Abstract
Explorations into the α6-containing nicotinic acetylcholine receptors (α6* nAChRs) as putative drug targets have been severely hampered by the inefficient functional expression of the receptors in heterologous expression systems. In this study, the molecular basis for the problem was investigated through the construction of chimeric α6/α3 and mutant α3 and α6 subunits and functional characterization of these co-expressed with β4 or β4β3 subunits in tsA201 cells in a fluorescence-based assay and in Xenopus oocytes using two-electrode voltage clamp electrophysiology. Substitution of a small C-terminal segment in the second intracellular loop or the Phe(223) residue in transmembrane helix 1 of α6 with the corresponding α3 segment or residue was found to enhance α6β4 functionality in tsA201 cells significantly, in part due to increased cell surface expression of the receptors. The gain-of-function effects of these substitutions appeared to be additive since incorporation of both α3 elements into α6 resulted in assembly of α6β4* receptors exhibiting robust functional responses to acetylcholine. The pharmacological properties exhibited by α6β4β3 receptors comprising one of these novel α6/α3 chimeras in oocytes were found to be in good agreement with those from previous studies of α6* nAChRs formed from other surrogate α6 subunits or concatenated subunits and studies of other heteromeric nAChRs. In contrast, co-expression of this α6/α3 chimera with β2 or β2β3 subunits in oocytes did not result in efficient formation of functional receptors, indicating that the identified molecular elements in α6 could be specific impediments for the expression of functional α6β4* nAChRs.
Highlights
Inefficient functional receptor expression in heterologous expression systems has hampered investigations of ␣6* nicotinic acetylcholine (ACh)2 receptors (nAChRs)
Expression of functional ␣623 and ␣6␣423 nAChRs in oocytes has recently been accomplished by linking subunits in pentameric constructs; this concatemerization somehow makes up for the absence of whatever cellular factors that enables the formation of functional wild type (WT) receptors in neurons [26]
In concordance with the literature [22, 24], ACh was found to elicit a robust functional response in tsA201 cells expressing the WT ␣34 nAChR in the FLIPR Membrane PotentialTM Blue (FMP) assay, whereas no significant response could be detected in WT ␣64-expressing cells (Tables 1 and 2 and Figs. 3 and 4)
Summary
Inefficient functional receptor expression in heterologous expression systems has hampered investigations of ␣6* nAChRs. Expression of functional ␣623 and ␣6␣423 nAChRs in oocytes has recently been accomplished by linking subunits in pentameric constructs; this concatemerization somehow makes up for the absence of whatever cellular factors that enables the formation of functional wild type (WT) receptors in neurons [26]. We further investigated the molecular determinants underlying the difficulties connected with in vitro expression of functional ␣6* nAChRs. A considerable number of novel ␣6/␣3 chimeras and several ␣6 and ␣3 mutants were constructed, and the functional properties of the receptors assembled from these subunits and various  subunits in mammalian cells and Xenopus oocytes were characterized. Two molecular elements in the ␣6 subunit were identified as important determinants, or rather impediments, of the expression of functional ␣64* nAChRs in heterologous expression systems
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