Detection and characterization of functional heteromeric receptors composed of the alpha7 nicotinic subunit and its duplicate form

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

San Miguel de Tucuman

Congreso; IIILatinAmerican Federationof BiophysicalSocieties (LAFeBS) IXIberoAmerican CongressofBiophysics XLVReunionAnual SAB2016; 2016

The α7 nicotinic receptor subunit gene, CHRNA7, codes for a subunit that forms the homomeric α7 receptor, which is involved in learning and memory. Exons 5-10 of CHRNA7 were duplicated and fused to the FAM7A gene. The product of the resulting chimeric gene, dupα7, is a truncated subunit that lacks part of the ACh binding site. We here combine cell expression and electrophysiological recordings in HEK cells to understand the functional role of the dupα7 subunit. By confocal microscopy and flow cytometry we found that cells transfected with dupα7 do not show cell surface binding of α-bungarotoxin, a specific antagonist of α7. The incorporation of dupα7 cDNA during cell transfection with α7 cDNA reduces α-bungarotoxin binding compared to that determine with α7 alone, indicating a negative modulatory role of the duplicated subunit. To determine if dupα7 forms functional pentamers, we recorded single-channel and macroscopic currents elicited by an allosteric agonist that binds to a transmembrane site that is conserved between α7 and dupα7. We found that, in contrast to cells expressing α7, currents are not detected in those transfected with dupα7 cDNA. To determine if dupα7 can co-assemble into functional receptors with α7 we used an α7 subunit carrying mutations in determinants of ion conductance (α7LC) as a reporter of receptor stoichiometry. Although α7LC forms functional receptors, single-channel openings cannot be detected due to their low conductance. Co-expression of α7LC with dupα7, which by itself does not form functional receptors, allows detection of single-channel openings elicited by ACh. This result unequivocally indicates that α7 and dupα7 subunits assemble into functional heteromeric receptors. The analysis shows that a minimum of two α7 subunits is required for forming functional receptors. Our results contribute to the understanding of the functional significance of the partial duplication of the α7 gene.