Detection and characterization of functional heteromeric receptors composed of the α7 nicotinic subunit and its duplicate form

LASALA M; CORRADI J.; DIONISIO, L.; ESANDI, M.C.; BOUZAT, C.

San Miguel de Tucuman

Congreso; III LAFeBS, IX Iberoamerican Congress of Biophisics, XLV Annual Meeting SAB; 2016

Sociedad Argentina de Biofísica, LatinAmericanFederationofBiophysicalSocieties

The a7 nicotinic receptor subunit gene, CHRNA7, codes for a subunitthat forms the homomeric a7 receptor, which is involved in learningand memory. Exons 5-10 of CHRNA7 were duplicated and fused tothe FAM7A gene. The product of the resulting chimeric gene, dupa7,is a truncated subunit that lacks part of the ACh binding site. We herecombine cell expression and electrophysiological recordings in HEKcells to understand the functional role of the dupa7 subunit. By confocal microscopy and flow cytometry we found that cells transfectedwith dupa7 do not show cell surface binding of a-bungarotoxin, a specific antagonist of a7. The incorporation of dupa7 cDNA during celltransfection with a7 cDNA reduces a-bungarotoxin binding comparedto that determine with a7 alone, indicating a negative modulatory roleof the duplicated subunit. To determine if dupa7 forms functional pentamers, we recorded single-channel and macroscopic currents elicitedby an allosteric agonist that binds to a transmembrane site that is conserved between a7 and dupa7. We found that, in contrast to cells ex-pressing a7, currents are not detected in those transfected with dupa7cDNA. To determine if dupa7 can co-assemble into functional receptors with a7 we used an a7 subunit carrying mutations in determinantsof ion conductance (a7LC) as a reporter of receptor stoichiometry. Although a7LC forms functional receptors, single-channel openings cannot be detected due to their low conductance. Co-expression of a7LCwith dupa7, which by itself does not form functional receptors, allowsdetection of single-channel openings elicited by ACh. This result unequivocally indicates that a7 and dupa7 subunits assemble into functional heteromeric receptors. The analysis shows that a minimum oftwo a7 subunits is required for forming functional receptors. Our results contribute to the understanding of the functional significance ofthe partial duplication of the a7 gene.