying GIRK5 and Delta5GIRK5 splicing variants do not express functional potassium channels. In contrast, Delta25GIRK5 forms functional homomultimers in Xenopus laevis oocytes. A tyrosine is present at the N-term of the non-functional isoforms. We studied the effect of endogenous tyrosine phosphorylation on the GIRK5 surface and functional expression. Unlike wild type channels, GIRK5Y16A and Delta5GIRK5Y16A mutants displayed inwardly rectifying currents and inhibitors of Src tyrosine kinase promoted the traffiking of GIRK5 to the cell surface. This is the first evidence that endogenous phosphorylation of a tyrosine residue in a GIRK channel inhibits its surface expression.     

Synergistic activation of G protein-gated inwardly rectifying potassium channels by the betagamma subunits of G proteins and Na(+) and Mg(2+) ions.

Native and recombinant G protein-gated inwardly rectifying potassium (GIRK) channels are directly activated by the betagamma subunits of GTP-binding (G) proteins. The presence of phosphatidylinositol-bis-phosphate (PIP(2)) is required for G protein activation. Formation (via hydrolysis of ATP) of endogenous PIP(2) or application of exogenous PIP(2) increases the mean open time of GIRK channels and sensitizes them to gating by internal Na(+) ions. In the present study, we show that the activity of ATP- or PIP(2)-modified channels could also be stimulated by intracellular Mg(2+) ions. In addition, Mg(2+) ions reduced the single-channel conductance of GIRK channels, independently of their gating ability. Both Na(+) and Mg(2+) ions exert their gating effects independently of each other or of the activation by the G(betagamma) subunits. At high levels of PIP(2), synergistic interactions among Na(+), Mg(2+), and G(betagamma) subunits resulted in severalfold stimulated levels of channel activity. Changes in ionic concentrations and/or G protein subunits in the local environment of these K(+) channels could provide a rapid amplification mechanism for generation of graded activity, thereby adjusting the level of excitability of the cells.     

Identification of critical residues controlling G protein-gated inwardly rectifying K(+) channel activity through interactions with the beta gamma subunits of G proteins.

G protein-sensitive inwardly rectifying potassium (GIRK) channels are activated through direct interactions of their cytoplasmic N- and C-terminal domains with the beta gamma subunits of G proteins. By using a combination of biochemical and electrophysiological approaches, we identified minimal N- and C-terminal G beta gamma -binding domains responsible for stimulation of GIRK4 channel activity. Within these domains one N-terminal residue, His-64, and one C-terminal residue, Leu-268, proved critical for G beta gamma-mediated GIRK4 activity. Moreover, mutations at these GIRK4 sites reduced significantly binding of the channel domains to G beta gamma . The corresponding residues in GIRK1 also showed a critical involvement in G beta gamma sensitivity. In GIRK4/GIRK1 heteromers the GIRK4 His-64 and Leu-268 residues showed greater contributions to G beta zeta sensitivity than did the corresponding GIRK1 His-57 and Leu-262 residues. These results identify functionally important channel interaction sites with the beta gamma subunits of G proteins, critical for channel activity.     

Identification of a potassium channel site that interacts with G protein betagamma subunits to mediate agonist-induced signaling

Activation of heterotrimeric GTP-binding (G) proteins by their coupled receptors, causes dissociation of the G protein alpha and betagamma subunits. Gbetagamma subunits interact directly with G protein-gated inwardly rectifying K+ (GIRK) channels to stimulate their activity. In addition, free Gbetagamma subunits, resulting from agonist-independent dissociation of G protein subunits, can account for a major component of the basal channel activity. Using a series of chimeric constructs between GIRK4 and a Gbetagamma-insensitive K+ channel, IRK1, we have identified a critical site of interaction of GIRK with Gbetagamma. Mutation of Leu339 to Glu within this site impaired agonist-induced sensitivity and decreased binding to Gbetagamma, without removing the Gbetagamma contribution to basal currents. Mutation of the corresponding residue in GIRK1 (Leu333) resulted in a similar phenotype. Both the GIRK1 and GIRK4 subunits contributed equally to the agonist-induced sensitivity of the heteromultimeric channel. Thus, we have identified a channel site that interacts specifically with Gbetagamma subunits released through receptor stimulation.     

Contributions of a negatively charged residue in the hydrophobic domain of the IRK1 inwardly rectifying K+ channel to K(+)-selective permeation.

Inwardly rectifying K+ channels are highly selective for K+ ions and show strong interaction with ions in the pore. Both features are important for the physiological functions of these channels and pose intriguing mechanistic questions of ion permeation. The aspartate residue in the second putative transmembrane segment of the IRK1 inwardly rectifying K+ channel, previously implicated in inward rectification gating due to cytoplasmic Mg2+ and polyamine block, is found in this study to be crucial for the channel's ability to distinguish between K+ and Rb+ ions. Mutation of this residue also perturbs the interaction between the channel pore and the Sr2+ blocking ion. Our studies suggest that this aspartate residue contributes to a selectivity filter near the cytoplasmic end of the pore.     

Pharmacological Characterization of 5-HT1A Autoreceptor-Coupled GIRK Channels in Rat Dorsal Raphe 5-HT Neurons

G protein-activated inwardly rectifying potassium (GIRK) channels in 5-HT neurons are assumed to be principal effectors of 5-hydroxytryptamine 1A (5-HT_1A) autoreceptors, but their pharmacology, subunit composition and the role in regulation of 5-HT neuron activity have not been fully elucidated. We sought for a pharmacological tool for assessing the functional role of GIRK channels in 5-HT neurons by characterizing the effects of drugs known to block GIRK channels in the submicromolar range of concentrations. Whole-cell voltage-clamp recording in brainstem slices were used to determine concentration-response relationships for the selected GIRK channel blockers on 5-HT_1A autoreceptor-activated inwardly rectifying K⁺ conductance in rat dorsal raphe 5-HT neurons. 5-HT_1A autoreceptor-activated GIRK conductance was completely blocked by the nonselective inwardly rectifying potassium channels blocker Ba²⁺ (EC₅₀ = 9.4 μM, full block with 100 μM) and by SCH23390 (EC₅₀ = 1.95 μM, full block with 30 μM). GIRK-specific blocker tertiapin-Q blocked 5-HT_1A autoreceptor-activated GIRK conductance with high potency (EC₅₀ = 33.6 nM), but incompletely, i.e. ~16% of total conductance resulted to be tertiapin-Q-resistant. U73343 and SCH28080, reported to block GIRK channels with submicromolar EC₅₀s, were essentially ineffective in 5-HT neurons. Our data show that inwardly rectifying K⁺ channels coupled to 5-HT_1A autoreceptors display pharmacological properties generally expected for neuronal GIRK channels, but different from GIRK1-GIRK2 heteromers, the predominant form of brain GIRK channels. Distinct pharmacological properties of GIRK channels in 5-HT neurons should be explored for the development of new therapeutic agents for mood disorders.     

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.     

Gating kinetics of GIRK channels are mediated by cytoplasmic residues adjacent to transmembrane domains

G-protein-coupled inwardly rectifying potassium channels (GIRK / Kir3.x) are involved in neurotransmission-mediated reduction of excitability. The gating mechanism following G protein activation of these channels likely proceeds from movement of inner transmembrane helices to allow K⁺ ions movement through the pore of the channel. There is limited understanding of how the binding of G-protein βγ subunits to cytoplasmic regions of the channel transduces the signal to the transmembrane regions. In this study, we examined the molecular basis that governs the activation kinetics of these channels, using a chimeric approach. We identified two regions as being important in determining the kinetics of activation. One region is the bottom of the outer transmembrane helix (TM1) and the cytoplasmic domain immediately adjacent (the slide helix); and the second region is the bottom of the inner transmembrane helix (TM2) and the cytoplasmic domain immediately adjacent. Interestingly, both of these regions are sufficient in mediating the kinetics of fast gating. This result suggests that there is a cooperative movement of both of these domains to allow fast and efficient gating of GIRK channels.     

Ion selectivity filter regulates local anesthetic inhibition of G-protein-gated inwardly rectifying K+ channels

The weaver mutation (G156S) in G-protein-gated inwardly rectifying K+ (GIRK) channels alters ion selectivity and reveals sensitivity to inhibition by a charged local anesthetic, QX-314, applied extracellularly. In this paper, disrupting the ion selectivity in another GIRK channel, chimera I1G1(M), generates a GIRK channel that is also inhibited by extracellular local anesthetics. I1G1(M) is a chimera of IRK1 (G-protein-insensitive) and GIRK1 and contains the hydrophobic domains (M1-pore-loop-M2) of GIRK1 (G1(M)) with the N- and C-terminal domains of IRK1 (I1). The local anesthetic binding site in I1G1(M) is indistinguishable from that in GIRK2(wv) channels. Whereas chimera I1G1(M) loses K+ selectivity, although there are no mutations in the pore-loop complex, chimera I1G2(M), which contains the hydrophobic domain from GIRK2, exhibits normal K+ selectivity. Mutation of two amino acids that are unique in the pore-loop complex of GIRK1 (F137S and A143T) restores K+ selectivity and eliminates the inhibition by extracellular local anesthetics, suggesting that the pore-loop complex prevents QX-314 from reaching the intrapore site. Alanine mutations in the extracellular half of the M2 transmembrane domain alter QX-314 inhibition, indicating the M2 forms part of the intrapore binding site. Finally, the inhibition of G-protein-activated currents by intracellular QX-314 appears to be different from that observed in nonselective GIRK channels. The results suggest that inward rectifiers contain an intrapore-binding site for local anesthetic that is normally inaccessible from extracellular charged local anesthetics.     

NMR analyses of the Gbetagamma binding and conformational rearrangements of the cytoplasmic pore of G protein-activated inwardly rectifying potassium channel 1 (GIRK1)

G protein-activated inwardly rectifying potassium channel (GIRK) plays crucial roles in regulating heart rate and neuronal excitability in eukaryotic cells. GIRK is activated by the direct binding of heterotrimeric G protein βγ subunits (Gβγ) upon stimulation of G protein-coupled receptors, such as M2 acetylcholine receptor. The binding of Gβγ to the cytoplasmic pore (CP) region of GIRK causes structural rearrangements, which are assumed to open the transmembrane ion gate. However, the crucial residues involved in the Gβγ binding and the structural mechanism of GIRK gating have not been fully elucidated. Here, we have characterized the interaction between the CP region of GIRK and Gβγ, by ITC and NMR. The ITC analyses indicated that four Gβγ molecules bind to a tetramer of the CP region of GIRK with a dissociation constant of 250 μM. The NMR analyses revealed that the Gβγ binding site spans two neighboring subunits of the GIRK tetramer, which causes conformational rearrangements between subunits. A possible binding mode and mechanism of GIRK gating are proposed.     

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.     

Single Channel Analysis of the Regulation of GIRK1/GIRK4 Channels by Protein Phosphorylation

G-Protein activated, inwardly rectifying potassium channels (GIRKs) are important effectors of G-protein β/γ-subunits, playing essential roles in the humoral regulation of cardiac activity and also in higher brain functions. G-protein activation of channels of the GIRK1/GIRK4 heterooligomeric composition is controlled via phosphorylation by cyclic AMP dependent protein kinase (PKA) and dephosphorylation by protein phosphatase 2A (PP₂A). To study the molecular mechanism of this unprecedented example of G-protein effector regulation, single channel recordings were performed on isolated patches of plasma membranes of Xenopus laevis oocytes. Our study shows that: (i) The open probability (P_o) of GIRK1/GIRK4 channels, stimulated by coexpressed m₂-receptors, was significantly increased upon addition of the catalytic subunit of PKA to the cytosolic face of an isolated membrane patch. (ii) At moderate concentrations of recombinant G_β1/γ2, used to activate the channel, P_o was significantly reduced in patches treated with PP₂A, when compared to patches with PKA-cs. (iii) Several single channel gating parameters, including modal gating behavior, were significantly different between phosphorylated and dephosphorylated channels, indicating different gating behavior between the two forms of the protein. Most of these changes were, however, not responsible for the marked difference in P_o at moderate G-protein concentrations. (iv) An increase of the frequency of openings (f_o) and a reduction of dwell time duration of the channel in the long-lasting C₅ state was responsible for facilitation of GIRK1/GIRK4 channels by protein phosphorylation. Dephosphorylation by PP₂A led to an increase of G_β1/γ2 concentration required for full activation of the channel and hence to a reduction of the apparent affinity of GIRK1/GIRK4 for G_β1/γ2. (v) Although possibly not directly the target of protein phosphorylation/dephosphorylation, the last 20 C-terminal amino acids of the GIRK1 subunit are required for the reduction of apparent affinity for the G-protein by PP₂A, indicating that they constitute an essential part of the off-switch.