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The minimum M3-M4 loop length of neurotransmitter-activated pentameric receptors is critical for the structural integrity of cytoplasmic portals.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2013 Jul 26; Vol. 288 (30), pp. 21558-68. Date of Electronic Publication: 2013 Jun 05. - Publication Year :
- 2013
-
Abstract
- The 5-HT3A receptor homology model, based on the partial structure of the nicotinic acetylcholine receptor from Torpedo marmorata, reveals an asymmetric ion channel with five portals framed by adjacent helical amphipathic (HA) stretches within the 114-residue loop between the M3 and M4 membrane-spanning domains. The positive charge of Arg-436, located within the HA stretch, is a rate-limiting determinant of single channel conductance (γ). Further analysis reveals that positive charge and volume of residue 436 are determinants of 5-HT3A receptor inward rectification, exposing an additional role for portals. A structurally unresolved stretch of 85 residues constitutes the bulk of the M3-M4 loop, leaving a >45-Å gap in the model between M3 and the HA stretch. There are no additional structural data for this loop, which is vestigial in bacterial pentameric ligand-gated ion channels and was largely removed for crystallization of the Caenorhabditis elegans glutamate-activated pentameric ligand-gated ion channels. We created 5-HT3A subunit loop truncation mutants, in which sequences framing the putative portals were retained, to determine the minimum number of residues required to maintain their functional integrity. Truncation to between 90 and 75 amino acids produced 5-HT3A receptors with unaltered rectification. Truncation to 70 residues abolished rectification and increased γ. These findings reveal a critical M3-M4 loop length required for functions attributable to cytoplasmic portals. Examination of all 44 subunits of the human neurotransmitter-activated Cys-loop receptors reveals that, despite considerable variability in their sequences and lengths, all M3-M4 loops exceed 70 residues, suggesting a fundamental requirement for portal integrity.
- Subjects :
- Amino Acid Sequence
Animals
Arginine chemistry
Arginine genetics
Arginine physiology
Binding Sites genetics
HEK293 Cells
Humans
Kinetics
Membrane Potentials drug effects
Membrane Potentials genetics
Membrane Potentials physiology
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Mutation
Patch-Clamp Techniques
Protein Multimerization
Receptors, Serotonin, 5-HT3 genetics
Sequence Homology, Amino Acid
Serotonin pharmacology
Serotonin Receptor Agonists pharmacology
Torpedo
Transfection
Protein Conformation
Protein Structure, Secondary
Receptors, Serotonin, 5-HT3 chemistry
Receptors, Serotonin, 5-HT3 physiology
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 288
- Issue :
- 30
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 23740249
- Full Text :
- https://doi.org/10.1074/jbc.M113.481689