Influence of the M3-M4 intracellular domain upon nicotinic acetylcholine receptor assembly, targeting and function.

ABSTRACT

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY 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 AND IMPLICATIONS 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.