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Abstract
Binding of agonists to nicotinic acetylcholine receptors generates a sequence of conformational changes resulting in channel opening. Previously, we have shown that the aspartate residue Asp-266 at the M2-M3 linker of the alpha7 nicotinic receptor is involved in connecting binding and gating. High resolution structural data suggest that this region could interact with the so-called loops 2 and 7 of the extracellular N-terminal region. In this case, certain charged amino acids present in these loops could integrate together with Asp-266 and other amino acids, a mechanism involved in channel activation. To test this hypothesis, all charged residues in these loops, Asp-42, Asp-44, Glu-45, Lys-46, Asp-128, Arg-130, and Asp-135, were substituted with other amino acids, and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Glu-45, Lys-46, and Asp-135 exhibited poor or null functional responses to different nicotinic agonists regardless of significant membrane expression, whereas D128A showed a gain of function effect. Because the double reverse charge mutant K46D/D266K did not restore receptor function, a gating mechanism controlled by the pairwise electrostatic interaction between these residues is not likely. Rather, a network of interactions formed by residues Lys-46, Asp-128, Asp-135, Asp-266, and possibly others appears to link agonist binding to channel gating.
Highlights
The nicotinic acetylcholine receptor1 is a member of the superfamily of ligand-gated ion channels that mediates fast synaptic transmission in nerve and muscle cells [1, 2]
We report here that mutations of amino acids Glu-45 and Lys-46 and Asp-135 of the ␣7 subunit caused dramatic reductions in the currents evoked by different agonists, whereas some mutants at Asp-128 showed the opposite effect suggesting that these residues play an important role in the transduction of agonist binding into channel activation
A Charged to Alanine Scanning Mutagenesis Study of Loops 2 and 7—As an initial approach to determine which of the charged residues present in loops 2 and 7 of the nicotinic acetylcholine receptor (nAChR) ␣7 subunit play a role in the mechanism of channel activation, an Ala-scanning mutagenesis study was carried out
Summary
The nicotinic acetylcholine receptor (nAChR) is a member of the superfamily of ligand-gated ion channels that mediates fast synaptic transmission in nerve and muscle cells [1, 2]. Charged amino acids located in the extracellular M2-M3 linker have been reported to affect coupling in glycinergic (GlyR) [4, 5], ␥-aminobutyric acid (GABAAR) [6, 7], and nicotinic receptors (nAChRs) [8, 9]. Kash et al [12] have demonstrated the relevance for channel gating of a direct electrostatic interaction between the positively charged Lys-279 residue in the M2-M3 linker of the GABAAR and negatively charged residues of loops 2 and 7. We report here that mutations of amino acids Glu-45 and Lys-46 (loop 2) and Asp-135 (loop 7) of the ␣7 subunit caused dramatic reductions in the currents evoked by different agonists, whereas some mutants at Asp-128 showed the opposite effect suggesting that these residues play an important role in the transduction of agonist binding into channel activation
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