Probing the G-protein Regulation of GIRK1 and GIRK4, the Two Subunits of the KACh Channel, Using Functional Homomeric Mutants

Vivaudou, Michel; Chan, Kim W.; Sui, Jinliang; Jan, Yuh Nung; Reuveny, Eitan; Logothetis, Diomedes E.

doi:10.1074/jbc.272.50.31553

Cited by 152 publications

(156 citation statements)

References 34 publications

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“…We tested the effect of oleoylCoA on several other members of the Kir channel family, which were as follows: Kir1.1, Kir2.2, Kir2.3, Kir4.1, Kir4.2, and Kir3.4* (the homomerically active GIRK4 mutant S143T; ref. 30). We found that none of these channels was activated by 10 M oleoyl-CoA (data not shown).…”

Section: Resultsmentioning

confidence: 80%

Specificity of activation by phosphoinositides determines lipid regulation of Kir channels

Rohács

Lopes

Jin

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Phosphoinositides are critical regulators of ion channel and transporter activity. Defects in interactions of inwardly rectifying potassium (Kir) channels with phosphoinositides lead to disease. ATP-sensitive K ؉ channels (KATP) are unique among Kir channels in that they serve as metabolic sensors, inhibited by ATP while stimulated by long-chain (LC) acyl-CoA. Here we show that KATP are the least specific Kir channels in their activation by phosphoinositides and we demonstrate that LC acyl-CoA activation of these channels depends on their low phosphoinositide specificity. We provide a systematic characterization of phosphoinositide specificity of the entire Kir channel family expressed in Xenopus oocytes and identify molecular determinants of such specificity. We show that mutations in the Kir2.1 channel decreasing phosphoinositide specificity allow activation by LC acyl-CoA. Our data demonstrate that differences in phosphoinositide specificity determine the modulation of Kir channel activity by distinct regulatory lipids.

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Section: Resultsmentioning

confidence: 80%

Specificity of activation by phosphoinositides determines lipid regulation of Kir channels

Rohács

Lopes

Jin

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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196

230

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“…Here, we use GIRK4(S143T) (denoted as GIRK4*), a homomeric-active GIRK4 channel with a mutation in the porehelix (11), to test the effectiveness of these G␤␥ mutants to enhance basal currents. We have previously shown that homomeric GIRK4* channels behave similarly to the GIRK1/GIRK4 heteromers and are therefore functionally interchangeable (15). Fig.…”

Section: G␤ Mutants Confer Functional But Not Binding Defects-wementioning

confidence: 99%

Distinct Sites on G Protein βγ Subunits Regulate Different Effector Functions

Mirshahi¹,

Mittal²,

Zhang³

et al. 2002

Journal of Biological Chemistry

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G proteins interact with effectors at multiple sites and regulate their activity. The functional significance of multiple contact points is not well understood. We previously identified three residues on distinct surfaces of G␤␥ that are crucial for G protein-coupled inward rectifier K ؉ (GIRK) channel activation. Here we show that mutations at these sites, S67K, S98T, and T128F, abolished or reduced direct GIRK current activation in inside-out patches, but, surprisingly, all mutants synergized with sodium in activating K ؉ currents. Each of the three G␤ 1 mutants bound the channel indicating that the defects reflected mainly functional impairments. We tested these mutants for functional interactions with effectors other than K ؉ channels. With N-type calcium channels, G␤␥ wild type and mutants all inhibited basal currents. A depolarizing pre-pulse relieved G␤␥ inhibition of Ca 2؉ currents by the wild type and the S98T and T128F mutants but not the S67K mutant. Both wild type and mutant G␤␥ subunits activated phospholipase C ␤ 2 with similar potencies; however, the S67K mutant showed reduced maximal activity. These data establish a pattern where mutations can alter the G␤␥ regulation of a specific effector function without affecting other G␤␥-mediated functions. Moreover, Ser-67 showed this pattern in all three effectors tested, suggesting that this residue participates in a common functional domain on G␤ 1 that regulates several effectors. These data show that distinct domains within G␤␥ subserve specific functional roles.G protein ␤␥ subunits interact with several effectors including G protein-gated K ϩ channels, N-type calcium channels and phospholipase C␤ (1). Several reports have investigated the molecular sites of interactions between G␤␥ and their effectors, but the exact molecular mechanism by which G␤␥ alters effector function remains unclear. In one case, the co-crystal structure of the G␤ 1 ␥ 2 and phosducin has been determined (2). This structure points to multiple interactions between the two proteins concentrated in at least two distinct domains. Other studies using indirect approaches including mutagenesis have corroborated this design for G␤␥ interaction with its effectors (2-5). Most of these studies have identified multiple sites on G␤␥ as well as on each effector, where such interactions may occur.We have recently reported multiple sites of interactions between GIRK channels and G␤␥ subunits based on chimeric analysis and mutagenesis studies (6 -8). First, we showed that there are at least three interaction sites on the GIRK channel that are crucial both for binding to G␤␥ and for transducing the G␤␥ effects on channel activity. Recently, we also identified eight residues on G␤ 1 that are important in functional interactions with GIRK 1 channels (8). These amino acids reside on distinct domains of the G protein as assessed from the known crystal structure (9, 10), consistent with the notion of multiple interactions between the two proteins.Although several interaction points between G␤␥ and ef...

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“…Deactivation kinetics were compared for Kir3.4(S143T) (a variant able to form functional homomers as described) (20) and Kir3.4(S143T/Y32F/Y53F) (a homomeric channel with both N-terminal tyrosine residues point-mutated to phenylalanines) (7). trkB activation accelerated channel deactivation in oocytes expressing Kir3.4(S143T) (vehicle, off ϭ 26 Ϯ 4 s, n ϭ 18 and BDNF, off ϭ 17 Ϯ 1 s, n ϭ 17).…”

Section: Fig 1 Trkb Accelerates Kir3 Deactivationmentioning

confidence: 99%

N-terminal Tyrosine Residues within the Potassium Channel Kir3 Modulate GTPase Activity of Gαi

Ippolito

Temkin

Rogalski

et al. 2002

Journal of Biological Chemistry

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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 (brainderived neurotrophic factor) accelerated Kir3 deactivation following termination of mu opioid receptor signaling. Similarly, overexpression of RGS4, a GTPaseactivating protein (GAP), accelerated Kir3 deactivation. Blocking GTPase activity with GTP␥S also prevented Kir3 deactivation, and the GTP␥S effect was not reversed by BDNF treatment. These results suggest that BDNF treatment did not reduce Kir3 affinity for G␤␥ 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.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) (1). 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 ϩ , eicosanoids, ATP, Mg 2ϩ , and phosphorylation (1-6). For example, in a previous study (7), 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. G␤␥, 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 G␤␥, 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 (8). 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 inhib...

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Probing the G-protein Regulation of GIRK1 and GIRK4, the Two Subunits of the KACh Channel, Using Functional Homomeric Mutants

Cited by 152 publications

References 34 publications

Specificity of activation by phosphoinositides determines lipid regulation of Kir channels

Specificity of activation by phosphoinositides determines lipid regulation of Kir channels

Distinct Sites on G Protein βγ Subunits Regulate Different Effector Functions

N-terminal Tyrosine Residues within the Potassium Channel Kir3 Modulate GTPase Activity of Gαi

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