Allosterically Potentiated α7 Nicotinic Acetylcholine Receptors: Reduced Calcium Permeability and Current-Independent Control of Intracellular Calcium

Abstract

The currents of a7 nicotinic acetylcholine receptors activated by acetylcholine (ACh) are brief. The channel has high permeability to calcium relative to monovalent cations and shows inward rectification. It has been previously noted that in the presence of positive allosteric modulators (PAMs), currents through the channels of a7 receptors differ from normal a7 currents both in sensitivity to specific channel blockers and their current-voltage (I-V) relationships, no longer showing inward rectification. Linear I-V functions are often associated with channels lacking calcium permeability, so we measured the I-V functions of a7 receptors activated by ACh when PAMs were bound to the allosteric binding site in the transmembrane domain. Currents were recorded in chloride-free Ringer's solution with low or high concentrations of extracellular calcium to determine the magnitude of the reversal potential shift in the two conditions as well as the I-V relationships. ACh-evoked currents potentiated by the ago-PAMs GAT107 and B-973B showed reduced inward rectification and calcium-dependent reversal potential shifts decreased by 80%, and 50%, respectively compared to currents activated by ACh alone, indicative of reduced calcium permeability. TQS-potentiated currents were also linear and showed no calciumdependent reversal potential shifts. The ago-PAMs GAT-107 and B-973B stimulated increases in intracellular calcium in stably transfected HEK293 cells. However, these calcium signals were delayed relative to channel activation produced by these agents and were insensitive to the channel blocker mecamylamine. Our results indicate that, although allosterically-activated a7 nAChR may affect intracellular calcium levels, such effects are not likely due to large channel-dependent calcium influx.

Therapeutic Targeting of α7 Nicotinic Acetylcholine Receptors

“…Because of its special properties discussed above, a7 nAChRs remain difficult to study with high-throughput cell-based assays, which has often led to compromised approaches, such as the use of nondesensitizing a7-5HT3 chimeric receptors (Craig et al, 2004;O'Donnell et al, 2010) or by amplifying responses with an allosteric modulator (Arunrungvichian et al, 2015;Kaczanowska et al, 2017). However, both of these approaches yield receptors with properties atypical of native a7 receptors activated by ACh (Dinklo et al, 2006;Gee et al, 2007;Miller et al, 2020;Papke and Lindstrom, 2020). Likewise, high-throughput Fluorescent Imaging Plate Reader (FLIPR) assays (Dunlop et al, 2007), which rely on calcium signals (Skidmore et al, 2012;Zanaletti et al, 2012b;Hill et al, 2016;Iwuagwu et al, 2017), are most likely reporting downstream signaling and not ion-channel currents (King et al, 2018;Miller et al, 2020) and may suggest a significantly higher potency than what may be obtained with traditional electrophysiological methods (Haydar et al, 2009).…”

“…However, both of these approaches yield receptors with properties atypical of native a7 receptors activated by ACh (Dinklo et al, 2006;Gee et al, 2007;Miller et al, 2020;Papke and Lindstrom, 2020). Likewise, high-throughput Fluorescent Imaging Plate Reader (FLIPR) assays (Dunlop et al, 2007), which rely on calcium signals (Skidmore et al, 2012;Zanaletti et al, 2012b;Hill et al, 2016;Iwuagwu et al, 2017), are most likely reporting downstream signaling and not ion-channel currents (King et al, 2018;Miller et al, 2020) and may suggest a significantly higher potency than what may be obtained with traditional electrophysiological methods (Haydar et al, 2009). Because of these limitations, many of both older studies (Horenstein et al, 2008) and more recent work (Tietje et al, 2008;Malysz et al, 2010;Marrero et al, 2010;Prickaerts et al, 2012;Yamauchi et al, 2012;Zanaletti et al, 2012a;Feuerbach et al, 2015;Tang et al, 2015) identifying a7-selective agonists rely on receptors expressed in Xenopus oocytes.…”

“…With a relatively large potentiating ligand bound within one or more of the transmembrane domains, it is perhaps not surprising that the ion conduction pathways that form in PAM-potentiated receptors are qualitatively different from the channels formed when the receptor is activated by ACh alone. Channels activated by ACh have relatively high calcium permeability and inward rectifying current-voltage relations, which are features that are not typical of PAM-potentiated currents (Sitzia et al, 2011;Miller et al, 2020). Specific PAMs may each generate their own unique conduction pathway (Miller et al, 2020), a differing set of full and subconductance states, and varying sensitivity to channel-blocking antagonists (Quadri et al, 2019).…”

Homomeric and Heteromeric α7 Nicotinic Acetylcholine Receptors in Health and Some Central Nervous System Diseases

“…Cytotoxicity leading to cell death was observed upon overactivation of α7 nAChRs in neuroblastoma cells treated with the PAM II drug PNU120596 [ 90 ]. ACh-evoked currents potentiated by GAT107 or B-973B showed reduced calcium permeability, although intracellular calcium was observed to rise [ 91 ]. Silent agonists can overcome this reduced calcium permeability under conditions that do not require ion flux for signaling [ 92 ].…”

Stable desensitization of α7 nicotinic acetylcholine receptors by NS6740 requires interaction with S36 in the orthosteric agonist binding site

“…Whereas the responses of wild-type  responses in the absence of allosteric modulators are strongly inward rectifying (Miller et al, 2020;Seguela et al, 1993), a feature that prevented us from measuring the effects of voltage directly on responses to NS6740 alone, currents potentiated by modulators like PNU-120596 have linear current-voltage relationships (Miller et al, 2020;Peng et al, 2013;Sitzia et al, 2011). Therefore, we were able to directly compare the response of both wild-type  (Fig.…”