Expression and functional characterisation of a human chimeric nicotinic receptor with α6β4 properties

Elucidation of Molecular Impediments in the α6 Subunit for in Vitro Expression of Functional α6β4* Nicotinic Acetylcholine Receptors

“…Furthermore, it is the nAChR subunit most homologous to ␣6, making it ideal to use in this study. In concordance with the literature (22,24), ACh was found to elicit a robust functional response in tsA201 cells expressing the WT ␣3␤4 nAChR in the FMP assay, whereas no significant response could be detected in WT ␣6␤4-expressing cells (Tables 1 and 2 and Figs. 3 and 4).…”

“…Electrophysiological Recordings-Electrophysiological recordings were performed using the two-electrode voltage clamp technique on Xenopus oocytes expressing the various receptors using a protocol adapted from previous studies (24,27,32). The oocytes were placed in a recording chamber continuously perfused with a saline solution (115 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 1.8 mM CaCl 2 , 0.1 mM MgCl 2 ).…”

“…As mentioned in the Introduction, ␣6 NTD /␣3 TMD/ICL and ␣6 NTD /␣4 TMD/ICL subunits form functional receptors together with ␤ subunits in heterologous expression systems (22)(23)(24)(25)(26)(27). In contrast, co-expression of ␣3 NTD /␣6 TMD/ICL and ␣4 NTD / ␣6 TMD/ICL chimeras with ␤2 or ␤2␤3 in oocytes does not result in functional receptors, and although the chimeras have been reported to form some functional receptors with ␤4, these are characterized by dramatically impaired functionalities compared with WT ␣3␤4 and ␣4␤4 nAChRs (23,27).…”

“…Several approaches have been applied to overcome this obstacle. First, co-expression of chimeric ␣6/␣3 or ␣6/␣4 subunits (␣6-NTD fused with ␣3-or ␣4-TMD/ICL) with ␤2, ␤2␤3, and ␤4 subunits results in formation of functional receptors in both mammalian cells and oocytes (22)(23)(24)(25)(26)(27). Second, the minute responses observed for ␣6␤2␤3 and ␣6␤4␤3 nAChRs in oocytes have been found to be dramatically enhanced by the introduction of a ␤3 V273S mutant in the receptors (28,29).…”

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

“…The construction of nAChR chimeras containing the C-terminal region of the 5-HT3A subunit has proved to be a powerful experimental technique and one that has been used subsequently with several other nAChR subunits including α1 [169], α4 [119], α8 [170] α9 [171], α10 [171] and β2 [119], as well as with insect nAChR subunits [116]. Other studies have exploited chimeras containing domains from two different nAChR subunits to overcome inefficient functional expression of, for example, the α6 subunit [172][173][174].…”