Activation of α7 Nicotinic Receptors by Orthosteric and Allosteric Agonists: Influence on Single-Channel Kinetics and Conductance

Abstract

Nicotinic acetylcholine receptors (nAChRs) are oligomeric transmembrane proteins in which five subunits coassemble to form a central ion channel pore. Conventional agonists, such as acetylcholine (ACh), bind to an orthosteric site, located at subunit interfaces in the extracellular domain. More recently, it has been demonstrated that nAChRs can also be activated by ligands binding to an allosteric transmembrane site. In the case of ␣7 nAChRs, ACh causes rapid activation and almost complete desensitization. In contrast, allosteric agonists such as 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c] quin oline-8-sulfonamide (4BP-TQS) activate ␣7 nAChRs more slowly and cause only low levels of apparent desensitization. In the present study, single-channel patch-clamp recording has been used to investigate differences in the mechanism of activation of ␣7 nAChRs by ACh and 4BP-TQS. The most striking difference between activation by ACh and 4BP-TQS is in single-channel kinetics. In comparison with activation by ACh, single-channel open times and burst lengths are substantially longer (ϳ160 -800-fold, respectively), and shut times are shorter (ϳ8-fold) when activated by 4BP-TQS. In addition, coapplication of ACh and 4BP-TQS results in a further increase in single-channel burst lengths. Mean burst lengths seen when the two agonists are coapplied (3099 Ϯ 754 ms) are ϳ2.5-fold longer than with 4BP-TQS alone and ϳ370-fold longer than with ACh alone. Intriguingly, the main single-channel conductance of ␣7 nAChRs, was significantly larger when activated by 4BP-TQS (100.3 Ϯ 2.4 pS) than when activated by ACh (90.0 Ϯ 2.7 pS), providing evidence that activation by allosteric and orthosteric agonists results in different ␣7 nAChRs openchannel conformations.

Molecular function of α7 nicotinic receptors as drug targets

“…In the presence of ACh, PNU-120596 (1-10 μM) gives rise to significantly prolonged openings, grouped in bursts of openings separated by brief closings (200-300 μs), which in turn coalesce into long activation periods, named clusters, that can last for several seconds (daCosta et al 2011(daCosta et al , 2015Palczynska et al 2012;Andersen et al 2016) (Fig. 3).…”

“…Overall, there is consensus that type II PAMs increase the open-channel duration, the number of detectable open states, the burst or cluster duration and the open probability (Hurst et al 2005;daCosta et al 2011daCosta et al , 2015Palczynska et al 2012;Andersen et al 2013). The suggested underlying mechanisms involve the increase in the energetic barrier for desensitization and/or reverse of some forms of desensitized states induced by agonists (Williams et al 2011;Szabo et al 2014;Bouzat & Sine, 2017).…”

Understanding the Bases of Function and Modulation of α7 Nicotinic Receptors: Implications for Drug Discovery

“…This cavity is also the site for allosteric agonists that mediate a7 activation in the absence of an orthosteric agonist (Gill et al, 2011(Gill et al, , 2012(Gill et al, , 2013Pałczy nska et al, 2012). Singlechannel activity of a7 in the presence of allosteric agonists resembles that of the receptor in the presence of ACh and a PAM.…”

“…Singlechannel activity of a7 in the presence of allosteric agonists resembles that of the receptor in the presence of ACh and a PAM. Both are characterized by long bursts instead of isolated brief ACh-elicited openings, indicating activation with significantly reduced desensitization (Pałczy nska et al, 2012).…”

An Unaltered Orthosteric Site and a Network of Long-Range Allosteric Interactions for PNU-120596 in α7 Nicotinic Acetylcholine Receptors

“…However, the high functional resolution of electrophysiological data allows other approaches to this question. For example, the structurally unrelated allosteric modulator 4PB-TQS has been shown to change the kinetics of gating as well as single-channel conductance of α7 nAChRs (Pałczyńska, et al, 2012), indicating that an allosteric modulator can change the structure of the conducting pore.…”