Role of the Cytoplasmatic M3-M4 Loop for the Homopentameric Assembly of a Chimeric Nicotinic Alpha 7 Receptor

Abstract

It is long known that the nicotinic acetylcholine receptor α7 (CHRNA7R) expresses weakly as a functional acetylcholine-gated ion channel in many cells. Using the blue native PAGE (BN-PAGE) technique for visualization of oligomers, we have previously shown that singly expressed CHRNA7R subunits assemble in X. laevis oocytes to lower order homooligomers, mostly tetramers; only a small fraction of the synthesized CHRNA7R subunits acquire the fully assembled homopentameric state, in which they can escape the endoplasmic reticulum to be routed to the plasma membrane (Nicke et al, FEBS Lett 575, 52-58. 2004). Here we show that a previously described chimeric receptor (Eiselé et al, Nature 366, 479-483, 1993) comprising the extracellular domain of the CHRNA7R fused to the transmembrane spanning and intracellular domains of the 5HT3A receptor (designated rCHRNA7V2015HT3AR chimera) assembled as efficiently to homopentamers as the 5HT3AR. The large cytoplasmatic M3-M4 loop of the 5HT3AR has previously been identified to represent an oligomerization domain that assembles into stable homopentamers when expressed as a soluble protein without flanking transmembrane helices (Pandhare et al, Sci. Rep. 1-9, 2016). To define the 5HT3AR sequence needed for efficient homopentameric assembly, we systematically varied the sequence encoding the M3-M4 loop of the rCHRNA7V2015HT3AR chimera from 5HT3A to CHRNA7R. We analyzed the resulting chimeras by BN-PAGE, SDS-PAGE and two-electrode voltage-clamp electrophysiology. We found that assembly of functional homopentameric receptors was largely abolished when the CHRNA7R sequence was present into the proximal half of the M3-M4 loop. By a detailed mapping of the first half of the M3-M4 loop we identified a sequence of six amino acids being indispensable for efficient subunit assembly. We will interpret these results on the basis of known 3D structures of ligand-gated ion channels.