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- W2005621748 abstract "The molluscan acetylcholine-binding protein (AChBP) is a soluble homopentameric homolog of the extracellular domain of various ligand-gated ion channels. Previous studies have reported that AChBP, when fused to the ion pore domain of the serotonin receptor (5HT3AR), can form a functional ligand-gated chimeric channel only if the AChBP loop regions between β-strands β1 and β2 (β1–β2), β6 and β7 (β6–β7), and β8 and β9 (β8–β9) are replaced with those of the 5HT3AR. To investigate further the potential interactions among these three important loop regions in a membrane- and detergent-free system, we designed AChBP constructs in which loops β1–β2, β6–β7, and β8–β9 of the AChBP were individually and combinatorially substituted in all permutations with the analogous loops of the 5HT3AR. These chimeras were expressed as secreted proteins using the Pichia pastoris yeast expression system. [125I]-α-Bungarotoxin-binding was detected in the culture media obtained from homologous recombinant clones expressing the wild-type AChBP, the β1–β2 loop-only chimera, and the chimera containing all three 5HT3AR loop substitutions. The remaining chimeras failed to show [125I]-α-bungarotoxin binding, and further analysis of cellular extracts allowed us to determine that these binding-negative chimeric constructs accumulated intracellularly and were not secreted into the culture medium. Our results demonstrate that coordinated interactions among loops β1–β2, β6–β7, and β8–β9 are essential for the formation of a functional ligand-binding site, as evidenced by [125I]-α-bungarotoxin-binding, and for efficient protein secretion. In addition, the constructs described here demonstrate the feasibility of utilizing soluble scaffolds to explore functionally important interactions within the extracellular domain of membrane-bound proteins." @default.
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- W2005621748 date "2009-10-01" @default.
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- W2005621748 title "Effect of homologous serotonin receptor loop substitutions on the heterologous expression in Pichia of a chimeric acetylcholine-binding protein with α-bungarotoxin-binding activity" @default.
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- W2005621748 doi "https://doi.org/10.1016/j.pep.2009.05.001" @default.
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