Biophysical and pharmacological characterization of α6-containing nicotinic acetylcholine receptors expressed in HEK293 cells

Nicotinic acetylcholine receptors (nAChR's) containing the alpha 6 subunit (alpha 6*) are putative drug targets of relevance to Parkinson's disease and nicotine addiction. However, heterologous expression of alpha 6* receptors has proven challenging which has stifled drug discovery efforts. Here, we investigate potential new avenues for achieving functional alpha 6* receptor expression. Combinations of chimeric and mutated alpha 6, beta 2 and beta 3 subunits were co-expressed in the human HEK293 cell line and receptor expression was assessed using Ca2+-imaging (FLIPRThi) and whole-cell patch-clamp electrophysiology. Transient transfections of a chimeric alpha 6/alpha 3 subunit construct in combination with beta 2 and beta 3(V9's) gave rise to significant acetylcholineevoked whole-cell currents. Increasing the beta(V9'S):beta 2:alpha 6/alpha 3 cDNA ratio, resulted in a significantly higher fraction of cells with robust current levels. Using an excess of wildtype beta 3, significant functional expression of alpha 6/a3 beta 2 beta 3 was also demonstrated. Comparing the acetylcholine concentration-response relationship of alpha 6/alpha 3 beta 2 beta 3(V9'S)to that of alpha 6/alpha 3 beta 2 beta 3 revealed the beta 3 point mutation to result in decreased current decay rate and increased ACh agonist potency. Ca2+-imaging experiments showed preservation of basic alpha 6* receptor pharmacology. Our results establish that alpha 6/alpha 3 beta 2 beta 3(V9'S) replicate several basic features of native alpha 6* receptors but also highlight several caveats associated with using this construct and may therefore provide guidance for future drug hunting efforts. (C) 2013 Elsevier B.V. All rights reserved.