Channel Openings Are Necessary but not Sufficient for Use-dependent Block of Cardiac Na+ Channels by Flecainide: Evidence from the Analysis of Disease-linked Mutations

Na+ channel blockers such as flecainide have found renewed usefulness in the diagnosis and treatment of two clinical syndromes arising from inherited mutations in SCN5A, the gene encoding the α subunit of the cardiac voltage–gated Na+ channel. The Brugada syndrome (BrS) and the LQT-3 variant of the Long QT syndrome are caused by disease-linked SCN5A mutations that act to change functional and pharmacological properties of the channel. Here we have explored a set of SCN5A mutations linked both to BrS and LQT-3 to determine what disease-modified channel properties underlie distinct responses to the Na+ channel blocker flecainide. We focused on flecainide block that develops with repetitive channel activity, so-called use-dependent block (UDB). Our results indicate that mutation-induced changes in the voltage-dependence of channel availability (inactivation) may act as determinants of flecainide block. The data further indicate that UDB by flecainide requires channel opening, but is not likely due to open channel block. Rather, flecainide appears to interact with inactivation states that follow depolarization-induced channel opening, and mutation-induced changes in channel inactivation will alter flecainide block independent of the disease to which the mutation is linked. Analysis of flecainide block of mutant channels linked to these rare disorders has provided novel insight into the molecular determinants of drug action

The Na+ Channel Inactivation Gate Is a Molecular Complex: A Novel Role of the COOH-terminal Domain

Electrical activity in nerve, skeletal muscle, and heart requires finely tuned activity of voltage-gated Na+ channels that open and then enter a nonconducting inactivated state upon depolarization. Inactivation occurs when the gate, the cytoplasmic loop linking domains III and IV of the α subunit, occludes the open pore. Subtle destabilization of inactivation by mutation is causally associated with diverse human disease. Here we show for the first time that the inactivation gate is a molecular complex consisting of the III-IV loop and the COOH terminus (C-T), which is necessary to stabilize the closed gate and minimize channel reopening. When this interaction is disrupted by mutation, inactivation is destabilized allowing a small, but important, fraction of channels to reopen, conduct inward current, and delay cellular repolarization. Thus, our results demonstrate for the first time that physiologically crucial stabilization of inactivation of the Na+ channel requires complex interactions of intracellular structures and indicate a novel structural role of the C-T domain in this process

Metabotropic glutamate receptors in GtoPdb v.2023.1

Metabotropic glutamate (mGlu) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Metabotropic Glutamate Receptors [351]) are a family of G protein-coupled receptors activated by the neurotransmitter glutamate [140]. The mGlu family is composed of eight members (named mGlu1 to mGlu8) which are divided in three groups based on similarities of agonist pharmacology, primary sequence and G protein coupling to effector: Group-I (mGlu1 and mGlu5), Group-II (mGlu2 and mGlu3) and Group-III (mGlu4, mGlu6, mGlu7 and mGlu8) (see Further reading).Structurally, mGlu are composed of three juxtaposed domains: a core G protein-activating seven-transmembrane domain (TM), common to all GPCRs, is linked via a rigid cysteine-rich domain (CRD) to the Venus Flytrap domain (VFTD), a large bi-lobed extracellular domain where glutamate binds. mGlu form constitutive dimers, cross-linked by a disulfide bridge. The structures of the VFTD of mGlu1, mGlu2, mGlu3, mGlu5 and mGlu7 have been solved [200, 275, 268, 403]. The structure of the 7 transmembrane (TM) domains of both mGlu1 and mGlu5 have been solved, and confirm a general helical organisation similar to that of other GPCRs, although the helices appear more compacted [88, 433, 62]. Recent advances in cryo-electron microscopy have provided structures of full-length mGlu receptor homodimers [217, 191] and heterodimers [91]. Studies have revealed the possible formation of heterodimers between either group-I receptors, or within and between group-II and -III receptors [89]. First characterised in transfected cells, co-localisation and specific pharmacological properties suggest the existence of such heterodimers in the brain [270, 440, 145, 283, 259, 218]. Beyond heteromerisation with other mGlu receptor subtypes, increasing evidence suggests mGlu receptors form heteromers and larger order complexes with class A GPCRs (reviewed in [140]). The endogenous ligands of mGlu are L-glutamic acid, L-serine-O-phosphate, N-acetylaspartylglutamate (NAAG) and L-cysteine sulphinic acid. Group-I mGlu receptors may be activated by 3,5-DHPG and (S)-3HPG [30] and antagonised by (S)-hexylhomoibotenic acid [235]. Group-II mGlu receptors may be activated by LY389795 [269], LY379268 [269], eglumegad [354, 434], DCG-IV and (2R,3R)-APDC [355], and antagonised by eGlu [170] and LY307452 [425, 105]. Group-III mGlu receptors may be activated by L-AP4 and (R,S)-4-PPG [130]. An example of an antagonist selective for mGlu receptors is LY341495, which blocks mGlu2 and mGlu3 at low nanomolar concentrations, mGlu8 at high nanomolar concentrations, and mGlu4, mGlu5, and mGlu7 in the micromolar range [185]. In addition to orthosteric ligands that directly interact with the glutamate recognition site, allosteric modulators that bind within the TM domain have been described. Negative allosteric modulators are listed separately. The positive allosteric modulators most often act as ‘potentiators’ of an orthosteric agonist response, without significantly activating the receptor in the absence of agonist

Metabotropic glutamate receptors in GtoPdb v.2021.3

Metabotropic glutamate (mGlu) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Metabotropic Glutamate Receptors [347]) are a family of G protein-coupled receptors activated by the neurotransmitter glutamate [138]. The mGlu family is composed of eight members (named mGlu1 to mGlu8) which are divided in three groups based on similarities of agonist pharmacology, primary sequence and G protein coupling to effector: Group-I (mGlu1 and mGlu5), Group-II (mGlu2 and mGlu3) and Group-III (mGlu4, mGlu6, mGlu7 and mGlu8) (see Further reading).Structurally, mGlu are composed of three juxtaposed domains: a core G protein-activating seven-transmembrane domain (TM), common to all GPCRs, is linked via a rigid cysteine-rich domain (CRD) to the Venus Flytrap domain (VFTD), a large bi-lobed extracellular domain where glutamate binds. mGlu form constitutive dimers, cross-linked by a disulfide bridge. The structures of the VFTD of mGlu1, mGlu2, mGlu3, mGlu5 and mGlu7 have been solved [198, 271, 264, 399]. The structure of the 7 transmembrane (TM) domains of both mGlu1 and mGlu5 have been solved, and confirm a general helical organization similar to that of other GPCRs, although the helices appear more compacted [87, 429, 61]. Recent advances in cryo-electron microscopy have provided structures of full-length mGlu receptor dimers [189]. Studies have revealed the possible formation of heterodimers between either group-I receptors, or within and between group-II and -III receptors [88]. First well characterized in transfected cells, co-localization and specific pharmacological properties also suggest the existence of such heterodimers in the brain [266].[436, 143, 279]. Beyond heteromerization with other mGlu receptor subtypes, increasing evidence suggests mGlu receptors form heteromers and larger order complexes with class A GPCRs (reviewed in [138]). The endogenous ligands of mGlu are L-glutamic acid, L-serine-O-phosphate, N-acetylaspartylglutamate (NAAG) and L-cysteine sulphinic acid. Group-I mGlu receptors may be activated by 3,5-DHPG and (S)-3HPG [30] and antagonized by (S)-hexylhomoibotenic acid [232]. Group-II mGlu receptors may be activated by LY389795 [265], LY379268 [265], eglumegad [350, 430], DCG-IV and (2R,3R)-APDC [351], and antagonised by eGlu [168] and LY307452 [421, 103]. Group-III mGlu receptors may be activated by L-AP4 and (R,S)-4-PPG [128]. An example of an antagonist selective for mGlu receptors is LY341495, which blocks mGlu2 and mGlu3 at low nanomolar concentrations, mGlu8 at high nanomolar concentrations, and mGlu4, mGlu5, and mGlu7 in the micromolar range [183]. In addition to orthosteric ligands that directly interact with the glutamate recognition site, allosteric modulators that bind within the TM domain have been described. Negative allosteric modulators are listed separately. The positive allosteric modulators most often act as ‘potentiators’ of an orthosteric agonist response, without significantly activating the receptor in the absence of agonist

Activation of AMPK-Regulated CRH Neurons in the PVH is Sufficient and Necessary to Induce Dietary Preference for Carbohydrate over Fat

Food selection is essential for metabolic homeostasis and is influenced by nutritional state, food palatability, and social factors such as stress. However, the mechanism responsible for selection between a high-carbohydrate diet (HCD) and a high-fat diet (HFD) remains unknown. Here, we show that activation of a subset of corticotropin-releasing hormone (CRH)-positive neurons in the rostral region of the paraventricular hypothalamus (PVH) induces selection of an HCD over an HFD in mice during refeeding after fasting, resulting in a rapid recovery from the change in ketone metabolism. These neurons manifest activation of AMP-activated protein kinase (AMPK) during food deprivation, and this activation is necessary and sufficient for selection of an HCD over an HFD. Furthermore, this effect is mediated by carnitine palmitoyltransferase 1c (CPT1c). Thus, our results identify the specific neurons and intracellular signaling pathway responsible for regulation of the complex behavior of selection between an HCD and an HFD

Metabotropic glutamate receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Metabotropic glutamate (mGlu) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Metabotropic Glutamate Receptors [334]) are a family of G protein-coupled receptors activated by the neurotransmitter glutamate. The mGlu family is composed of eight members (named mGlu1 to mGlu8) which are divided in three groups based on similarities of agonist pharmacology, primary sequence and G protein coupling to effector: Group-I (mGlu1 and mGlu5), Group-II (mGlu2 and mGlu3) and Group-III (mGlu4, mGlu6, mGlu7 and mGlu8) (see Further reading).Structurally, mGlu are composed of three juxtaposed domains: a core G protein-activating seven-transmembrane domain (TM), common to all GPCRs, is linked via a rigid cysteine-rich domain (CRD) to the Venus Flytrap domain (VFTD), a large bi-lobed extracellular domain where glutamate binds. The structures of the VFTD of mGlu1, mGlu2, mGlu3, mGlu5 and mGlu7 have been solved [190, 262, 255, 386]. The structure of the 7 transmembrane (TM) domains of both mGlu1 and mGlu5 have been solved, and confirm a general helical organization similar to that of other GPCRs, although the helices appear more compacted [85, 415, 59]. mGlu form constitutive dimers crosslinked by a disulfide bridge. Recent studies revealed the possible formation of heterodimers between either group-I receptors, or within and between group-II and -III receptors [86]. Although well characterized in transfected cells, co-localization and specific pharmacological properties also suggest the existence of such heterodimers in the brain [422, 257]. The endogenous ligands of mGlu are L-glutamic acid, L-serine-O-phosphate, N-acetylaspartylglutamate (NAAG) and L-cysteine sulphinic acid. Group-I mGlu receptors may be activated by 3,5-DHPG and (S)-3HPG [29] and antagonized by (S)-hexylhomoibotenic acid [223]. Group-II mGlu receptors may be activated by LY389795 [256], LY379268 [256], eglumegad [337, 416], DCG-IV and (2R,3R)-APDC [338], and antagonised by eGlu [161] and LY307452 [408, 100]. Group-III mGlu receptors may be activated by L-AP4 and (R,S)-4-PPG [125]. An example of an antagonist selective for mGlu receptors is LY341495, which blocks mGlu2 and mGlu3 at low nanomolar concentrations, mGlu8 at high nanomolar concentrations, and mGlu4, mGlu5, and mGlu7 in the micromolar range [176]. In addition to orthosteric ligands that directly interact with the glutamate recognition site, allosteric modulators that bind within the TM domain have been described. Negative allosteric modulators are listed separately. The positive allosteric modulators most often act as 'potentiators' of an orthosteric agonist response, without significantly activating the receptor in the absence of agonist

Gi/o-coupled muscarinic receptors co-localize with GIRK channel for efficient channel activation.

G protein-gated inwardly rectifying K+ (GIRK) channel regulates cellular excitability upon activation of Gi/o-coupled receptors. In Gi/o-coupled muscarinic M2R, the intracellular third loop (i3) is known as a key domain for Gi/o coupling, because replacement of i3 of Gq-coupled muscarinic M1R with that of M2R enables the chimeric receptor (MC9) to activate the GIRK channel. In the present study, we showed that MC9, but not M1R, co-localizes with the GIRK channel and Gαi1 by Förster resonance energy transfer (FRET) analysis. When M1R was forced to stay adjacent to the channel through ligation with short linkers, M1R activated the GIRK channel. FRET analysis further suggested that the efficacy of channel activation is correlated with the linker length between M1R and the GIRK channel. The results show that co-localization is an important factor for activating the GIRK channel. In contrast, for MC9 and M2R, the GIRK channel was activated even when they were connected by long linkers, suggesting the formation of a molecular complex even in the absence of a linker. We also observed that replacement of 13 amino acid residues at the N-terminal end of i3 of MC9 with those of M1R impaired the co-localization with the GIRK channel as well as channel activation. These results show that localization of the receptor near the GIRK channel is a key factor in efficiently activating the channel and that the N-terminal end of i3 of M2R plays an important role in co-localization

Regulation of the two-pore domain potassium channel, THIK-1 and THIK-2, by G protein coupled receptors.

A member of THIK (two pore domain halothane-inhibited K+) channels, THIK-1, was reported as a target of Gi/o-coupled receptors (Gi/o-Rs) in neurons and microglia. We confirmed that in HEK293T cells the THIK-1 channel is activated by Gi/o-Rs and found that Gq-coupled receptors (Gq-Rs) also activates the channel. The effects of Gi/o-Rs and Gq-Rs were inhibited by the Gi/o inhibitor pertussis toxin and phospholipase C (PLC) inhibitor, respectively. The effects of Gi/o-Rs were attenuated when consensus Gβγ binding motif at the C-tail of the THIK-1 channel was mutated, suggesting that Gβγ serves as a THIK-1 channel activator upon the stimulation of Gi/o-Rs. As to the effects of Gq-Rs on the THIK-1 channel, a protein kinase C inhibitor and calcium chelators failed to inhibit the effect of a Gq coupled muscarinic M1R. Neither the hydrolysis of phosphatidyl inositol bisphosphate induced by voltage sensitive phosphatase nor the application of a diacylglycerol analogue, OAG, increased the channel current. The mediator of Gq-dependent activation of the THIK-1 channel remained unsolved. The effects of Gi/o- and Gq-Rs on the THIK-2 channel were also investigated, by using a THIK-2 mutant channel whose N-terminal domain is deleted to improve the surface membrane expression. We observed that Gi/o- and Gq-Rs activate the mutated THIK-2 channel, similarly to the THIK-1 channel. Interestingly, heterodimeric channels of THIK-1 and THIK-2 responded to Gi/o-R and Gq-R stimulation. Taken together, Gi/o- or Gq-Rs activates the THIK-1 and THIK-2 channels in a Gβγ or PLC dependent manner, respectively

Regulation of the two-pore domain potassium channel, THIK-1 and THIK-2, by G protein coupled receptors

A member of THIK (two pore domain halothane-inhibited K+) channels, THIK-1, was reported as a target of Gi/o-coupled receptors (Gi/o-Rs) in neurons and microglia. We confirmed that in HEK293T cells the THIK-1 channel is activated by Gi/o-Rs and found that Gq-coupled receptors (Gq-Rs) also activates the channel. The effects of Gi/o-Rs and Gq-Rs were inhibited by the Gi/o inhibitor pertussis toxin and phospholipase C (PLC) inhibitor, respectively. The effects of Gi/o-Rs were attenuated when consensus Gβγ binding motif at the C-tail of the THIK-1 channel was mutated, suggesting that Gβγ serves as a THIK-1 channel activator upon the stimulation of Gi/o-Rs. As to the effects of Gq-Rs on the THIK-1 channel, a protein kinase C inhibitor and calcium chelators failed to inhibit the effect of a Gq coupled muscarinic M1R. Neither the hydrolysis of phosphatidyl inositol bisphosphate induced by voltage sensitive phosphatase nor the application of a diacylglycerol analogue, OAG, increased the channel current. The mediator of Gq-dependent activation of the THIK-1 channel remained unsolved. The effects of Gi/o- and Gq-Rs on the THIK-2 channel were also investigated, by using a THIK-2 mutant channel whose N-terminal domain is deleted to improve the surface membrane expression. We observed that Gi/o- and Gq-Rs activate the mutated THIK-2 channel, similarly to the THIK-1 channel. Interestingly, heterodimeric channels of THIK-1 and THIK-2 responded to Gi/o-R and Gq-R stimulation. Taken together, Gi/o- or Gq-Rs activates the THIK-1 and THIK-2 channels in a Gβγ or PLC dependent manner, respectively