Mapping the G -binding Sites in GIRK1 and GIRK2 Subunits of the G Protein-activated K+ Channel (original) (raw)
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Gαi Controls the Gating of the G Protein-Activated K+ Channel, GIRK
Neuron, 2002
region, altering open channel kinetics and bursting be-Sackler School of Medicine havior (Sadja et al., 2001; Yi et al., 2001). Tel Aviv University G␣ subunits play an important role in determining Ramat Aviv, 69978 the specificity of GPCR-GIRK signaling. In cardiac and Israel neuronal cells, only GPCRs coupled to pertussis toxin 2 Department of Integrative Biology (PTX)-sensitive G␣ i/o proteins activate GIRK (Wickman and Pharmacology and Clapham, 1995); the preferred donors of G␥ are University of Texas G␣ i2 and G␣ i3 but not G␣ o (Kozasa et al., 1996; Sowell Houston Medical School et al., 1997). We have previously found that, in excised Houston, Texas 77030 patches of Xenopus oocytes, G ␥ -induced GIRK activation is antagonized by GTP␥S-activated G␣ i1 (but not by G␣ i2 or G␣ i3 ), and proposed a role for this antagonism in ensuring the specificity of signaling (Schreibmayer et Summary al., 1996). However, later studies showed that, in intact cells, G ␥ released from G i1 can activate GIRK (Leaney GIRK (Kir3) channels are activated by neurotransmitet al., 2000; Leaney and Tinker, 2000). In all, the role of ters coupled to G proteins, via a direct binding of G ␥ . G␣ i in GIRK gating remains uncertain. Also, it is not clear The role of G␣ subunits in GIRK gating is elusive. Here exactly how G␣ subunits help to determine signaling we demonstrate that G␣ i is not only a donor of G␥ specificity. Upon overexpression of various components but also regulates GIRK gating. When overexpressed of signaling pathways, such as G␣ or G, GIRK can be in Xenopus oocytes, GIRK channels show excessive
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.
The Journal of Physiology, 2009
G protein activated K + channels (GIRK, Kir3) are switched on by direct binding of Gβγ following activation of G i/o proteins via G protein-coupled receptors (GPCRs). Although Gα i subunits do not activate GIRKs, they interact with the channels and regulate the gating pattern of the neuronal heterotetrameric GIRK1/2 channel (composed of GIRK1 and GIRK2 subunits) expressed in Xenopus oocytes. Coexpressed Gα i3 decreases the basal activity (I basal ) and increases the extent of activation by purified or coexpressed Gβγ. Here we show that this regulation is exerted by the 'inactive' GDP-bound Gα i3 GDP and involves the formation of Gα i3 βγ heterotrimers, by a mechanism distinct from mere sequestration of Gβγ 'away' from the channel. The regulation of basal and Gβγ-evoked current was produced by the 'constitutively inactive' mutant of Gα i3 , Gα i3 G203A, which strongly binds Gβγ, but not by the 'constitutively active' mutant, Gα i3 Q204L, or by Gβγ-scavenging proteins. Furthermore, regulation by Gα i3 G203A was unique to the GIRK1 subunit; it was not observed in homomeric GIRK2 channels. In vitro protein interaction experiments showed that purified Gβγ enhanced the binding of Gα i3 GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a 'classical' Gβγ effector, showing low I basal and strong Gβγ-dependent activation. Expression of Gα i3 G203A did not affect either I basal or Gβγ-induced activation. In contrast, homomeric GIRK1 * (a pore mutant able to form functional homomeric channels) exhibited large I basal and was poorly activated by Gβγ. Expression of Gα i3 GDP reduced I basal and restored the ability of Gβγ to activate GIRK1 * , like in GIRK1/2. Transferring the unique distal segment of the C terminus of GIRK1 to GIRK2 rendered the latter functionally similar to GIRK1 * . These results demonstrate that GIRK1 containing channels are regulated by both Gα i3 GDP and Gβγ, while GIRK2 is a Gβγ-effector insensitive to Gα i3 GDP .
Journal of Biological Chemistry, 1997
In heart, G-protein-activated channels are complexes of two homologous proteins, GIRK1 and GIRK4. Expression of either protein alone results in barely active or non-active channels, making it difficult to assess the individual contribution of each subunit to the channel complex. The residue Phe 137 , located within the H5 region of GIRK1, is critical to the synergy between GIRK1 and GIRK4 (Chan, K. W., Sui, J. L., Vivaudou, M., and Logothetis, D. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14193-14198). By modifying this residue or the matching residue of GIRK4, Ser 143 , we have been able to generate mutant proteins that produced large inwardly rectifying, G-protein-modulated currents when expressed alone in Xenopus oocytes. The enhanced activity of the heterologous expression of each of two active mutants, GIRK1(F137S) and GIRK4(S143T), was not caused by association with an endogenous oocyte channel subunit, and these mutants did not display apparent differences in the ability to localize to the cell surface compared with their wild-type counterparts. When these functional mutant channels were compared individually with wild-type heteromeric channels, they responded with only small differences to a number of maneuvers involving coexpression with muscarinic receptors, G-protein ␥ subunits, wild-type or mutated G-protein ␣ subunits, and active protomers of pertussis toxin. These experiments, which confirmed the crucial, though not exclusive, role of G␥ in regulating channel activity, demonstrated that GIRK1(F137S) and GIRK4(S143T), and by extrapolation their wild-type counterparts, interact in a qualitatively similar way with G-protein subunits. These findings suggest that functionally important sites of interaction with G-proteins are likely to be located within the homologous regions of GIRK1 and GIRK4 rather than within the divergent terminal regions. They also raise the question of the functional advantage of a heteromeric over homomeric design for G-protein-gated channels.
The Journal of Physiology, 1997
1. In order to find out the functional roles of cytosolic regions of a G protein-activated, inwardly rectifying potassium channel subunit we studied block of GIRK channels, expressed in Xenopus laevis oocytes, by synthetic peptides in isolated inside-out membrane patches. 2. A peptide (DS6) derived from the very end of the C-terminus of GIRK1 reversibly blocked GIRK activity with IC50 values of 7 9 + 2'0 or 3 5 + 0' 5,ug ml-1 (corresponding to 3-7 + 09 or 1-7 + 0-2 #smol F1) for GIRK1/GIRK5 or GIRK1/GIRK4 channels, respectively.
The Journal of Physiology, 2009
G protein activated K + channels (GIRK, Kir3) are switched on by direct binding of Gβγ following activation of G i/o proteins via G protein-coupled receptors (GPCRs). Although Gα i subunits do not activate GIRKs, they interact with the channels and regulate the gating pattern of the neuronal heterotetrameric GIRK1/2 channel (composed of GIRK1 and GIRK2 subunits) expressed in Xenopus oocytes. Coexpressed Gα i3 decreases the basal activity (I basal) and increases the extent of activation by purified or coexpressed Gβγ. Here we show that this regulation is exerted by the 'inactive' GDP-bound Gα i3 GDP and involves the formation of Gα i3 βγ heterotrimers, by a mechanism distinct from mere sequestration of Gβγ 'away' from the channel. The regulation of basal and Gβγ-evoked current was produced by the 'constitutively inactive' mutant of Gα i3 , Gα i3 G203A, which strongly binds Gβγ, but not by the 'constitutively active' mutant, Gα i3 Q204L, or by Gβγ-scavenging proteins. Furthermore, regulation by Gα i3 G203A was unique to the GIRK1 subunit; it was not observed in homomeric GIRK2 channels. In vitro protein interaction experiments showed that purified Gβγ enhanced the binding of Gα i3 GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a 'classical' Gβγ effector, showing low I basal and strong Gβγ-dependent activation. Expression of Gα i3 G203A did not affect either I basal or Gβγ-induced activation. In contrast, homomeric GIRK1 * (a pore mutant able to form functional homomeric channels) exhibited large I basal and was poorly activated by Gβγ. Expression of Gα i3 GDP reduced I basal and restored the ability of Gβγ to activate GIRK1 * , like in GIRK1/2. Transferring the unique distal segment of the C terminus of GIRK1 to GIRK2 rendered the latter functionally similar to GIRK1 *. These results demonstrate that GIRK1 containing channels are regulated by both Gα i3 GDP and Gβγ, while GIRK2 is a Gβγ-effector insensitive to Gα i3 GDP .
G i1 and G i3 Differentially Interact with, and Regulate, the G Protein-activated K+ Channel
Journal of Biological Chemistry, 2004
G protein-activated K ؉ channels (GIRKs; Kir3) are activated by direct binding of G␥ subunits released from heterotrimeric G proteins. In native tissues, only pertussis toxin-sensitive G proteins of the G i/o family, preferably G␣ i3 and G␣ i2 , are donors of G␥ for GIRK. How this specificity is achieved is not known. Here, using a pulldown method, we confirmed the presence of G␣ 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 G␣ i3 by a factor of 2-4. G␣ i1-GDP bound to GIRK1 and GIRK2 much weaker than G␣ 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 G␥ in excised membrane patches was strongly augmented by coexpression of G␣ i3 and less by G␣ i1 . Differences in physical interactions of GIRK with GDP-bound G␣ subunits, or G␣␥ heterotrimers, may dictate different extents of G␣␥ anchoring, influence the efficiency of GIRK activation by G␥, and play a role in determining signaling specificity.
Single Channel Analysis of the Regulation of GIRK1/GIRK4 Channels by Protein Phosphorylation
Biophysical Journal, 2003
G-Protein activated, inwardly rectifying potassium channels (GIRKs) are important effectors of G-protein b/gsubunits, 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 (PP 2 A). 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 m 2 -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 b1/g2 , used to activate the channel, P o was significantly reduced in patches treated with PP 2 A, 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 C 5 state was responsible for facilitation of GIRK1/GIRK4 channels by protein phosphorylation. Dephosphorylation by PP 2 A led to an increase of G b1/g2 concentration required for full activation of the channel and hence to a reduction of the apparent affinity of GIRK1/GIRK4 for G b1/g2 . (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 PP 2 A, indicating that they constitute an essential part of the off-switch.
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.
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. ...