N-terminal Tyrosine Residues within the Potassium Channel Kir3 Modulate GTPase Activity of Gαi
2002; Elsevier BV; Volume: 277; Issue: 36 Linguagem: Inglês
10.1074/jbc.m204407200
ISSN1083-351X
AutoresDanielle L. Ippolito, Paul Temkin, Sherri L. Rogalski, Charles Chavkin,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumotrkB activation results in tyrosine phosphorylation of N-terminal Kir3 residues, decreasing channel activation. To determine the mechanism of this effect, we reconstituted Kir3, trkB, and the mu opioid receptor in Xenopus oocytes. Activation of trkB by BDNF (brain-derived neurotrophic factor) accelerated Kir3 deactivation following termination of mu opioid receptor signaling. Similarly, overexpression of RGS4, a GTPase-activating protein (GAP), accelerated Kir3 deactivation. Blocking GTPase activity with GTPγS also prevented Kir3 deactivation, and the GTPγS effect was not reversed by BDNF treatment. These results suggest that BDNF treatment did not reduce Kir3 affinity for Gβγ but rather acted to accelerate GTPase activity, like RGS4. Tyrosine phosphatase inhibition by peroxyvanadate pretreatment reversibly mimicked the BDNF/trkB effect, indicating that tyrosine phosphorylation of Kir3 may have caused the GTPase acceleration. Tyrosine to phenylalanine substitution in the N-terminal domain of Kir3.4 blocked the BDNF effect, supporting the hypothesis that phosphorylation of these tyrosines was responsible. Like other GAPs, Kir3.4 contains a tyrosine-arginine-glutamine motif that is thought to function by interacting with G protein catalytic domains to facilitate GTP hydrolysis. These data suggest that the N-terminal tyrosine hydroxyls in Kir3 normally mask the GAP activity and that modification by phosphorylation or phenylalanine substitution reveals the GAP domain. Thus, BDNF activation of trkB could inhibit Kir3 by facilitating channel deactivation. trkB activation results in tyrosine phosphorylation of N-terminal Kir3 residues, decreasing channel activation. To determine the mechanism of this effect, we reconstituted Kir3, trkB, and the mu opioid receptor in Xenopus oocytes. Activation of trkB by BDNF (brain-derived neurotrophic factor) accelerated Kir3 deactivation following termination of mu opioid receptor signaling. Similarly, overexpression of RGS4, a GTPase-activating protein (GAP), accelerated Kir3 deactivation. Blocking GTPase activity with GTPγS also prevented Kir3 deactivation, and the GTPγS effect was not reversed by BDNF treatment. These results suggest that BDNF treatment did not reduce Kir3 affinity for Gβγ but rather acted to accelerate GTPase activity, like RGS4. Tyrosine phosphatase inhibition by peroxyvanadate pretreatment reversibly mimicked the BDNF/trkB effect, indicating that tyrosine phosphorylation of Kir3 may have caused the GTPase acceleration. Tyrosine to phenylalanine substitution in the N-terminal domain of Kir3.4 blocked the BDNF effect, supporting the hypothesis that phosphorylation of these tyrosines was responsible. Like other GAPs, Kir3.4 contains a tyrosine-arginine-glutamine motif that is thought to function by interacting with G protein catalytic domains to facilitate GTP hydrolysis. These data suggest that the N-terminal tyrosine hydroxyls in Kir3 normally mask the GAP activity and that modification by phosphorylation or phenylalanine substitution reveals the GAP domain. Thus, BDNF activation of trkB could inhibit Kir3 by facilitating channel deactivation. G protein-coupled inwardly rectifying potassium channel G protein-coupled receptor brain-derived neurotrophic factor [d-Ala2,N-MePhe4-Gly-Ol5]enkephalin mu opioid receptor low potassium vehicle high potassium buffer regulator of G protein signaling, subtype 4 GTPase-activating protein tyrosine kinase receptor for BDNF The family of G protein-activated potassium channels (Kir3 or GIRK)1 is a principal effector mediating the actions of a wide range of pertussis toxin-sensitive, G protein-coupled receptors (GPCR) (1Dascal N. Cell. Signal. 1997; 9: 551-573Crossref PubMed Scopus (268) Google Scholar). Channel activation provides key regulation of both cardiac and neuronal excitability. The activity of this channel is controlled by a large number of modulators including phosphatidylinositol bisphosphate, Na+, eicosanoids, ATP, Mg2+, and phosphorylation (1Dascal N. Cell. Signal. 1997; 9: 551-573Crossref PubMed Scopus (268) Google Scholar, 2Yi B.A. Minor D.L., Jr. Lin Y.F. Jan Y.N. Jan L.Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11016-11023Crossref PubMed Scopus (71) Google Scholar, 3Ishii M. Inanobe A. Kurachi Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4325-4330Crossref PubMed Scopus (71) Google Scholar, 4Kobrinsky E. Mirshahi T. Zhang H. Jin T. Logothetis D.E. Nat. Cell Biol. 2000; 2: 507-514Crossref PubMed Scopus (203) Google Scholar, 5Sui J.L. Petit-Jacques J. Logothetis D.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1307-1312Crossref PubMed Scopus (213) Google Scholar, 6Lesage F. Guillemare E. Fink M. Duprat F. Heurteaux C. Fosset M. Romey G. Barhanin J. Lazdunski M. J. Biol. Chem. 1995; 270: 28660-28667Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). For example, in a previous study (7Rogalski S. Appleyard S. Pattillo A. Terman G. Chavkin C. J. Biol. Chem. 2000; 275: 25082-25088Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), we found that tyrosine phosphorylation of Kir3 resulted in channel inhibition. Brain-derived neurotrophic factor (BDNF) activation of trkB receptors caused the phosphorylation of specific tyrosine residues in the N-terminal domain of Kir3.1 and Kir3.4, reducing basal channel conductance. Gβγ, released by opioid receptor activation, overcame the inhibition and evoked the original maximal effect. The results suggest that channel phosphorylation regulates the specific interaction with Gβγ, but the mechanism of this effect is unclear. However, the finding may be physiologically significant because tyrosine kinase cascades initiated by neurotrophic factors such as BDNF and nerve growth factor are up-regulated under conditions such as inflammation (8Woolf C.J. Costigan M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7723-7730Crossref PubMed Scopus (468) Google Scholar). It is possible that tyrosine phosphorylation of Kir3 may mediate neuronal excitability in conjunction with GPCRs under conditions of inflammation. The goal of the present study was to define the mechanism responsible for BDNF inhibition of G protein regulation of Kir3 functioning. Recent evidence shows that both arms of the heterotrimeric G protein complex, Gαi and Gβγ, interact with Kir3 (9Peleg S. Varon D. Ivanina T. Dessauer C.W. Dascal N. Neuron. 2002; 33: 87-99Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Binding of the Gβγ subunit activates Kir3, whereas Gαibinding suppresses basal Kir3 activity and enhances G protein activation (9Peleg S. Varon D. Ivanina T. Dessauer C.W. Dascal N. Neuron. 2002; 33: 87-99Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). This action of Gαi binding resembles the effect of BDNF-induced tyrosine phosphorylation on Kir3. Both reduce basal channel activity without blocking GPCR activation of Kir3, suggesting the possibility of mechanistic similarity. Thus, we explored the hypothesis that the underlying mechanisms were related and that tyrosine phosphorylation controls Gαi regulation of Kir3 response to GPCR activation. Understanding the basis for the modulation of Kir3 by tyrosine phosphorylation would provide additional insight into the processes regulating the activity of this physiologically significant channel and a molecular basis for interaction between GPCR and tyrosine kinase receptor signaling. To test this hypothesis, we reconstituted the mu opioid receptor (MOR, a GPCR), Kir3, and trkB in Xenopus oocytes and investigated channel activation and deactivation kinetics. When current traces were fit to a simple exponential, trkB accelerated deactivation of Kir3. The kinetics resembled GTPase-activating protein (GAP)-mediated acceleration of channel deactivation, suggesting that trkB might stimulate GAP activity. Sequence alignment showed that Kir3 contains two signature GAP residues (glutamine and arginine) near both N-terminal domain tyrosines. These residues have been shown to promote catalytic activity of the GTPase domain (10Scheffzek K. Ahmadian M.R. Kabsch W. Wiesmuller L. Lautwein A. Schmitz F. Wittinghofer A. Science. 1997; 277: 333-338Crossref PubMed Scopus (1202) Google Scholar, 11Rittinger K. Walker P.A. Eccleston J.F. Smerdon S.J. Gamblin S.J. Nature. 1997; 389: 758-762Crossref PubMed Scopus (355) Google Scholar, 12Nassar N. Hoffman G.R. Manor D. Clardy J.C. Cerione R.A. Nat. Struct. Biol. 1998; 5: 1047-1052Crossref PubMed Scopus (176) Google Scholar, 13Kovoor A. Lester H.A. Neuron. 2002; 33: 6-8Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 14Sprang S.R. Science. 1997; 277: 329-330Crossref PubMed Scopus (16) Google Scholar, 15Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1384) Google Scholar). Phenylalanine substitution of the two N-terminal tyrosine residues within these tyrosine-arginine-glutamine sequences resulted in Kir3 with constitutively faster kinetics of deactivation. Further, BDNF treatment no longer accelerated deactivation kinetics of the mutant channels. These results suggest that phosphorylation of Kir3 unmasks a GAP domain embedded in the sequence of Kir3 itself, promoting GAP activity. Plasmid vectors containing cDNA for the following GenBank accession numbers were obtained from Drs. Lei Yu, Cesar Lebarca, Henry Lester, John Adelman, Mark Bothwell, and Nathan Dascal, respectively: MOR (L13069), Kir3.1 (U01071), Kir3.4 (X83584), trkB (M55293), and RGS4 (AF117211). Kir3.4 was point-mutated by PCR-based site-directed mutagenesis to create the functional homomers Kir3.4(S143T) and Kir3.4(S143T/Y32F/Y53F) (7Rogalski S. Appleyard S. Pattillo A. Terman G. Chavkin C. J. Biol. Chem. 2000; 275: 25082-25088Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). cRNA was synthesized from linearized plasmid vectors using RNA polymerases provided by the commercially available mMessage mMachine kit (Ambion Corporation, Austin, TX). The cRNA was subsequently micro-injected into stage V and VI oocytes harvested from mature anesthetizedXenopus laevis frogs. Oocytes were maintained in high salt buffer (ND96: 96 mm NaCl, 2 mm KCl, 1 mm CaCl2, 1 mm MgCl2, 5 mm HEPES, 2.5 mm sodium pyruvate, 50 μg/ml gentamicin, pH 7.5). Following 3–5 days of expression, the two-electrode voltage clamp technique was used to assess coupling efficiency of MOR to Kir3 in oocytes pretreated in BDNF or vehicle. Briefly, oocyte membranes were voltage-clamped at −80 mV by a microelectrode containing 3 m KCl (resistance between 0.4 and 2 mΩ) connected to a feedback amplifier (Axon Instruments, Inc.). A second microelectrode delivered current necessary to maintain the preset voltage. High potassium buffer (HK+: 2 mm NaCl, 96 mm KCl, 1 mmCaCl2, 1 mm MgCl2, 5 mmHEPES, pH 7.5) and the MOR-agonist [d-Ala2,N-MePhe4-Gly-ol5]enkephalin (DAMGO, diluted in high potassium buffer) were sequentially washed onto the oocytes, and changes in current were recorded and analyzed using pCLAMP 6 software. DAMGO was obtained from Sigma and Peninsula Labs (San Carlos, CA). Naloxone was from Sigma. Sodium orthovanadate (Sigma) was activated in 3% hydrogen peroxide for 4–5 h prior to use. BDNF was a gift from AMGEN Corporation. All reagents were dissolved in water and then diluted in ND96 or high potassium buffer. Kinetics of Kir3 deactivation were calculated as reported previously by measuring time constant to reversal (τoff, 16, 17). Exponentials were fit to the naloxone reversal phases of the DAMGO-induced Kir3 currents. Cursors were positioned at ∼20 and 80% of the maximal equilibrium conductance from current initiation and termination, respectively. Current traces between these points were fit to exponential Equation 1. F(t)=∑i=1nAi*e−t/τi+CEquation 1 Time constants exceeding two standard deviations of the mean were excluded from the analysis. The membrane time constant (τoff) was used to estimate kinetics of Kir3 deactivation. Statistical significance was determined using an unpaired Student's t test, in which a probability ofp < 0.05 was considered statistically significant. We found previously that tyrosine kinase phosphorylation of Kir3 modulates its conductance (7Rogalski S. Appleyard S. Pattillo A. Terman G. Chavkin C. J. Biol. Chem. 2000; 275: 25082-25088Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). To investigate how tyrosine phosphorylation cascades initiated by trkB might regulate Kir3 activation by G protein receptors, we coexpressed cRNA for Kir3.1, Kir3.4, trkB, and MOR in Xenopus oocytes. Oocytes were pretreated in either vehicle (ND96) or 0.2 μg/ml BDNF. Plasma membranes were clamped at −80 mV in two-electrode voltage clamp configuration. Perfusion of 96 mm potassium buffer (HK+) revealed an inwardly rectifying basal current (Fig.1A). Pretreatment in BDNF suppressed basal current to 77 ± 8% (p < 0.05,n = 35) of vehicle controls in agreement with previous studies (Fig. 1B). Basal current was potentiated when the MOR agonist (DAMGO, 1 μm) was perfused into the recording chamber. BDNF had no effect on the amplitude of the DAMGO-induced response (104 ± 1% of vehicle-treated controls,n = 35, Fig. 1B). MOR antagonist perfusion (naloxone, 1 μm) returned current back to basal amplitude, and ND96 perfusion returned the basal current to baseline (Fig. 1A). The rate of channel deactivation during naloxone perfusion appeared to increase after BDNF treatment. To quantify channel deactivation kinetics, we fit the portion of the trace between naloxone and final ND96 applications to a simple exponential. The time constant of deactivation (τoff) was calculated using pCLAMP software as described under "Experimental Procedures." BDNF significantly accelerated channel deactivation rate; τoffwas reduced to 71 ± 3% of vehicle control (p < 0.05, n = 35, Fig. 1, A and B). Channel activation rate, as measured by τon, was not significantly accelerated in BDNF-treated oocytes (data not shown). The decrease in the τoff parameter was not a consequence of the reduced basal current amplitude; a regression analysis showed no correlation between DAMGO current amplitude and τoff(r2 < 0.1). Oocytes lacking the trkB receptor did not show significant acceleration of Kir3 deactivation or decrease in basal channel conductance following BDNF treatment (data not shown). Thus, trkB activation by BDNF reduced the basal channel conductance and accelerated the deactivation kinetics of Kir3 without reducing the maximal DAMGO-evoked effect. Because Kir3 conductance is controlled predominately by binding to free Gβγ, these results suggest that tyrosine phosphorylation affected the interaction between channel and Gβγ. The kinetic profile we describe for Kir3 deactivation resembles GAP-mediated acceleration of deactivation without concomitant decrease in DAMGO current amplitude or increase in τon(18Doupnik C. Davidson N. Lester H. Kofuji P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10461-10466Crossref PubMed Scopus (297) Google Scholar). To illustrate the kinetic similarity, we injected 10 ng of cRNA for the GAP RGS4 into oocytes expressing Kir3, MOR, and trkB 12–18 h before recording. When traces were fit to a simple exponential, Kir3 deactivation (as measured by τoff) was accelerated from 39 ± 3 s (Kir3.1/3.4, n = 28) to 27 ± 3 s (Kir3.1/3.4, n = 36) when oocytes not expressing RGS4 were pretreated in BDNF (Fig.2). Injection of RGS4 accelerated channel deactivation to 12 ± 2 s (τoff,n = 7). BDNF treatment reduced τoff to 7 ± 1 s (n = 11). These results show that trkB modulation of deactivation kinetics is similar to the effect of the GAP RGS4, leading to the speculation that both operate by similar mechanisms. To test the hypothesis that trkB activation increased GAP activity (rather than decreasing Gβγ binding affinity for Kir3) we inhibited GTP hydrolysis by injecting GTPγS, a non-hydrolyzable analogue of GTP (19Dascal N. Ifune C. Hopkins R. Snutch T.P. Lubbert H. Davidson N. Simon M.I. Lester H.A. Brain Res. 1986; 387: 201-209PubMed Google Scholar). If Kir3 deactivation rate was controlled solely by Gβγ dissociation and not Gβγ sequestration by Gαi·GDP, then BDNF-accelerated deactivation could result from a reduction in Gβγ affinity for the channel. Alternatively, if the deactivation rate was controlled by Gβγ sequestration by Gαi·GDP, then the accelerated deactivation rate would result from a BDNF-induced increased GTPase activity that would be blocked by GTPγS. In oocytes expressing MOR, trkB, and Kir3, GTPγS virtually eliminated Kir3 deactivation whether or not RGS4 was injected (Fig. 3,a and b). Pretreatment with BDNF did not accelerate deactivation kinetics of GTPγS-injected oocytes (Fig.3c). Because BDNF pretreatment did not even partially recover deactivation in the presence of GTPγS, trkB-mediated acceleration of deactivation kinetics was probably not a result of a reduction in Gβγ affinity for the channel. These results suggest that trkB accelerates channel deactivation by facilitating GTP hydrolysis and subsequent Gβγ sequestration by Gαi. Previously, we identified tyrosine residues in the Kir3 N-terminal tail that were phosphorylated by trkB and modulated Kir3 gating parameters (7Rogalski S. Appleyard S. Pattillo A. Terman G. Chavkin C. J. Biol. Chem. 2000; 275: 25082-25088Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). To investigate whether phosphorylation state of Kir3 controlled channel deactivation, we pretreated oocytes with the phosphatase inhibitor peroxyvanadate (100 μm) before voltage clamp experimentation. Peroxyvanadate pretreatment reduced τoff from 39 ± 3 s (n= 4) to 23 ± 5 s (n = 4). A 3-min perfusion with ND96 returned τoff to 34 ± 2 s (n = 4), approaching vehicle-treated controls (Fig.4, A and C). Conversely, when oocytes were pretreated first in vehicle followed by a 3-min treatment in peroxyvanadate, channel deactivation was accelerated (Fig. 4B). These results suggest that constitutive phosphorylation of Kir3 may result in enhanced GTP hydrolysis. We hypothesized that BDNF/trkB-induced phosphorylation of specific N-terminal tyrosine residues accelerated Kir3 deactivation kinetics. Deactivation kinetics were compared for Kir3.4(S143T) (a variant able to form functional homomers as described) (20Vivaudou M. Chan K.W. Sui J.L. Jan L.Y. Reuveny E. Logothetis D.E. J. Biol. Chem. 1997; 272: 31553-31560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) and Kir3.4(S143T/Y32F/Y53F) (a homomeric channel with both N-terminal tyrosine residues point-mutated to phenylalanines) (7Rogalski S. Appleyard S. Pattillo A. Terman G. Chavkin C. J. Biol. Chem. 2000; 275: 25082-25088Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). trkB activation accelerated channel deactivation in oocytes expressing Kir3.4(S143T) (vehicle, τoff = 26 ± 4 s, n = 18 and BDNF, τoff = 17 ± 1 s,n = 17). However, trkB no longer accelerated deactivation in the double tyrosine to phenylalanine mutant (vehicle, τoff = 15 ± 1 s, n = 30 and BDNF, τoff = 15 ± 1 s, n = 40, Fig. 5A). These results suggest that trkB-mediated phosphorylation of these tyrosine residues is important in facilitating GAP activity of the channel. Interestingly, we found that the Kir3.4(S143T/Y32F/Y53F) mutations constitutively accelerated Kir3.4 kinetics (compare Kir3.4(S143T), τoff = 26 ± 4 s, n = 18 with Kir3.4(S143T/Y32F/Y53F), τoff = 15 ± 1 s,n = 30, Fig. 5A). These results suggest that the hydroxyl group in the tyrosine dampens GAP activity because phenylalanine substitution for tyrosine promotes GAP activity. A direct measure of GTPase activity of Kir3 was not possible because we would not have attained the high level of channel expression required (21Mosser V.A. Amana I.J. Schimerlik M.I. J. Biol. Chem. 2002; 277: 922-931Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Recently, the crystal structures of G protein·GAP combinations such as Ras-Ras·GAP, Rho-Rho·GAP, and Gαi·RGS have been solved (10Scheffzek K. Ahmadian M.R. Kabsch W. Wiesmuller L. Lautwein A. Schmitz F. Wittinghofer A. Science. 1997; 277: 333-338Crossref PubMed Scopus (1202) Google Scholar, 11Rittinger K. Walker P.A. Eccleston J.F. Smerdon S.J. Gamblin S.J. Nature. 1997; 389: 758-762Crossref PubMed Scopus (355) Google Scholar, 12Nassar N. Hoffman G.R. Manor D. Clardy J.C. Cerione R.A. Nat. Struct. Biol. 1998; 5: 1047-1052Crossref PubMed Scopus (176) Google Scholar,14Sprang S.R. Science. 1997; 277: 329-330Crossref PubMed Scopus (16) Google Scholar, 22Srinivasa S.P. Watson N. Overton M.C. Blumer K.J. J. 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To determine whether trkB worked cooperatively with RGS4 to accelerate channel deactivation, we investigated the effect of BDNF pretreatment on the τoff parameter in oocytes expressing RGS4, trkB, Kir3 mutants, and MOR. RGS4 accelerated channel deactivation in the Kir3.4(S143T/Y32F/Y53F) mutants (τoff= 10 ± 2 s, n = 20, Fig. 5A), but BDNF had no additional effect (τoff = 15 ± 2 s, n = 18). In fact, time constants resembled BDNF-pretreated oocytes lacking RGS4 (Fig. 5A). Similar acceleration of deactivation was seen in Kir3.4(S143T) mutants (compare RGS4, τoff = 13 ± 3 s, n = 37 with RGS4+BDNF, τoff = 19 ± 3 s,n = 8). These results suggest an independent mechanism of Kir3 GAP activity for RGS4- and trkB-mediated Kir3 phosphorylation. We conclude that Kir3.4 contains critical GAP-like amino acids and probably acts as a GAP for Gαi. GAP activity is kept in check by the tyrosine hydroxyl. Either elimination of the hydroxyl by phenyalanine substitution or phosphorylation of the hydroxyl by tyrosine kinase signaling cascades results in potentiation of GAP activity. These results suggest a novel control mechanism of channel deactivation. The principal finding of this study is that tyrosine phosphorylation of Kir3 by trkB/BDNF reduces channel activation by accelerating GTPase kinetics. Our data did not support the alternative hypothesis that the binding affinity of channel for Gβγ was reduced by channel phosphorylation. The concept that the channel regulates the GTPase activity of the Gαi subunit is novel, although the principle of an effector providing feedback regulation of its activator is well established (15Vetter I.R. Wittinghofer A. Science. 2001; 294: 1299-1304Crossref PubMed Scopus (1384) Google Scholar, 25Nissen P. Kjeldgaard M. Thirup S. Polekhina G. Reshetnikova L. Clark B.F. Nyborg J. Science. 1995; 270: 1464-1472Crossref PubMed Scopus (803) Google Scholar). 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Further, the arginine, glutamine, and tyrosine residues in the portion of Kir3 contacting Gαi are residues found in GTPase-activating proteins shown by crystallography to be critical in stabilizing the transition state of Gαi, favoring catalysis (10Scheffzek K. Ahmadian M.R. Kabsch W. Wiesmuller L. Lautwein A. Schmitz F. Wittinghofer A. Science. 1997; 277: 333-338Crossref PubMed Scopus (1202) Google Scholar, 14Sprang S.R. Science. 1997; 277: 329-330Crossref PubMed Scopus (16) Google Scholar, 23Sprang S.R. Curr. Opin. Struct. Biol. 1997; 7: 849-856Crossref PubMed Scopus (125) Google Scholar). Glutamine and arginine residues in GAPs such as Ras·GAP, p50Rho·GAP, cdc42·GAP, and the yeast Ypt/Rab·GAP·Gyp1p are critical in mimicking residues in their respective G proteins to stabilize the transition phase of the Gαi binding pocket (10Scheffzek K. Ahmadian M.R. Kabsch W. Wiesmuller L. Lautwein A. Schmitz F. Wittinghofer A. Science. 1997; 277: 333-338Crossref PubMed Scopus (1202) Google Scholar, 11Rittinger K. Walker P.A. Eccleston J.F. Smerdon S.J. Gamblin S.J. Nature. 1997; 389: 758-762Crossref PubMed Scopus (355) Google Scholar, 12Nassar N. Hoffman G.R. Manor D. Clardy J.C. Cerione R.A. Nat. Struct. Biol. 1998; 5: 1047-1052Crossref PubMed Scopus (176) Google Scholar, 14Sprang S.R. Science. 1997; 277: 329-330Crossref PubMed Scopus (16) Google Scholar, 23Sprang S.R. Curr. Opin. Struct. Biol. 1997; 7: 849-856Crossref PubMed Scopus (125) Google Scholar, 39Rak A. Fedorov R. Alexandrov K. Albert S. Goody R.S. Gallwitz D. Scheidig A.J. EMBO J. 2000; 19: 5105-5113Crossref PubMed Scopus (83) Google Scholar). The contribution of the tyrosine residue is less clear, although some studies imply that a tyrosine hydroxyl on the G protein itself (11Rittinger K. Walker P.A. Eccleston J.F. Smerdon S.J. Gamblin S.J. Nature. 1997; 389: 758-762Crossref PubMed Scopus (355) Google Scholar, 12Nassar N. Hoffman G.R. Manor D. 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Thus, regulation of Kir3 functioning by tyrosine kinase cascades may provide an important means for physiologically regulating cardiac and neuronal excitability during processes such as development, inflammatory responses, synaptic plasticity, and cardiac excitability.
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