Influence of the M3-M4 intracellular domain upon nicotinic acetylcholine receptor assembly, targeting and function.
OBJECTIVE
The aim of this study was to investigate the influence of the intracellular domain of nicotinic acetylcholine receptor (nAChR) subunits upon receptor assembly, targeting and functional properties.


METHODS
Because most nAChR subunits form functional receptors only as heteromeric complexes, it can be difficult to examine the influence of individual subunits or subunit domains in isolation. A series of subunit chimaeras was constructed which contain the intracellular loop region (located between the M3 and M4 transmembrane domains) from nAChR subunits alpha1-alpha10 or beta1-beta4. All of these chimaeras contain common extracellular and transmembrane domains (from the nAChR alpha7 subunit and the 5-hydroxytryptamine receptor 5-HT(3A) subunit, respectively), thereby facilitating both homomeric receptor assembly and detection with radiolabelled or fluorescent alpha-bungarotoxin.


RESULTS
The nAChR M3-M4 intracellular loop domain had no significant effect upon levels of total subunit protein detected in transfected cells but had a significant influence upon levels of both cell surface and intracellular assembled receptors. Comparisons of functional properties revealed a significant influence of the intracellular loop domain upon both single-channel conductance and receptor desensitization. In addition, studies conducted in polarized epithelial cells demonstrate that the nAChR loop can influence receptor targeting, resulting in either polarized (apical) or non-polarized distribution.


CONCLUSIONS
Evidence has been obtained which demonstrates that the large intracellular loop domain of nAChR subunits can exert a profound influence upon receptor assembly, targeting and ion channel properties.
Kracun
S
S
Department of Pharmacology, University College London, London, UK.
Harkness
P C
PC
Gibb
A J
AJ
Millar
N S
NS
D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism*; D002478:Cells, Cultured; D002678:Chimera / Q000378:metabolism; D004285:Dogs; D016503:Drug Delivery Systems; D004594:Electrophysiology; D006801:Humans; D047468:Immunoprecipitation; D007473:Ion Channels / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D021122:Protein Subunits / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000378:metabolism*; D044406:Receptors, Serotonin, 5-HT3 / Q000378:metabolism; D014162:Transfection
D002038:Bungarotoxins; D007473:Ion Channels; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D044406:Receptors, Serotonin, 5-HT3