The nicotine metabolite, cotinine, alters the assembly and trafficking of a subset of nicotinic acetylcholine receptors.
Exposure to nicotine alters the trafficking and assembly of nicotinic receptors (nAChRs), leading to their up-regulation on the plasma membrane. Although the mechanism is not fully understood, nicotine-induced up-regulation is believed to contribute to nicotine addiction. The effect of cotinine, the primary metabolite of nicotine, on nAChR trafficking and assembly has not been extensively investigated. We utilize a pH-sensitive variant of GFP, super ecliptic pHluorin, to differentiate between intracellular nAChRs and those expressed on the plasma membrane to quantify changes resulting from cotinine and nicotine exposure. Similar to nicotine, exposure to cotinine increases the number of α4β2 receptors on the plasma membrane and causes a redistribution of intracellular receptors. In contrast to this, cotinine exposure down-regulates α6β2β3 receptors. We also used single molecule fluorescence studies to show that cotinine and nicotine both alter the assembly of α4β2 receptors to favor the high sensitivity (α4)2(β2)3 stoichiometry.
From the Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506.
Ashley M
AM
Fox
From the Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506.
Faruk H
FH
Moonschi
From the Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506 chris.richards@uky.edu.
Christopher I
CI
Richards
D000818:Animals; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003367:Cotinine / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008856:Microscopy, Fluorescence; D009538:Nicotine / Q000737:chemistry; D021122:Protein Subunits / Q000737:chemistry; D021381:Protein Transport; D011978:Receptors, Nicotinic / Q000737:chemistry*; D014029:Tobacco Use Disorder / Q000235:genetics; D015854:Up-Regulation
D021122:Protein Subunits; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; D049452:Green Fluorescent Proteins; D009538:Nicotine; D003367:Cotinine
cell surface receptor; cotinine; fluorescence; fluorescence microscopy; in vivo imaging; membrane protein; membrane trafficking; microscopy; nicotine; nicotinic acetylcholine receptors (nAChR); single-molecule biophysics; up-regulation