Received for publication November 8, 2004.
Revised January 11, 2005.
Accepted for publication January 11, 2005.

Asparagine, valine, and threonine in the third extracellular loop of muscarinic receptor have essential role in the positive cooperativity of strychnine-like allosteric modulators

Abstract

We have investigated allosteric interactions of four closely related strychnine-like substances: Wieland Gumlich Aldehyde (WGA), propargyl Wieland Gumlich Aldehyde, strychnine and brucine with N-methylscopolamine (NMS) on M₃ subtype of muscarinic receptor genetically modified in the second or the third extracellular loop to corresponding loops of M₂ subtype (M₃o2 and M₃o3 chimera). The M₃o2 chimeric receptor exhibited no change in either affinity of strychnine, brucine, and WGA, or cooperativity of brucine or WGA while both parameters for propargyl-WGA changed. In contrast, there was a change in affinity of all tested modulators (except for brucine) and in their cooperativity in the M₃o3 chimera. Directions of affinity changes in both chimeras were always towards values of the donor M₂ subtype but changes in cooperativity were variable. Compared to the native M₃ receptor, strychnine displayed a slight increase in positive cooperativity and propargyl-WGA a robust decrease in negative cooperativity at M₃o2 chimera. Similar changes were found in the M₃o3 chimera. Interestingly, cooperativity of brucine and WGA at the M₃o3 chimera changed from negative to positive. This is the first evidence of constitution of positive cooperativity of WGA by switching sequences of two parental receptors, both exhibiting negative cooperativity. Gradual replacement of individual amino acids revealed that only three residues (NVT of the o3 loop of the M₂ receptor) are involved in this effect. Data suggest that these amino acids are essential for propagation of a conformation change resulting in positive cooperativity induced by these modulators.

Key words: acetylcholine, allosteric, modulation, muscarinic, receptors, strychnine

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