Heteromeric Neuronal Nicotinic Acetylcholine Receptors with Mutant β Subunits Acquire Sensitivity to α7-Selective Positive Allosteric Modulators

Abstract

Homomeric a7 nicotinic acetylcholine receptors (nAChR) have an intrinsically low probability of opening that can be overcome by a7-selective positive allosteric modulators (PAMs), which bind at a site involving the second transmembrane domain (TM2). Mutation of a methionine that is unique to a7 at the 159 position of TM2 to leucine, the residue in most other nAChR subunits, largely eliminates the activity of such PAMs. We tested the effect of the reverse mutation (L159M) in heteromeric nAChR receptors containing a4 and b2, which are the nAChR subunits that are most abundant in the brain. Receptors containing these mutations were found to be strongly potentiated by the a7 PAM 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c] quinoline-8-sulfonamide (TQS) but insensitive to the alternative PAM 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3yl)-urea. The presence of the mutation in the b2 subunit was necessary and sufficient for TQS sensitivity. The primary effect of the mutation in the a4 subunit was to reduce responses to acetylcholine applied alone. Sensitivity to TQS required only a single mutant b subunit, regardless of the position of the mutant b subunit within the pentameric complex. Similar results were obtained when b2L159M was coexpressed with a2 or a3 and when the L159M mutation was placed in b4 and coexpressed with a2, a3, or a4. Functional receptors were not observed when b1L159M subunits were coexpressed with other muscle nAChR subunits. The unique structure-activity relationship of PAMs and the a4b2L159M receptor compared with a7 and the availability of high-resolution a4b2 structures may provide new insights into the fundamental mechanisms of nAChR allosteric potentiation. SIGNIFICANCE STATEMENT Heteromeric neuronal nAChRs have a relatively high initial probability of channel activation compared to receptors that are homomers of a7 subunits but are insensitive to PAMs, which greatly increase the open probability of a7 receptors. These features of heteromeric nAChR can be reversed by mutation of a single residue present in all neuronal heteromeric nAChR subunits to the sequence found in a7. Specifically, the mutation of the TM2 159 leucine to methionine in a subunits reduces heteromeric receptor channel activation, while the same mutation in neuronal b subunits allows heteromeric receptors to respond to select a7 PAMs. The results indicate a key role for this residue in the functional differences in the two main classes of neuronal nAChRs.

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

“…Scale bars in Figure 1 of averaged traces reflect the scaling factor relative to the average peak current amplitude of the ACh controls used for the normalization procedures. These plots (Stokes et al, 2019) illustrate the differences in peak currents, net charge, the kinetics of the responses, and the variability throughout the entire time course of the responses.…”

Differing Activity Profiles of the Stereoisomers of 2,3,5,6TMP-TQS, a Putative Silent Allosteric Modulator of α7 nAChR

“…Racemic 2,3,5,6TMP-TQS blocks this activity, but in the course of our experiments, we also noted that 2,3,5,6TMP-TQS could additionally potentiate the responses to low concentrations of ACh, contrary to initial reports. Since we have previously documented large differences in the activity profiles of the stereoisomers of 4BP-TQS and TQS (Stokes et al, 2019), we hypothesized that the two stereoisomers of 2,3,5,6TMP-TQS might discriminate between the AA and PAM binding sites. Our results confirm that only (+)2,3,5,6TMP-TQS is a PAM and that while (-)2,3,5,6TMP-TQS is not a PAM, it is a more potent allosteric antagonist than (+)2,3,5,6TMP-TQS.…”

“…The a7C190A mutant was made as previously described with a C116S double mutation to prevent spurious disulfide bond formation with the free cysteine (Papke et al, 2011). The b2L15'M mutant was prepared as previously described (Stokes et al, 2019) and co-expressed with a b2-a4 concatamer (Zhou et al, 2003) to obtain receptors with a single mutant b subunit in the accessory subunit position, outside the ACh binding sites.…”

“…In order to test the hypothesis that PAM and allosteric antagonist activities of 2,3,5,6TMP-TQS might be associated with specific enantiomers, we separated the (+) and (-) enantiomers and resolved their crystal structures ( Figure 3). Also shown in Figure 3 are the structures of the enantiomers of TQS and 4BP-TQS that we have previously shown to be the active α7 PAMs (Stokes et al, 2019;. All of these compounds have three chiral centers, two that join the cyclopentenyl ring to the quinoline, and a third that connects to the aromatic rings at the base.…”

“…PAMs is within the receptor's transmembrane domains (Young et al, 2008) and requires the presence of a methionine residue that is unique to a7 in the pore-forming second transmembrane domain (Stokes et al, 2019).…”