Heterologous expression of human {alpha}6{beta}4{beta}3{alpha}5 nicotinic acetylcholine receptors: binding properties consistent with their natural expression require quaternary subunit assembly including the {alpha}5 subunit.
Heterologous expression and lesioning studies were conducted to identify possible subunit assembly partners in nicotinic acetylcholine receptors (nAChR) containing alpha6 subunits (alpha6(*) nAChR). SH-EP1 human epithelial cells were transfected with the requisite subunits to achieve stable expression of human alpha6beta2, alpha6beta4, alpha6beta2beta3, alpha6beta4beta3, or alpha6beta4beta3alpha5 nAChR. Cells expressing subunits needed to form alpha6beta4beta3alpha5 nAChR exhibited saturable [(3)H]epibatidine binding (K(d) = 95.9 +/- 8.3 pM and B(max) = 84.5 +/- 1.6 fmol/mg of protein). The rank order of binding competition potency (K(i)) for prototypical nicotinic compounds was alpha-conotoxin MII (6 nM) > nicotine (156 nM) approximately methyllycaconitine (200 nM) > alpha-bungarotoxin (>10 microM), similar to that for nAChR in dopamine neurons displaying a distinctive pharmacology. 6-Hydroxydopamine lesioning studies indicated that beta3 and alpha5 subunits are likely partners of the alpha6 subunits in nAChR expressed in dopaminergic cell bodies. Similar to findings in rodents, quantitative real-time reverse transcription-polymerase chain reactions of human brain indicated that alpha6 subunit mRNA expression was 13-fold higher in the substantia nigra than in the cortex or the rest of the brain. Thus, heterologous expression studies suggest that the human alpha5 subunit makes a critical contribution to alpha6beta4beta3alpha5 nAChR assembly into a ligand-binding form with native alpha6(*)-nAChR-like pharmacology and of potential physiological and pathophysiological relevance.
Grinevich
Vladimir P
VP
Targacept, Inc., 200 East First Street, Suite 300, Winston-Salem, NC 27101, USA.
Letchworth
Sharon R
SR
Lindenberger
Kari A
KA
Menager
Jean
J
Mary
Veronique
V
Sadieva
Khalima A
KA
Buhlman
Lori M
LM
Bohme
Georg Andrees
GA
Pradier
Laurent
L
Benavides
Jesus
J
Lukas
Ronald J
RJ
Bencherif
Merouane
M
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D011978:Receptors, Nicotinic / Q000096:biosynthesis* / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013329:Structure-Activity Relationship; D013378:Substantia Nigra / Q000187:drug effects / Q000378:metabolism
D018076:DNA, Complementary; D011978:Receptors, Nicotinic; C497723:alpha6beta4beta3alpha5 nicotinic receptor, human; D012313:RNA