Identification of critical residues controlling G protein-gated inwardly rectifying K+ channel activity through interactions with the βγ subunits of G proteins (original) (raw)
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Proceedings of the …, 2000
G protein-coupled inwardly rectifying potassium (GIRK) channels can be activated or inhibited by different classes of receptors, suggesting a role for G proteins in determining signaling specificity. Because G protein ␥ subunits containing either 1 or 2 with multiple G␥ subunits activate GIRK channels, we hypothesized that specificity might be imparted by 3, 4, or 5 subunits. We used a transfection assay in cell lines expressing GIRK channels to examine effects of dimers containing these G subunits. Inwardly rectifying K ؉ currents were increased in cells expressing 3 or 4, with either ␥2 or ␥11. Purified, recombinant 3␥2 and 4␥2 bound directly to glutathione-S-transferase fusion proteins containing Nor C-terminal cytoplasmic domains of GIRK1 and GIRK4, indicating that 3 and 4, like 1, form dimers that bind to and activate GIRK channels. By contrast, 5containing dimers inhibited GIRK channel currents. This inhibitory effect was obtained with either 5␥2 or 5␥11, was observed with either GIRK1,4 or GIRK1,2 channels, and was evident in the context of either basal or agonist-induced currents, both of which were mediated by endogenous G␥ subunits. In cotransfection assays, 5␥2 suppressed 1␥2-activated GIRK currents in a dose-dependent manner consistent with competitive inhibition. Moreover, we found that 5␥2 could bind to the same GIRK channel cytoplasmic domains as other, activating G␥ subunits. Thus, 5-containing dimers inhibit G␥-stimulated GIRK channels, perhaps by directly binding to the channels. This suggests that 5-containing dimers could act as competitive antagonists of other G␥ dimers on GIRK channels. P otassium channels that are active near resting membrane potentials are key determinants of cellular excitability. The G protein-coupled inwardly rectifying K ϩ (GIRK; Kir3.x) channels are particularly interesting in that they are differentially regulated by receptors that couple to different classes of heterotrimeric G proteins: GIRK channels are activated by receptors that couple to G␣i͞o and inhibited by receptors that couple to G␣q (1, 2). This dual up-and down-regulation of GIRK channels by different receptor classes has been described in atrial cells (3), aminergic brainstem neurons (4, 5), and enteric neurons of the peripheral nervous system (6). Mechanisms underlying inhibition of GIRK channels are not well understood. By contrast, the characteristics of receptormediated activation of GIRK channels have been worked out in detail. It is now clear that G␥ subunits liberated from G protein heterotrimers bind directly to GIRK channels to enhance channel activity (reviewed in refs. 1 and 2). This mechanism raises an interesting conundrum: If all G protein-coupled receptors release G␥ subunits when activated and all G␥ subunits tested to date activate GIRK channels (7), how is signaling specificity obtained such that different classes of receptor can activate or inhibit GIRK channels? One possibility is that specificity derives from associations of different receptors with particular combinations of G protein subunits, which either activate or inhibit GIRK channels. Indeed, exquisite specificity in receptor-G protein subunit interactions has been demonstrated by using antisense approaches in a number of test systems (reviewed in ref. 8), but there is currently no direct evidence for specificity of G␥ effects on GIRK channels (1, 2, 7). However, of the five G subunits identified to date by molecular cloning, only 1 and 2 have been systematically tested for effects on GIRK channels (7). Limited functional evidence indicates that dimers containing 3 and 4 can activate GIRK channels (9, 10), but yeast two-hybrid assays suggested that 3 and 4 do not interact with GIRK channels (11). The most structurally divergent G subunit is 5 (12). It is expressed in a number of tissues (e.g., brain, heart, and kidney; refs. 12-14), and forms G␥ dimers that interact preferentially with G␣q-coupled receptors (15, 16), the same receptors that mediate GIRK channel inhibition (1, 3-6). Effects on GIRK channels of 5-containing G␥ dimers have not been previously examined. Here, by using transfection and glutathione-S-transferase (GST) pull-down assays, we show that 3 and 4 are like 1, inasmuch as they form G␥ dimers that bind directly to GIRK channel proteins and activate GIRK currents in mammalian cells. By contrast, the 5 subunit is distinctly different in its effects on GIRK channels; it forms dimers that inhibit G␥activated GIRK channel currents. Because 5-containing dimers bind to the same cytoplasmic GIRK channel domains as the activating G␥ subunits, we suggest that 5-mediated GIRK channel inhibition results from competitive interactions between 5 and other subunits for active sites on the channels. Methods Stable Cell Lines Expressing GIRK Channels. We obtained GIRK1 in pBSII (KSϪ) and GIRK4 in pcDNA3 from N. Davidson (California Institute of Technology, Pasadena, CA) and subcloned GIRK1 into pcDNA3 (Invitrogen). HEK 293 cells were transfected with pcDNA3-GIRK1 and pcDNA3-GIRK4, maintained under G418 selection (400 g͞ml; GIBCO͞BRL), and a G418-resistant cell line (G1,4 cell line) was chosen for study based on robust expression of inwardly rectifying K ϩ currents. Western blots of crude cell lysates and immunocytochemistry with GIRK1 antisera (Alomone Labs, Jerusalem, Israel) verified GIRK expression in G 1,4 cells that was absent in HEK 293 cells and diminished by antigenic peptide (data not shown). An additional HEK 293 cell line that stably expresses GIRK1 and GIRK2 together with the m4-muscarinic receptor (G1,2m4) was provided by L.
Mapping the G -binding Sites in GIRK1 and GIRK2 Subunits of the G Protein-activated K+ Channel
Journal of Biological Chemistry, 2003
G protein-activated K ؉ channels (Kir3 or GIRK) are activated by direct binding of G␥. The binding sites of G␥ in the ubiquitous GIRK1 (Kir3.1) subunit have not been unequivocally charted, and in the neuronal GIRK2 (Kir3.2) subunit the binding of G␥ has not been studied. We verified and extended the map of G␥-binding sites in GIRK1 by using two approaches: direct binding of G␥ to fragments of GIRK subunits (pull down), and competition of these fragments with the G␣ i1 subunit for binding to G␥. We also mapped the G␥-binding sites in GIRK2. In both subunits, the N terminus binds G␥. In the C terminus, the G␥-binding sites in the two subunits are not identical; GIRK1, but not GIRK2, has a previously unrecognized G␥-interacting segments in the first half of the C terminus. The main C-terminal G␥-binding segment found in both subunits is located approximately between amino acids 320 and 409 (by GIRK1 count). Mutation of C-terminal leucines 262 or 333 in GIRK1, recognized previously as crucial for G␥ regulation of the channel, and of the corresponding leucines 273 and 344 in GIRK2 dramatically altered the properties of K ؉ currents via GIRK1/GIRK2 channels expressed in Xenopus oocytes but did not appreciably reduce the binding of G␥ to the corresponding fusion proteins, indicating that these residues are mainly important for the regulation of G␥-induced changes in channel gating rather than G␥ binding.
Journal of Biological Chemistry, 2000
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 G␥ 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 toxinresistant G protein ␣ subunits and chimeras between G i1 and G s indicate a central role for the G protein ␣ subunits in determining receptor specificity of coupling to, but not activation of, G protein-gated inwardly rectifying K ؉ channels.
The Journal of physiology, 2014
The G-protein coupled inwardly rectifying potassium (GIRK, or Kir3) channels are important mediators of inhibitory neurotransmission via activation of G-protein coupled receptors (GPCRs). GIRK channels are tetramers comprising combinations of subunits (GIRK1-4), activated by direct binding of the Gβγ subunit of Gi/o proteins. Heterologously expressed GIRK1/2 exhibit high, Gβγ-dependent basal currents (Ibasal) and a modest activation by GPCR or coexpressed Gβγ. Inversely, the GIRK2 homotetramers exhibit low Ibasal and strong activation by Gβγ. The high Ibasal of GIRK1 seems to be associated with its unique distal C terminus (G1-dCT), which is not present in the other subunits. We investigated the role of G1-dCT using electrophysiological and fluorescence assays in Xenopus laevis oocytes and protein interaction assays. We show that expression of GIRK1/2 increases the plasma membrane level of coexpressed Gβγ (a phenomenon we term 'Gβγ recruitment') but not of coexpressed Gαi3. ...
Proceedings of the National Academy of Sciences, 1996
G protein-gated inwardly rectifying K ؉ (GIRK) channels, which are important regulators of membrane excitability both in heart and brain, appear to function as heteromultimers. GIRK1 is unique in the GIRK channel family in that although it is by itself inactive, it can associate with the other family members (GIRK2-GIRK5) to enhance their activity and alter their single-channel characteristics. By generating a series of chimeras, we identified a phenylalanine residue, F137, in the pore region of GIRK1 that critically controls channel activity. F137 is found only in GIRK1, while the remaining GIRK channels possess a conserved serine residue in the analogous position. The single-point mutant GIRK4(S143F) behaved as a GIRK1 analog, forming multimers with GIRK2, GIRK4, or GIRK5 channels that exhibited prolonged single-channel open-time duration and enhanced activity compared with that of homomultimers. Expression of the corresponding GIRK1(F137S) mutant alone resulted in appreciable channel activity with novel characteristics that was further enhanced upon coexpression with other GIRK subunits. Thus, although the F137 residue renders the GIRK1 subunit inactive, when combined with other GIRK heteromeric partners it alters their gating and contributes to their enhanced activity.
Journal of Biological Chemistry
Stable complexes among G proteins and effectors are an emerging concept in cell signaling. The prototypical Gβγ effector G protein-activated K+ channel (GIRK; Kir3) physically interacts with Gβγ but also with Gαi/o. Whether and how Gαi/o subunits regulate GIRK in vivo is unclear. We studied triple interactions among GIRK subunits 1 and 2, Gαi3 and Gβγ. We used in vitro protein interaction assays and in vivo intramolecular Förster resonance energy transfer (i-FRET) between fluorophores attached to N and C termini of either GIRK1 or GIRK2 subunit. We demonstrate, for the first time, that Gβγ and Gαi3 distinctly and interdependently alter the conformational states of the heterotetrameric GIRK1/2 channel. Biochemical experiments show that Gβγ greatly enhances the binding of GIRK1 subunit to Gαi3GDP and, unexpectedly, to Gαi3GTP. i-FRET showed that both Gαi3 and Gβγ induced distinct conformational changes in GIRK1 and GIRK2. Moreover, GIRK1 and GIRK2 subunits assumed unique, distinct con...
Redox-dependent Gating of G Protein-coupled Inwardly Rectifying K+ Channels
Journal of Biological Chemistry, 2001
G protein-coupled inwardly rectifying K ؉ channels (GIRK) play a major role in inhibitory signaling in excitable and endocrine tissues. The gating mechanism of these channels is mediated by a direct interaction of the G␥ subunits of G protein, which are released upon inhibitory neurotransmitter receptor activation. This gating mechanism is further manifested by intracellular factors such as anionic phospholipids and Na ؉ and Mg 2؉ ions. In addition to the essential role of these components for channel function, phosphorylation events can also modulate channel activity. In this study we explored the involvement of redox modulation on GIRK channel function. Extracellular application of the reducing agent dithiothreitol (DTT), but not reduced glutathione, activated GIRK channels without affecting their permeation or rectification properties. The DTTdependent activation was found to mimic receptor activation and to act directly on the channel in a membrane delimited fashion. A critical cysteine residue located in the N-terminal cytoplasmic domain was found to be essential for DTT-dependent activation in hetero-and homotetrameric contexts. Interestingly, when mutating this cysteine residue, DTT-dependent activation was abolished, but receptor-mediated channel activation was not affected. These results suggest that intracellular redox potential can play a major role in tuning GIRK channel activity in a receptor-independent manner. This sort of redox modulation can be part of an important cellular protective mechanism against ischemic or hypoxic insults.
Gβγ-dependent and Gβγ-independent Basal Activity of G Protein-activated K+ Channels
Journal of Biological Chemistry, 2005
Cardiac and neuronal G protein-activated K ؉ channels (GIRK; Kir3) open following the binding of G␥ subunits, released from G i/o proteins activated by neurotransmitters. GIRKs also possess basal activity contributing to the resting potential in neurons. It appears to depend largely on free G␥, but a G␥-independent component has also been envisaged. We investigated G␥ dependence of the basal GIRK activity (A GIRK,basal) quantitatively, by titrated expression of G␥ scavengers, in Xenopus oocytes expressing GIRK1/2 channels and muscarinic m2 receptors. The widely used G␥ scavenger, myristoylated C terminus of -adrenergic kinase (m-cARK), reduced A GIRK,basal by 70-80% and eliminated the acetylcholine-evoked current (I ACh). However, we found that m-cARK directly binds to GIRK, complicating the interpretation of physiological data. Among several newly constructed G␥ 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 G␥ and G␣ in the plasma membrane, supporting the existence of preformed complexes of GIRK with G protein subunits. Increased expression of G␥ 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 cARK. These results demonstrate that a major part of I basal is G␥-dependent at all levels of channel expression, and only a small fraction (<10%) may be G␥-independent. G protein-activated, inwardly rectifying K ϩ channels (GIRK, Kir3) 1 mediate postsynaptic inhibitory effects of various neu