Multiple Roles of the Conserved Key Residue Arginine 209 in Neuronal Nicotinic Receptors

Vicente-Agullo, Francisco; Rovira, José Carlos; Sala, Salvador; Sala, Francisco; Rodríguez-Ferrer, Carmen R.; Campos‐Caro, Antonio; Criado, Manuel; Ballesta, Juan J.

doi:10.1021/bi010087g

Cited by 19 publications

(25 citation statements)

References 21 publications

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“…This functionally important interaction can be restored by the proposed disulfide bond in the Asp-148 3 Cys,Arg-218 3 Cys double mutant. Second, and in agreement with previous studies on the equivalent residue of different nAChRs (38,51) and ␣1 GlyR Arg-218 (26), ␣1 GlyR Arg-218 is important for channel expression. Our data show that in contrast to the functionally relevant salt bridge with Asp-148, which can be mimicked by the proposed disulfide bond, the positive charge of Arg-218 is required for efficient expression, as the Asp-148 3 Cys,Arg-218 3 Cys double mutant, although functionally similar to WT, shows dramatically reduced expression levels ( Table 1).…”

Section: Discussionsupporting

confidence: 90%

“…The idea of an electrostatic interaction between Asp-148 and Arg-218 is further supported by another study that suggested an interaction between the equivalent residues in the ␤2 subunit of GABA A receptors (12). The proposed interaction between Asp-148 and Arg-218 in the ␣1 GlyR is of particular interest as mutations at side chains equivalent to ␣1 GlyR Arg-218 have been shown to be crucial for channel function in different pLGICs (12,15,38,(51)(52)(53) and especially because the ␣1 GlyR Arg-218 3 Gln mutation can give rise to the inherited hyperekplexia or startle disease (26). The idea that Arg-218 contributes to a salt bridge crucial for channel gating in the native receptor provides an explanation for molecular basis for a pathophysiologically relevant mutant; the neutral Arg-218 3 Gln mutation would prevent the formation of a strong salt bridge and, hence, give rise to the drastic effect on gating observed with this mutant.…”

Section: Discussionmentioning

confidence: 86%

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Contributions of Conserved Residues at the Gating Interface of Glycine Receptors

Pless

Leung

Galpin

et al. 2011

Journal of Biological Chemistry

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Glycine receptors (GlyRs) are chloride channels that mediate fast inhibitory neurotransmission and are members of the pentameric ligand-gated ion channel (pLGIC) family. The interface between the ligand binding domain and the transmembrane domain of pLGICs has been proposed to be crucial for channel gating and is lined by a number of charged and aromatic side chains that are highly conserved among different pLGICs. However, little is known about specific interactions between these residues that are likely to be important for gating in ␣1 GlyRs. Here we use the introduction of cysteine pairs and the in vivo nonsense suppression method to incorporate unnatural amino acids to probe the electrostatic and hydrophobic contributions of five highly conserved side chains near the interface, Glu-53, Phe-145, Asp-148, Phe-187, and Arg-218. Our results suggest a salt bridge between Asp-148 in loop 7 and Arg-218 in the pre-M1 domain that is crucial for channel gating. We further propose that Phe-145 and Phe-187 play important roles in stabilizing this interaction by providing a hydrophobic environment. In contrast to the equivalent residues in loop 2 of other pLGICs, the negative charge at Glu-53 ␣1 GlyRs is not crucial for normal channel function. These findings help decipher the GlyR gating pathway and show that distinct residue interaction patterns exist in different pLGICs. Furthermore, a salt bridge between Asp-148 and Arg-218 would provide a possible mechanistic explanation for the pathophysiologically relevant hyperekplexia, or startle disease, mutant Arg-218 3 Gln. The glycine receptor (GlyR)2 chloride channel is a member of the Cys-loop receptor family, a subfamily of the pentameric ligand-gated ion channel (pLGIC) superfamily (1). GlyRs mediate fast inhibitory neurotransmission in the nervous system, and recent studies have provided a wealth of insight into the structure and function of the GlyR and other pLGICs. One of the more studied regions, the N-terminal ligand binding domain (LBD), is composed of a 10-strand ␤-sheet sandwich interconnected by 9 loops and the ligand binding pocket situated at the interface between adjacent subunits (2-8). The transmembrane domain (TMD) contains four ␣-helical segments (M1-M4), including the pore-lining M2 helices.How is the binding of an agonist molecule in the LBD communicated to the channel gate in the TMD, almost 60 Å away? This question has gained considerable attention in the past (9 -11), and previous studies have identified a number of charged residues likely to couple the LBD to the TMD via electrostatic interactions in different pLGICs (12)(13)(14)(15)(16)(17). A number of critical residues have been identified at the interface of LBD and TMD of ␣1 GlyRs (18 -22), but evidence for direct electrostatic interactions is thus far missing. The interface of LBD and TMD in ␣1 GlyRs is of interest not only because of its proposed role in channel gating but also because inherited mutations of side chains near this interface cause hyperekplexia or startle disease (23): Ala-52 ...

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Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 86%

Contributions of Conserved Residues at the Gating Interface of Glycine Receptors

Pless

Leung

Galpin

et al. 2011

Journal of Biological Chemistry

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“…This motif starts at a position equivalent to Pro 207 in V201 and, therefore, is located at a 5-amino acid distance from Ile 202 downward from the C terminus. Moreover, we have previously shown that an arginine (Arg 205 according to the numbering system used here) located between Ile 202 and the latter motif and conserved in all ligand-gated channel subunits is required for transport of assembled ␣7 receptors to the cell surface (31). Also in this region certain domains have been found that affect surface expression of ␣7-5HT 3 chimeric receptors (32).…”

Section: Discussionmentioning

confidence: 97%

Dual Role of the RIC-3 Protein in Trafficking of Serotonin and Nicotinic Acetylcholine Receptors

Castillo

Mulet

Gutiérrez

et al. 2005

Journal of Biological Chemistry

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176

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The ric-3 gene is required for maturation of nicotinic acetylcholine receptors in Caenorhabditis elegans. The human homolog of RIC-3, hRIC-3, enhances expression of ␣7 nicotinic receptors in Xenopus laevis oocytes, whereas it totally abolishes expression of ␣4␤2 nicotinic and 5-HT 3 serotonergic receptors. Both the N-terminal region of hRIC-3, which contains two transmembrane segments, and the C-terminal region are needed for these differential effects. hRIC-3 inhibits receptor expression by hindering export of mature receptors to the cell membrane. By using chimeric proteins made of ␣7 and 5-HT 3 receptors, we have shown that the presence of an extracellular isoleucine close to the first transmembrane receptor fragment is responsible for the transport arrest induced by hRIC-3. Enhancement of ␣7 receptor expression occurs, at least, at two levels: by increasing the number of mature receptors and facilitating its transport to the membrane. Certain amino acids of a putative amphipathic helix present at the large cytoplasmic region of the ␣7 subunit are required for these actions. Therefore, hRIC-3 can act as a specific regulator of receptor expression at different levels.

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“…In particular, ␤ 2 R216 appears to be a key player. This residue is absolutely conserved in every member of the Cys-loop receptor superfamily, and its mutation has been shown to alter agonist-induced channel gating in other Cys-loop receptors (Vicente-Agullo et al, 2001;Hu et al, 2003;Castaldo et al, 2004). A model of the GABA A receptor shows ␤ 2 R216 in close proximity to ␤ 2 E52 (Fig.…”

Section: Discussionmentioning

confidence: 99%

Charged Residues in the α₁and β₂Pre-M1 Regions Involved in GABA_AReceptor Activation

Mercado¹,

Czajkowski²

2006

J. Neurosci.

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For Cys-loop ligand-gated ion channels (LGIC), the protein movements that couple neurotransmitter binding to channel gating are not well known. The pre-M1 region, which links the extracellular agonist-binding domain to the channel-containing transmembrane domain, is in an ideal position to transduce binding site movements to gating movements. A cluster of cationic residues in this region is observed in all LGIC subunits, and in particular, an arginine residue is absolutely conserved. We mutated charged pre-M1 residues in the GABA A receptor ␣ 1 (K219, R220, K221) and ␤ 2 (K213, K215, R216) subunits to cysteine and expressed the mutant subunits with wild-type ␤ 2 or ␣ 1 in Xenopus oocytes. Cysteine substitution of ␤ 2 R216 abolished channel gating by GABA without altering the binding of the GABA agonist [3 H]muscimol, indicating that this residue plays a key role in coupling GABA binding to gating. Tethering thiol-reactive methanethiosulfonate (MTS) reagents onto ␣ 1 K219C, ␤ 2 K213C, and ␤ 2 K215C increased maximal GABA-activated currents, suggesting that structural perturbations of the pre-M1 regions affect channel gating. GABA altered the rates of sulfhydryl modification of ␣ 1 K219C, ␤ 2 K213C, and ␤ 2 K215C, indicating that the pre-M1 regions move in response to channel activation. A positively charged MTS reagent modified ␤ 2 K213C and ␤ 2 K215C significantly faster than a negatively charged reagent, and GABA activation eliminated modification of ␤ 2 K215C by the negatively charged reagent. Overall, the data indicate that the pre-M1 region is part of the structural machinery coupling GABA binding to gating and that the transduction of binding site movements to channel movements is mediated, in part, by electrostatic interactions.

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Multiple Roles of the Conserved Key Residue Arginine 209 in Neuronal Nicotinic Receptors

Cited by 19 publications

References 21 publications

Contributions of Conserved Residues at the Gating Interface of Glycine Receptors

Contributions of Conserved Residues at the Gating Interface of Glycine Receptors

Dual Role of the RIC-3 Protein in Trafficking of Serotonin and Nicotinic Acetylcholine Receptors

Charged Residues in the α₁and β₂Pre-M1 Regions Involved in GABA_AReceptor Activation

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Multiple Roles of the Conserved Key Residue Arginine 209 in Neuronal Nicotinic Receptors

Cited by 19 publications

References 21 publications

Contributions of Conserved Residues at the Gating Interface of Glycine Receptors

Contributions of Conserved Residues at the Gating Interface of Glycine Receptors

Dual Role of the RIC-3 Protein in Trafficking of Serotonin and Nicotinic Acetylcholine Receptors

Charged Residues in the α1and β2Pre-M1 Regions Involved in GABAAReceptor Activation

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Charged Residues in the α₁and β₂Pre-M1 Regions Involved in GABA_AReceptor Activation