Rapid desensitization of G protein-gated inwardly rectifying K(+) currents is determined by G protein cycle

Activation of G protein-gated inwardly rectifying K⁺ (GIRK) channels, found in the brain, heart, and endocrine tissue, leads to membrane hyperpolarization that generates neuronal inhibitory postsynaptic potentials, slows the heart rate, and inhibits hormone release. During stimulation of G_i/o-coupled receptors and subsequent channel activation, it has been observed that the current desensitizes. In this study we examined mechanisms underlying fast desensitization of cloned heteromeric neuronal Kir3.1+3.2A and atrial Kir3.1+3.4 channels and also homomeric Kir3.0 currents in response to stimulation of several G_i/o G protein-coupled receptors (GPCRs) expressed in HEK-293 cells (adenosine A₁, adrenergic _2A, dopamine D_2S, M₄ muscarinic, and GABA_B1b/2 receptors). We found that all agonist-induced currents displayed a similar degree of desensitization except the adenosine A₁ receptor, which exhibits an additional desensitizing component. Using the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPS), we found that this is due to a receptor-dependent, G protein-independent process. Using Ca²⁺ imaging we showed that desensitization is unlikely to be accounted for solely by phospholipase C activation and phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolysis. We examined the contribution of the G protein cycle and found the following. First, agonist concentration is strongly correlated with degree of desensitization. Second, competitive inhibition of GDP/GTP exchange by using nonhydrolyzable guanosine 5'-O-(2-thiodiphosphate) (GDPS) has two effects, a slowing of channel activation and an attenuation of the fast desensitization phenomenon. Finally, using specific G subunits we showed that ternary complexes with fast activation rates display more prominent desensitization than those with slower activation kinetics. Together our data suggest that fast desensitization of GIRK currents is accounted for by the fundamental properties of the G protein cycle. G protein-coupled receptor; potassium channel; inward rectifier; kinetics     

Molecular determinants responsible for differential cellular distribution of G protein-gated inwardly rectifying K+ channels

Activation of the heteromeric G protein-gated inwardly rectifying K(+) channel (GIRK) GIRK1 and GIRK4 subunits gives rise to I(KACh), which controls excitability in atrial tissue. Although homomeric GIRK4 channels localize to the plasma membrane and display moderate function, GIRK1 channels fail to localize to the cell surface and do not exhibit significant function as homomers. Using oocytes to express GFP-tagged GIRK1 and GIRK4 and chimeras between these two proteins, we have identified two regions, one in the proximal C terminus and another in the distal N terminus that are critical for their subcellular localization. Replacement of both of these regions in GIRK1 with corresponding regions from GIRK4 was required for efficient expression of GIRK1 on the plasma membrane. Replacement of either region by itself was ineffective. The distal N terminus and proximal C terminus have been previously suggested to play important roles in ER-export and subunit co-assembly respectively in this family of channels. Our data indicate for the first time that both of these regions need to work in concert to mediate efficient targeting of these channels to the plasma membrane.     

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.     

Agonist-induced localization of Gq-coupled receptors and G protein-gated inwardly rectifying K+ (GIRK) channels to caveolae determines receptor specificity of phosphatidylinositol 4,5-bisphosphate signaling

G protein-gated inwardly rectifying K(+) (GIRK) channels are parasympathetic effectors in cardiac myocytes that act as points of integration of signals from diverse pathways. Neurotransmitters and hormones acting on the Gq protein regulate GIRK channels by phosphatidylinositol 4,5-bisphosphate (PIP(2)) depletion. In previous studies, we found that endothelin-1, but not bradykinin, inhibited GIRK channels, even though both of them hydrolyze PIP(2) in cardiac myocytes, showing receptor specificity. The present study assessed whether the spatial organization of the PIP(2) signal into caveolar microdomains underlies the specificity of PIP(2)-mediated signaling. Using biochemical analysis, we examined the localization of GIRK and Gq protein-coupled receptors (GqPCRs) in mouse atrial myocytes. Agonist stimulation induced a transient co-localization of GIRK channels with endothelin receptors in the caveolae, excluding bradykinin receptors. Such redistribution was eliminated by caveolar disruption with methyl-β-cyclodextrin (MβCD). Patch clamp studies showed that the specific response of GIRK channels to GqPCR agonists was abolished by MβCD, indicating the functional significance of the caveolae-dependent spatial organization. To assess whether low PIP(2) mobility is essential for PIP(2)-mediated signaling, we blocked the cytoskeletal restriction of PIP(2) diffusion by latrunculin B. This abolished the GIRK channel regulation by GqPCRs without affecting their targeting to caveolae. These data suggest that without the hindered diffusion of PIP(2) from microdomains, PIP(2) loses its signaling efficacy. Taken together, these data suggest that specific targeting combined with restricted diffusion of PIP(2) allows the PIP(2) signal to be compartmentalized to the targets localized closely to the GqPCRs, enabling cells to discriminate between identical PIP(2) signaling that is triggered by different receptors.     

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.     

Importance of the G protein gamma subunit in activating G protein-coupled inward rectifier K(+) channels

Arachidonic acid (AA) is generated via Rac-mediated phospholipase A2 (PLA2) activation in response to growth factors and cytokines and is implicated in cell growth and gene expression. In this study, we show that AA activates the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in a time- and dose-dependent manner. Indomethacin and nordihydroguaiaretic acid, potent inhibitors of cyclooxygenase and lipoxygenase, respectively, did not exert inhibitory effects on AA-induced SAPK/JNK activation, thereby indicating that AA itself could activate SAPK/JNK. As Rac mediates SAPK/JNK activation in response to a variety of stressful stimuli, we examined whether the activation of SAPK/JNK by AA is mediated by Rac1. We observed that AA-induced SAPK/JNK activation was significantly inhibited in Rat2-Rac1N17 dominant-negative mutant cells. Furthermore, treatment of AA induced membrane ruffling and production of hydrogen peroxide, which could be prevented by Rac1N17. These results suggest that AA acts as an upstream signal molecule of Rac, whose activation leads to SAPK/JNK activation, membrane ruffling and hydrogen peroxide production.     

The role of G proteins in assembly and function of Kir3 inwardly rectifying potassium channels

Kir3 channels (also known as GIRK channels) are important regulators of electrical excitability in both cardiomyocytes and neurons. Much is known regarding the assembly and function of these channels and the roles that their interacting proteins play in controlling these events. Further, they are one of the best-studied effectors of heterotrimeric G proteins in general and Gβγ subunits in particular. However, our understanding of the roles of multiple Gβγ binding sites on Kir3 channels is still rudimentary. We discuss potential roles for Gβγ in channel assembly and trafficking in addition to their known role in cellular signaling.Key words: Kir3 channels, G proteins, trafficking, neurons, cardiomyocytes     

Dynamic integration of alpha-adrenergic and cholinergic signals in the atria: role of G protein-regulated inwardly rectifying K+ channels

Numerous heptahelical receptors use activation of heterotrimeric G proteins to convey a multitude of extracellular signals to appropriate effector molecules in the cell. Both high specificity and correct integration of these signals are required for reliable cell function. Yet the molecular machineries that allow each cell to merge information flowing across different receptors are not well understood. Here we demonstrate that G protein-regulated inwardly rectifying K(+) (GIRK) channels can operate as dynamic integrators of alpha-adrenergic and cholinergic signals in atrial myocytes. Acting at the last step of the cholinergic signaling cascade, these channels are activated by direct interactions with betagamma subunits of the inhibitory G proteins (G betagamma), and efficiently translate M(2) muscarinic acetylcholine receptor (M2R) activation into membrane hyperpolarization. The parallel activation of alpha-adrenergic receptors imposed a distinctive "signature" on the function of M2R-activated GIRK1/4 channels, affecting both the probability of G betagamma binding to the channel and its desensitization. This modulation of channel function was correlated with a parallel depletion of G beta and protein phosphatase 1 from the oligomeric GIRK1 complexes. Such plasticity of the immediate GIRK signaling environment suggests that multireceptor integration involves large protein networks undergoing dynamic changes upon receptor activation.     

Recombinant alpha i-3 subunit of G protein activates Gk-gated K+ channels

G proteins, particularly those sensitive to pertussis toxin, are difficult to separate biochemically, creating uncertainty in functional assignments. For this reason the cDNAs encoding G alpha i-3 and two of the G alpha s splice variants were expressed as fusion proteins in Escherichia coli using a T7 promoter-based expression system. These proteins were denoted r alpha i-3 and r alpha s (short and long) and accumulated in bacteria to as much as 5-10% of total cellular protein, of which 5-10% was soluble in lysates. Soluble r alpha subunits were tested for stimulation of K+ channel activity in inside-out atrial membrane patches and for reconstitution of cyc- adenylyl cyclase activity. r alpha i-3, activated either by guanosine 5'-(3-thio)triphosphate (GTP gamma S) or AlF-4, stimulated in a concentration-dependent manner single channel K+ currents in isolated atrial membrane patches of three species: guinea pigs, neonatal rats, and embryonic chick. In contrast, GTP gamma S-activated r alpha s did not. In agreement with a similar study by Graziano et al. (Graziano, M. P., Casey, P. J. and Gilman, A. G. (1987) J. Biol. Chem. 262, 11375-11381), both r alpha s forms reconstituted GTP gamma S-stimulated cyc- adenylyl cyclase activity, albeit at concentrations 50-100 times higher than those needed with native Gs. The concentrations of r alpha i-3 needed to stimulate the K+ channels were also higher than needed with native human erythrocyte Gk, in this case 30-50 times. Single K+ channel currents stimulated by r alpha i-3 were indistinguishable from those stimulated by the natural effector acetylcholine. Thus, bacterial expression of G alpha subunits provided the means to demonstrate unequivocally that Gi-3 has intrinsic Gk activity.     

The role of Mg2+ in the inactivation of inwardly rectifying K+ channels in aortic endothelial cells

We have studied the role of Mg2+ in the inactivation of inwardly rectifying K+ channels in vascular endothelial cells. Inactivation was largely eliminated in Mg(2+)-free external solutions and the extent of inactivation was increased by raising Mg2+o. The dose-response relation for the reduction of channel open probability showed that Mg2+o binds to a site (KD = approximately 25 microM at -160 mV) that senses approximately 38% of the potential drop from the external membrane surface. Analysis of the single-channel kinetics showed that Mg2+ produced a class of long-lived closures that separated bursts of openings. Raising Mg2+o reduced the burst duration, but less than expected for an open-channel blocking mechanism. The effects of Mg2+o are antagonized by K+o in manner which suggests that K+ competes with Mg2+ for the inactivation site. Mg2+o also reduced the amplitude of the single-channel current at millimolar concentrations by a rapid block of the open channel. A mechanism is proposed in which Mg2+ binds to the closed channel during hyperpolarization and prevents it from opening until it is occupied by K+.     

The role of inwardly rectifying potassium channels in the relaxation of rat hind-limb arteries

An increase in the extracellular K⁺ concentration, which causes relaxation of arteries due to the activation of inwardly rectifying potassium channels, can occur in some organs under intensive metabolism, as well as endothelium-dependent hyperpolarization. The aim of this work was a comparison of the contribution of these channels in the regulation of the tone of arteries that supply skeletal muscles and the skin. The reactions of skin-region arteries (a subcutaneous artery and its branch) and gastrocnemius muscle arteries were recorded in the isometric mode. During the contraction caused by α₁-adrenoceptor agonist, the relaxation reactions upon an increase in extracellular K⁺ concentration and on acetylcholine in the presence of inhibitors of NO-synthase and cyclooxygenase were recorded (to detect the effects of endothelium-dependent hyperpolarization). The muscle arteries at both effects showed a pronounced relaxation, which was strongly suppressed by Ba²⁺ ions (blockers of inwardly rectifying potassium channels); both reactions did not exceed 20% in the skin arteries. Thus, the regulatory effect of inwardly rectifying potassium channels in the muscle arteries is much higher than in the skin arteries which is consistent with the idea about the functioning of these arteries in the organism.     

G protein beta2 subunit-derived peptides for inhibition and induction of G protein pathways. Examination of voltage-gated Ca2+ and G protein inwardly rectifying K+ channels

Voltage-gated Ca2+ channels of the N-, P/Q-, and R-type and G protein inwardly rectifying K+ channels (GIRK) are modulated via direct binding of G proteins. The modulation is mediated by G protein betagamma subunits. By using electrophysiological recordings and fluorescence resonance energy transfer, we characterized the modulatory domains of the G protein beta subunit on the recombinant P/Q-type channel and GIRK channel expressed in HEK293 cells and on native non-L-type Ca2+ currents of cultured hippocampal neurons. We found that Gbeta2 subunit-derived deletion constructs and synthesized peptides can either induce or inhibit G protein modulation of the examined ion channels. In particular, the 25-amino acid peptide derived from the Gbeta2 N terminus inhibits G protein modulation, whereas a 35-amino acid peptide derived from the Gbeta2 C terminus induced modulation of voltage-gated Ca2+ channels and GIRK channels. Fluorescence resonance energy transfer (FRET) analysis of the live action of these peptides revealed that the 25-amino acid peptide diminished the FRET signal between G protein beta2gamma3 subunits, indicating a reorientation between G protein beta2gamma3 subunits in the presence of the peptide. In contrast, the 35-amino acid peptide increased the FRET signal between GIRK1,2 channel subunits, similarly to the Gbetagamma-mediated FRET increase observed for this GIRK subunit combination. Circular dichroism spectra of the synthesized peptides suggest that the 25-amino acid peptide is structured. These results indicate that individual G protein beta subunit domains can act as independent, separate modulatory domains to either induce or inhibit G protein modulation for several effector proteins.     

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.     

Receptor-mediated inhibition of G protein-coupled inwardly rectifying potassium channels involves G(alpha)q family subunits, phospholipase C, and a readily diffusible messenger

G protein-coupled inwardly rectifying K+ (GIRK) channels can be activated or inhibited by distinct classes of receptor (G(alpha)i/o- and G(alpha)q-coupled), providing dynamic regulation of cellular excitability. Receptor-mediated activation involves direct effects of G(beta)gamma subunits on GIRK channels, but mechanisms involved in GIRK channel inhibition have not been fully elucidated. An HEK293 cell line that stably expresses GIRK1/4 channels was used to test G protein mechanisms that mediate GIRK channel inhibition. In cells transiently or stably cotransfected with 5-HT1A (G(alpha)i/o-coupled) and TRH-R1 (G(alpha)q-coupled) receptors, 5-HT (5-hydroxytryptamine; serotonin) enhanced GIRK channel currents, whereas thyrotropin-releasing hormone (TRH) inhibited both basal and 5-HT-activated GIRK channel currents. Inhibition of GIRK channel currents by TRH primarily involved signaling by G(alpha)q family subunits, rather than G(beta)gamma dimers: GIRK channel current inhibition was diminished by Pasteurella multocida toxin, mimicked by constitutively active members of the G(alpha)q family, and reduced by minigene constructs that disrupt G(alpha)q signaling, but was completely preserved in cells expressing constructs that interfere with signaling by G(beta)gamma subunits. Inhibition of GIRK channel currents by TRH and constitutively active G(alpha)q was reduced by, an inhibitor of phospholipase C (PLC). Moreover, TRH- R1-mediated GIRK channel inhibition was diminished by minigene constructs that reduce membrane levels of the PLC substrate phosphatidylinositol bisphosphate, further implicating PLC. However, we found no evidence for involvement of protein kinase C, inositol trisphosphate, or intracellular calcium. Although these downstream signaling intermediaries did not contribute to receptor-mediated GIRK channel inhibition, bath application of TRH decreased GIRK channel activity in cell-attached patches. Together, these data indicate that receptor-mediated inhibition of GIRK channels involves PLC activation by G(alpha) subunits of the G(alpha)q family and suggest that inhibition may be communicated at a distance to GIRK channels via unbinding and diffusion of phosphatidylinositol bisphosphate away from the channel.     

The role of G proteins in assembly and function of Kir3 inwardly rectifying potassium channels

Kir3 channels (also known as GIRK channels) are important regulators of electrical excitability in both cardiomyocytes and neurons. Much is known regarding the assembly and function of these channels and the roles that their interacting proteins play in controlling these events. Further, they are one of the best studied effectors of heterotrimeric G proteins in general and Gβγ subunits in particular. However, our understanding of the roles of multiple Gβγ binding sites on Kir3 channels is still rudimentary. We discuss potential roles for Gβγ in channel assembly and trafficking in addition to their known role in cellular signaling.     

The pore helix is involved in stabilizing the open state of inwardly rectifying K+ channels

Ion channels can be gated by various extrinsic cues, such as voltage, pH, and second messengers. However, most ion channels display extrinsic cue-independent transitions as well. These events represent spontaneous conformational changes of the channel protein. The molecular basis for spontaneous gating and its relation to the mechanism by which channels undergo activation gating by extrinsic cue stimulation is not well understood. Here we show that the proximal pore helix of inwardly rectifying (Kir) channels is partially responsible for determining spontaneous gating characteristics, affecting the open state of the channel by stabilizing intraburst openings as well as the bursting state itself without affecting K(+) ion-channel interactions. The effect of the pore helix on the open state of the channel is qualitatively similar to that of two well-characterized mutations at the second transmembrane domain (TM2), which stabilize the channel in its activated state. However, the effects of the pore helix and the TM2 mutations on gating were additive and independent of each other. Moreover, in sharp contrast to the two TM2 mutations, the pore helix mutation did not affect the functionality of the agonist-responsive gate. Our results suggest that in Kir channels, the bottom of the pore helix and agonist-induced conformational transitions at the TM2 ultimately stabilize via different pathways the open conformation of the same gate.     

Alpha i-3 cDNA encodes the alpha subunit of Gk, the stimulatory G protein of receptor-regulated K+ channels

cDNA cloning has identified the presence in the human genome of three genes encoding alpha subunits of pertussis toxin substrates, generically called "Gi." They are named alpha i-1, alpha i-2 and alpha i-3. However, none of these genes has been functionally identified with any of the alpha subunits of several possible G proteins, including pertussis toxin-sensitive Gp's, stimulatory to phospholipase C or A2, Gi, inhibitory to adenylyl cyclase, or Gk, stimulatory to a type of K+ channels. We now report the nucleotide sequence and the complete predicted amino acid sequence of human liver alpha i-3 and the partial amino acid sequence of proteolytic fragments of the alpha subunit of human erythrocyte Gk. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of alpha i-3, thus identifying it as alpha k. The probable identity of alpha i-1 with alpha p and possible roles for alpha i-2, as well as additional roles for alpha i-1 and alpha i-3 (alpha k) are discussed.