There are 55 SNPs causing missense mutations in the coding region of the human α7 nAChR gene in the NCBI SNP database. In this study, we selected and characterized 14 SNPs causing missense mutations in the agonist binding region and the coupling region between the amino-terminal domain and the channel gate in the transmembrane domain. In the oocyte expression system, we demonstrate that 6 out of 14 mutations made the receptors unresponsive to ACh and or nicotine in this expression system. Among remaining 8 mutants, 4 of them had reduced current expression, and one had a dramatic increase in ACh response but a dramatic decrease in nicotine response. Interestingly, some nonfunctional mutations could be rescued by α7 nAChR PAM, PNU-120596 or agonist-PAM, 4BP-TQS. Finally, when nonfunctional mutants coexpressed with the wild type, they could modify the receptor function in expression level or agonist sensitivity, suggesting a potential impact of these 6 SNPs on synaptic transmission even in heterozygous condition.

Impact of the mutations on current level

Among 14 mutants tested, we found 4 of them showed reduced maximal whole-cell current expression and 6 of them were totally nonfunctional in the Xenopus oocyte system. Interestingly, W55G mutation dramatically increased ACh-induced current. Thus, except for two mutants (G212S and G212V) in the extracellular end of M1 domain and one conserved mutant in binding loop C, K192R, all the other mutants in the binding domain and coupling region exhibited different extent of alteration in whole-cell current level. Interestingly, while most of the mutants exhibited similar changes in ACh and nicotine responses, the W55G mutant exhibited an opposite change in ACh and nicotine response. Alteration of whole cell current could be due to a change in the number of channels expressed in the plasma membrane. Alternatively, it may result from the altered channel opening probability (gating efficiency) or changes in single channel conductance. In addition, it is also possible that the change in current level could be due to alteration in receptor channel kinetics, such as desensitization.

For nonfunctional mutants, the question is whether they are expressed on the cell surface. The function of the Y93C, E173C, C191Y, D197N, and Y211C could be rescued in the presence of a PAM for α7 nAChR, suggesting that they are expressed on the cell surface, but their function is disrupted by the mutations. Y93C and C191Y are the mutations in the binding site located in binding loop A and loop C. These two mutations most likely decrease the binding energy to such an extent that the channel cannot be opened upon agonist binding. However, in the presence of the positive allosteric modulator PNU-120596, the energy barrier for channel opening is reduced. Thus, the agonist can reopen the channel despite of the reduced binding energy for these mutant channels. Tyr93 is essential for orthosteric activation, and the mutation of this residue to cysteine makes the receptor insensitive to ACh, but still can be activated by an allosteric agonist [38]. Our results with 4BP-TQS direct activation of Y93C are consistent with that finding. Interestingly, for the C191Y mutant, rescuing effects of the PAM with ACh or nicotine are different. PNU-120596 could only rescue the nicotine effect but not the ACh effect. This phenomenon suggests that the binding energy loss for ACh is much higher than that for nicotine in this mutant. In the crystal structures of ACh binding protein (AChBP), a soluble protein homologous to the amino-terminal domain of nicotinic receptor, the homologous cysteine (CYS188) does not form the disulfide bridge with the neighboring cysteine (CYS187) when ACh bound to the receptor, but this cysteine residue can coordinate with another binding residue (TYR192) in the same loop C to interact with ACh (Fig 10A, [39]). Note that in the same pentameric structure of AChBP (3WIP chains A-E), chains A, D, E do not have a disulfide bond, but chains B and C do have a disulfide bond. This suggests that the receptor can adopt two different conformations when binding ACh. In contrast, when nicotine is in the binding site, the homologous cysteine can form a disulfide bridge with the neighboring cysteine in all five subunits of the pentameric structure and indirectly interacts with nicotine (Fig 10B, [40]). For the Y211C mutation, it is likely that the mutation decreases the coupling between the M1 and M2-M3 domains, the outward tilting of the latter is proposed to be the mechanism for channel activation for this receptor family [41]. In fact, a computational study predict that the activation pathway of this receptor family is via pre-M1 region [42]. In our homology model, this residue is facing the beginning of M3 domain and is likely to be interacting with Met261. The mutation of Y211C probably disrupts the coupling between pre-M1 to M2-M3 domain, making the receptor nonfunctional. However, weakening of gating energy by PN-120596 would allowed the weakened coupling to transduce the binding energy enough to open the channel. For D197N nonfunctional mutation, the nicotine but not the ACh effect could be partially rescued by PNU-120596. However, 4BP-TQS could partially rescue ACh and nicotine effect. This differential effect again suggests that nicotine and acetylcholine gate the channel differently, and PNU-120596 and 4BP-TQS modulate the channel differently. Asp197 is likely a key residue in coordinating the binding loops B and C. In fact, it forms a functionally important triad with Tyr188 and Lys145 (loop B) [43]. The homologous residue (Asp194) along with Lys139 (loop B) and Tyr185 in the α1 muscle type nicotinic receptor has been shown to be functionally important triad. Dynamic interaction of this triad is an important mechanism for channel activation [44]. For nonfunctional E173K mutant, Glu173 is located in loop 9. It has been reported that the mutation of this residue (E173A) could completely abolish the current, but preserve the surface expression of the mutant receptor [45]. Other mutations of this residue have similar effects, suggesting that Glu173 is an important coupling residue. Mutation of this residue can completely uncouple the agonist binding to the channel gating.

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larger image TIFF original image Download: Fig 10. Different interactions of ACh and nicotine with the AChBP binding residues. A. The binding pocket between chain A and chain B of the AChBP co-crystalized with ACh (PBDID: 3WIP); B. The binding pocket between chain A and chain B of the AChBP co-crystalized with nicotine (PBDID: 1UW6). The residues of Cys191 and Trp55 in the human α7nAChR are labeled next to their homologous residues in the AChBP. Arrows indicate different interactions. https://doi.org/10.1371/journal.pone.0137588.g010

An alternative explanation of the PAM effect is that it makes the desensitized state conducting as proposed by Williams et al [46]. With this mechanism, the rescuable nonfunctional mutants can directly go to desensitized state upon agonist binding. However, in the presence of a PAMII, the desensitized channel is converted to a conducting state with different gating kinetics and even with different single channel conductance. Thus, they lack normal activation state, but still can be desensitized and converted to a conducting desensitized state by a PAMII. Regardless the mechanism, functional rescue of nonfunctional channel would have potential clinical applications.

4BP-TQS is an allosteric agonist as well as a PAM for α7nAChR. For the nonfunctional mutations in the orthosteric binding site or coupling region, if their surface expression is preserved, we expect that their response to 4BP-TQS direct activation through the allosteric site in the M2 domain should be normal. Indeed, 4BP-TQS directly activated Y93C, C191Y, and Y211C mutant receptors. The concentration response of 4BP-TQS for these mutants were similar to that for the wild type receptor, suggesting that mutations in the binding loop C tip, loop A, and M1 do not influence 4BP-TQS binding and function. However, in additional to sensitivity, we have noticed that the amplitude of the 4BP-TQS-induced currents in these 3 mutants was also different. With near saturation concentration, 4BP-TQS induced-current in Y211C had similar amplitude as the wild type. In contrast, Y93C and C191Y had the 4BP-TQS-induced currents with significantly lower amplitude, suggesting that these mutations may reduce the receptor surface expression. Alternatively, the mutations in the binding sites could allosterically influence channel gating efficiency. In GABA A/C receptors, mutations of residues in the binding loops A, B, or E created spontaneously opening channels [47, 48]. Thus, it is possible that mutation of a residue in the binding site can have an impact on the channel gating. Perhaps Y93C and C191Y mutations not only influence binding, but also allosterically inhibit channel gating, resulting in lower gating efficiency by the allosteric agonist 4BP-TQS. In contrast, Y211C has no direct effect on channel gating as reflected by the full efficacy of 4BP-TQS when compared to the wild type. Its effect on the activation by orthosteric agonist likely due to uncoupling of the N-terminal orthosteric binding domain to the channel gating domain. This explanation is further supported by its similar 4BP-TQS sensitivity to that of the wild type. In comparison, the 4BP-TQS sensitivity of the Y93C and C191Y mutants were slightly reduced. For E173K and D197N mutants, unexpectedly, they could not be activated by 4BP-TQS alone, but their function could be restored with the collaborative effort of an allosteric agonist and an orthosteric agonist. Thus, the perturbation in the orthosteric binding loop C arm (D197N) or the coupling region (E173K) not only severely impair the coupling between the orthosteric binding site and the channel gate, but also completely abolish direct gating by allosteric agonist. The non-rescuable mutants could influence receptor assembly or might have a larger influence in binding or coupling energy. The conserved arginine at the middle position of the pre-M1 RRR motif of nicotinic receptor subunits is required for the transport of assembled α7 nAChR to the cell surface [49]. Thus, the corresponding R206C mutation would likely prevent surface expression of the receptor, most probably by disrupting interactions between pre-M1, loop2 and M2-M3 linker as suggested by molecular dynamics simulation in α7 nAChR [50] or experimental evidence in other subunits [51].

For the functional W55G mutant, our findings of its impact on ACh response are consistent with previous reports [30, 52]. In addition, our observation of differential impact of this mutant on ACh and nicotine efficacy is interesting. By examining the crystal structures of AChBP with ACh or nicotine, we found that nicotine can directly interact with TRP55 through a Pi-Alkyl interaction (Fig 10B). In contrast, ACh does not directly interact with TRP55 (Fig 10A). That could be an important mechanism for the differential impact of the mutation on the efficacy of ACh or nicotine