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Mutagenic analysis of the intracellular portals of the human 5-HT3A receptor.

Carland JE, Cooper MA, Livesey MR, Hales TG, Peters JA, Lambert JJ - J. Biol. Chem. (2013)

Bottom Line: Structural models of Cys-loop receptors based on homology with the Torpedo marmorata nicotinic acetylcholine receptor infer the existence of cytoplasmic portals within the conduction pathway framed by helical amphipathic regions (termed membrane-associated (MA) helices) of adjacent intracellular M3-M4 loops.Numerous residues, prominently those at the 435, 436, 439, and 440 positions, were found to markedly influence γ.This approach yielded a functional map of the 5-HT3A receptor portals, which agrees well with the homology model.

View Article: PubMed Central - PubMed

Affiliation: From the Division of Neuroscience, Medical Research and Medical Education Institutes, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom.

ABSTRACT
Structural models of Cys-loop receptors based on homology with the Torpedo marmorata nicotinic acetylcholine receptor infer the existence of cytoplasmic portals within the conduction pathway framed by helical amphipathic regions (termed membrane-associated (MA) helices) of adjacent intracellular M3-M4 loops. Consistent with these models, two arginine residues (Arg(436) and Arg(440)) within the MA helix of 5-hydroxytryptamine type 3A (5-HT3A) receptors act singularly as rate-limiting determinants of single-channel conductance (γ). However, there is little conservation in primary amino acid sequences across the cytoplasmic loops of Cys-loop receptors, limiting confidence in the fidelity of this particular aspect of the 5-HT3A receptor model. We probed the majority of residues within the MA helix of the human 5-HT3A subunit using alanine- and arginine-scanning mutagenesis and the substituted cysteine accessibility method to determine their relative influences upon γ. Numerous residues, prominently those at the 435, 436, 439, and 440 positions, were found to markedly influence γ. This approach yielded a functional map of the 5-HT3A receptor portals, which agrees well with the homology model.

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The putative structure of the 5-HT3 receptor ion conduction pathway.A, the complete homology model of the 5-HT3A receptor based on the Torpedo nACh receptor structure. The pentameric protein is surface-rendered with foreground subunits made transparent. Arrows highlight the putative conduction pathway, one pointing to the outer vestibule, the others pointing out three of the five cytoplasmic portals. B, five 5-HT3A(QDA) receptor MA helices viewed from above (top panel) and from the cytoplasm (bottom panel). These structures are depicted with both transparent surface rendering and ribbons. The residues substituted by mutagenesis in the current study are indicated in color. Differing colors were used to distinguish each of the five subunits. C, the amino acid numbering is that of the human 5-HT3A subunit (h5-HT3A). The arginine residues that are collectively responsible for the sub-picosiemen single-channel conductance of the human 5-HT3A receptor are boxed together with the homologous residues within the 5-HT3B subunit sequence.
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Figure 1: The putative structure of the 5-HT3 receptor ion conduction pathway.A, the complete homology model of the 5-HT3A receptor based on the Torpedo nACh receptor structure. The pentameric protein is surface-rendered with foreground subunits made transparent. Arrows highlight the putative conduction pathway, one pointing to the outer vestibule, the others pointing out three of the five cytoplasmic portals. B, five 5-HT3A(QDA) receptor MA helices viewed from above (top panel) and from the cytoplasm (bottom panel). These structures are depicted with both transparent surface rendering and ribbons. The residues substituted by mutagenesis in the current study are indicated in color. Differing colors were used to distinguish each of the five subunits. C, the amino acid numbering is that of the human 5-HT3A subunit (h5-HT3A). The arginine residues that are collectively responsible for the sub-picosiemen single-channel conductance of the human 5-HT3A receptor are boxed together with the homologous residues within the 5-HT3B subunit sequence.

Mentions: By comparison with the extracellular and membrane spanning domains, the intracellular M3-M4 loop displays the lowest degree of sequence homology across different subunits of the same receptor family or between receptor families and is predicted to be largely unstructured (16–18). Notably, the large intracellular loop is virtually absent from prokaryotic pLGIC subunits (9), and almost complete deletion of this region, among others, was necessary to optimize crystallization of GLC-1, once more suggesting it to be largely unstructured (10). This region of Cys-loop receptors is an established target for phosphorylation (19, 20), and it exerts important influences on, for example, the assembly, maturation, targeting, and gating kinetics of the nACh receptor (18, 21–26). The M3-M4 loop has also been shown to be a critical determinant of single-channel conductance (γ) (27). Cryo-electron microscopic studies of the nACh receptor of Torpedo marmorata have revealed the presence of a helical amphipathic stretch, referred to as the membrane-associated (MA) helix, at the C-terminal end of the loop (15) (see Fig. 1). Five such helices extend below the ion channel pore, forming a vestibule that is perforated by five narrow openings, or portals (see Fig. 1). Structure-function studies (28–30) demonstrate that specific residues within human 5-HT3A and rat α4β2 nACh receptors positioned within these portals, as inferred by structural models based on homology with the T. marmorata nACh receptor, exert a strong influence on γ that is additional to that of the extensively characterized M2 domain and flanking sequences (31, 32). Furthermore, equivalent residues within MA helices of human α1 glycine receptors also influence γ (33). The same region in the 5-HT3A receptor additionally impacts upon divalent versus monovalent cation permeability, channel gating, and the kinetics of desensitization (34–36). However, ion size selectivity for monovalent cations appears to depend upon the M2 domain (37), and the M3-M4 loop is not essential for receptor function (38).


Mutagenic analysis of the intracellular portals of the human 5-HT3A receptor.

Carland JE, Cooper MA, Livesey MR, Hales TG, Peters JA, Lambert JJ - J. Biol. Chem. (2013)

The putative structure of the 5-HT3 receptor ion conduction pathway.A, the complete homology model of the 5-HT3A receptor based on the Torpedo nACh receptor structure. The pentameric protein is surface-rendered with foreground subunits made transparent. Arrows highlight the putative conduction pathway, one pointing to the outer vestibule, the others pointing out three of the five cytoplasmic portals. B, five 5-HT3A(QDA) receptor MA helices viewed from above (top panel) and from the cytoplasm (bottom panel). These structures are depicted with both transparent surface rendering and ribbons. The residues substituted by mutagenesis in the current study are indicated in color. Differing colors were used to distinguish each of the five subunits. C, the amino acid numbering is that of the human 5-HT3A subunit (h5-HT3A). The arginine residues that are collectively responsible for the sub-picosiemen single-channel conductance of the human 5-HT3A receptor are boxed together with the homologous residues within the 5-HT3B subunit sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814755&req=5

Figure 1: The putative structure of the 5-HT3 receptor ion conduction pathway.A, the complete homology model of the 5-HT3A receptor based on the Torpedo nACh receptor structure. The pentameric protein is surface-rendered with foreground subunits made transparent. Arrows highlight the putative conduction pathway, one pointing to the outer vestibule, the others pointing out three of the five cytoplasmic portals. B, five 5-HT3A(QDA) receptor MA helices viewed from above (top panel) and from the cytoplasm (bottom panel). These structures are depicted with both transparent surface rendering and ribbons. The residues substituted by mutagenesis in the current study are indicated in color. Differing colors were used to distinguish each of the five subunits. C, the amino acid numbering is that of the human 5-HT3A subunit (h5-HT3A). The arginine residues that are collectively responsible for the sub-picosiemen single-channel conductance of the human 5-HT3A receptor are boxed together with the homologous residues within the 5-HT3B subunit sequence.
Mentions: By comparison with the extracellular and membrane spanning domains, the intracellular M3-M4 loop displays the lowest degree of sequence homology across different subunits of the same receptor family or between receptor families and is predicted to be largely unstructured (16–18). Notably, the large intracellular loop is virtually absent from prokaryotic pLGIC subunits (9), and almost complete deletion of this region, among others, was necessary to optimize crystallization of GLC-1, once more suggesting it to be largely unstructured (10). This region of Cys-loop receptors is an established target for phosphorylation (19, 20), and it exerts important influences on, for example, the assembly, maturation, targeting, and gating kinetics of the nACh receptor (18, 21–26). The M3-M4 loop has also been shown to be a critical determinant of single-channel conductance (γ) (27). Cryo-electron microscopic studies of the nACh receptor of Torpedo marmorata have revealed the presence of a helical amphipathic stretch, referred to as the membrane-associated (MA) helix, at the C-terminal end of the loop (15) (see Fig. 1). Five such helices extend below the ion channel pore, forming a vestibule that is perforated by five narrow openings, or portals (see Fig. 1). Structure-function studies (28–30) demonstrate that specific residues within human 5-HT3A and rat α4β2 nACh receptors positioned within these portals, as inferred by structural models based on homology with the T. marmorata nACh receptor, exert a strong influence on γ that is additional to that of the extensively characterized M2 domain and flanking sequences (31, 32). Furthermore, equivalent residues within MA helices of human α1 glycine receptors also influence γ (33). The same region in the 5-HT3A receptor additionally impacts upon divalent versus monovalent cation permeability, channel gating, and the kinetics of desensitization (34–36). However, ion size selectivity for monovalent cations appears to depend upon the M2 domain (37), and the M3-M4 loop is not essential for receptor function (38).

Bottom Line: Structural models of Cys-loop receptors based on homology with the Torpedo marmorata nicotinic acetylcholine receptor infer the existence of cytoplasmic portals within the conduction pathway framed by helical amphipathic regions (termed membrane-associated (MA) helices) of adjacent intracellular M3-M4 loops.Numerous residues, prominently those at the 435, 436, 439, and 440 positions, were found to markedly influence γ.This approach yielded a functional map of the 5-HT3A receptor portals, which agrees well with the homology model.

View Article: PubMed Central - PubMed

Affiliation: From the Division of Neuroscience, Medical Research and Medical Education Institutes, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom.

ABSTRACT
Structural models of Cys-loop receptors based on homology with the Torpedo marmorata nicotinic acetylcholine receptor infer the existence of cytoplasmic portals within the conduction pathway framed by helical amphipathic regions (termed membrane-associated (MA) helices) of adjacent intracellular M3-M4 loops. Consistent with these models, two arginine residues (Arg(436) and Arg(440)) within the MA helix of 5-hydroxytryptamine type 3A (5-HT3A) receptors act singularly as rate-limiting determinants of single-channel conductance (γ). However, there is little conservation in primary amino acid sequences across the cytoplasmic loops of Cys-loop receptors, limiting confidence in the fidelity of this particular aspect of the 5-HT3A receptor model. We probed the majority of residues within the MA helix of the human 5-HT3A subunit using alanine- and arginine-scanning mutagenesis and the substituted cysteine accessibility method to determine their relative influences upon γ. Numerous residues, prominently those at the 435, 436, 439, and 440 positions, were found to markedly influence γ. This approach yielded a functional map of the 5-HT3A receptor portals, which agrees well with the homology model.

Show MeSH