alpha7 at the single-channel level

BOUZAT C

Conferencia; Nicotinic acetylcholine receptors 2017; 2017

The nicotinic receptor triannual meeting

α7 Nicotinic Receptors at the Single-Channel LevelCecilia Bouzat11Instituto de Investigaciones Bioquímicas de Bahía Blanca, INIBIBB (CONICET-UNS), Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, Camino La Carrindanga km 7, 8000 Bahía Blanca, Argentina.The α7 nicotinic receptor (nAChR) is expressed in brain and in non-neuronal cells. It is emerging as a drug target for cognitive deficits and inflammatory processes. We have focused on understanding α7 function at the single-channel level. We identified novel positive allosteric modulators (PAMs) and analyzed the activity of type I PAMs, which enhance macroscopic currents, and type II PAMs, which also decrease current decay rates. Regardless of their macroscopic behavior, all PAMs enhance open-channel lifetime and induce episodes of successive openings, indicating that both types affect α7 kinetics. We determined the temperature sensitivity and structural determinants for potentiation. To establish functional stoichiometry, we used our electrical fingerprinting strategy in which an α7 subunit is tagged with a mutation that decreases single-channel current amplitude to undetectable levels (α7LC). With this strategy, we analyzed function of heteromeric receptors composed of α7 and dupα7 or β2. The subunit dupα7, which is the product of a chimeric gene formed as a partial duplication of the α7 gene, lacks part of the ACh binding site. We found that dupα7 cannot form functional channels. However, co-expression of α7LC with dupα7 allows detection of single-channel openings, indicating the presence of functional heteromeric receptors. The analysis of single-channel current amplitudes shows that at least two α7 subunits are required for functional receptors. Heteromeric α7β2 receptors have been detected in brain but their function remains unknown. By co-expressing α7LC with β2 we detected the presence of α7β2 receptor channels. Single-channel amplitude analysis reveals receptors with three different stoichiometries, containing one, two or three β2 subunits and requiring the α7/α7 interface for activation. To control stoichiometry, α7 and β2 subunits were concatenated by synthetic linkers into pentameric constructs. The increasing number of β2 subunits in the concatamer produces a progressive increase in open-channel lifetime and burst duration, indicating that β2 contributes to channel kinetics. Overall, by examining function from a different perspective, our results provide foundations for understanding cholinergic signaling mediated by α7.