Sodium ions allosterically modulate the M2 muscarinic receptor

G protein coupled receptors (GPCRs) play a key role in the vast majority of cellular signal transduction processes. Previous experimental evidence has shown that sodium ion (Na+) allosterically modulate several class A GPCRs and theoretical studies suggested that the same also holds true for muscarinic receptors. Here we examined, using Xenopus oocytes as an expression system, the effect of Na+ on a prototypical GPCR, the M2 muscarinic receptor (M2R). We found that removal of extracellular Na+ resulted in a decrease in the potency of ACh toward the M2R and that a conserved aspartate in transmembrane domain 2 is crucial for this effect. We further show that this allosteric effect of Na+ does not underlie the voltage-dependence of this receptor.

Scientific RepoRtS | (2020) 10:11177 | https://doi.org/10.1038/s41598-020-68133-9 www.nature.com/scientificreports/ solution, ND96 or ND96-Na + free, and basal GIRK current, I K , was developed upon replacement of the ND96 by a 24 mM K + solution, either containing 72 mM Na + (Fig. 1a) or Na + -free (Fig. 1b). The two levels of [Na + ] o were selected in a random order, i.e. first 72 mM Na + and then Na + free or vice versa. Then, 3 concentrations of ACh were applied sequentially (10 nM-10 µM), each leading to an evolvement of currents denoted I Ach . The amplitude of I ACh was used as a measure for M2R activation. I ACh was terminated upon washout of the ACh from the bath. Employing this basic experimental protocol with several concentrations of ACh, DR curves at the two levels of [Na + ] o were constructed. To be able to compare between oocytes at different conditions and across different oocytes we measured fractional I ACh . That is, for each [Na + ] o , I ACh at any particular ACh concentration was normalized to I ACh obtained in the same recording at a saturating concentration of ACh.
The results of all of these experiments are depicted in Fig. 1c where DR curves obtained at 72 mM Na + (black) and at Na + -free solution (red) are shown. As illustrated in the figure, the curve shifts to the right when [Na + ] o is removed from the extracellular solution. Specifically, the EC 50 is 70 nM in 72 mM Na + and 110 nM at Na + free conditions. The two values are significantly different (p < 0.05). These results suggest that the potency of ACh toward the M2R is allosterically modulated by Na + . The removal of Na + from the extracellular solution did not affect the maximal current evoked by the receptor in our experimental system. The average amplitude of I ACh evoked by the maximal ACh concentration at Na + free solution was similar to that obtained at 72 mM Na solution (Fig. 1d, the two conditions are not significantly different; p = 0.74).
GIRK channels were reported to be modulated by Na +31-33 . To verify that the results of Fig. 1c reflect a genuine effect of Na + on the M2R rather than an effect on the GIRK channels, we measured, in oocytes that express the GIRK channel alone, the effect on [Na + ] o on the basal GIRK current. This current was proposed 34 to be due to activation of the GIRK channel by free βγ subunits in the oocyte. Figure 2a shows a representative recording where I K was measured at 72 mM Na + and then at Na + free solution. The figure illustrates that the removal of [Na + ] o did not affect the amplitude of I K . This conclusion was supported by the results of Fig. 2b, where I K was measured first at one of [Na + ] o and then at the other of [Na + ] o . The order of the recordings was selected randomly. As mentioned above, to ensure full removal of Na + from the extracellular solution, Na + free solution was applied at least 5 min prior to the recording. In these recordings, and in the experiment described in Fig. 2c, I K was measured by subtracting the current amplitude in the presence of 1 mM Ba 2+ (GIRK channel blocker 35 ) from the current in its absence. It was found that in all 10 oocytes tested I K measured in 72 mM Na + was not significantly different from I K at Na + free solution (paired t test, p = 0.16).
To further examine the effect of Na + on the GIRK channels themselves we measured the voltage-current relationship (I-V) of I K . To this end, the oocyte was voltage clamped to − 80 mV and the currents after depolarizing Recordings of ACh-induced GIRK currents in 72 mM Na + solution (a) and in Na + free solution (b) from the same oocyte. 3 concentrations of ACh were applied sequentially (1, 2 and 3 stand for 10, 100 and 10,000 nM, respectively). (c) dose response curves assembled from various experiments conducted at 72 mM Na + solution (black circles) and in Na + free solution (red circles). Each point represents mean (± SEM) from 7-21 oocytes from 6 batches of oocytes. The solid black and red lines were generated by fitting a 3 parameters equation to the data (see "Methods"). (d) The maximal amplitude of I ACh , evoked by 100 µM ACh in 72 mM Na + solution (black) and in Na + free solution (red). The two bars are not significantly different (unpaired t test, p = 0.71). www.nature.com/scientificreports/ pulses to various holding potentials from -80 to + 40 mV at 10 mV increments were measured in 24 mM K + solution that contained either 72 mM (black) and Na + free solution (red). In order extract I K from these recordings, the same protocol was repeated in the presence of 1 mM Ba 2+ , and these currents were subtracted from the currents in the absence of barium. The results indicate (Fig. 2c) that [Na + ] o did not affect either the size or the voltage-current relationship of the GIRK channel. Therefore, the effect of [Na + ] o on I ACh is likely due to its effect on the M2R. Extracellular Na + affect the dissociation of ACh from the M2R. The observed Na + -dependent shift in potency of ACh toward the M2R may be due to change in the association rate constant (k on ) and/or due to an effect on the dissociation rate constant (k off ) of the ligand from the receptor. Here, we confine our study to k off and examine whether Na + affect the dissociation rate constant. The actual dissociation rate of ACh from the M2R Figure 2. Extracellular Na + does not affect the basal GIRK current (I K ). (a) Recording of I K from an oocyte that expresses the GIRK channel but not the M2R. Removing Na + from the extracellular solution did not affect I K .
(b) Collected results from 10 oocytes. Each two circles connected with a dashed line represent the amplitude of I K from one oocyte at 72 mM Na + (black) and at Na + free solution (red). The order of recordings was selected randomly. Both here and in (c), I K was measured by subtracting the current at 24 mM K + solution in the presence of 1 mM Ba 2+ from the current in its absence. (c) Current-voltage relationship of I K at 72 mM Na + solution (black circles) and in Na + free solution (red circles). Results are given as mean (± SEM) from 11 oocytes.
Scientific RepoRtS | (2020) 10:11177 | https://doi.org/10.1038/s41598-020-68133-9 www.nature.com/scientificreports/ could not be measured directly at our experimental system. However, we have previously shown that the deactivation rate of M2R-induced GIRK currents may serve as a measure for the dissociation of ACh from the M2R 36 . Figure 3a shows a representative recording from one oocyte where the decay of I ACh following the washout of the ACh can be fitted well to a single exponential equation (dashed red line) from which the time constant of the decay can be extracted (see "Methods"). Figure 3b depicts the decay of I ACh following washout with ACh-free solution from the same oocyte at two levels of [Na + ] o . It is seen that I ACh decays more rapidly in Na + free solution (black) than in 72 mM Na + (red). The cumulative results are shown in Fig. 3c. In 9 out of 10 oocytes the time constant of the decay was smaller in Na + free solution than in 72 mM Na + , and the mean time constant of the decay at 72 mM Na + was significantly higher than that at Na + free solution (59.1 ± 7.4 and 34.7 ± 5.6 s, respec- www.nature.com/scientificreports/ tively) These results suggest that Na + affects the potency of ACh toward the M2R by modulating the dissociation of the agonist from the receptor.
Asp69 is involved in the effect of Na + on the M2R. Several structural and functional studies implicated an aspartate residue in transmembrane domain II (Asp2.50) as being involved in the allosteric effect of Na + on class A GPCRs. These studies suggest that Na + acts via binding at a specific binding site within the helical bundle. Mutating this residue to an uncharged residue results in the abolishment of the Na + dependence in other GPCRs [9][10][11][12] . To validate the role of this residue, Asp69 in the M2R, in Na + binding we mutated it to asparagine (Asp69Asn) and repeated the experiments described in Figs. 1 and 3. The results are depicted in Fig. 4. We found (Fig. 4a), that the potency of ACh toward the Asp69Asn is reduced and it the does not depend on [Na + ] o ; As in Fig. 1c, we fitted the data to a three-parameter equation (see "Methods"). Although the fit was not as good as in Fig. 1c (probably due to the complex effect of the Asp69Asn on both agonist binding and G protein activation 37,38 ), an EC 50 values at both Na + concentration could be extracted. The EC 50 was 920 nM in 72 mM Na + and 1,078 nM at Na + free conditions. The two values are not significantly different (p = 0.66). Corollary to this change in potency, the dissociation of ACh from the Asp69Asn mutant was faster than that of wt M2R, and it was similar at the two levels of [Na + ] o (Fig. 4b). The collected data from 10 oocytes are shown in Fig. 4c Does the allosteric effect of Na + underlie the voltage-dependence of ACh binding to the M2R?. We have previously reported that the affinity of the M2R is voltage-dependent; it is higher at resting potential than under depolarization 19 . We further showed that charge movement associated currents are correlated with the change in affinity and may underlie this voltage-dependence 26 . A recent theoretical work proposes that the measured charge movement associated currents may arise from voltage-dependent movement of a Na + from its binding pocket 17 . This movement was suggested to have functional consequences. In light of this, we examined the role of Na + in the voltage-dependence of the affinity of the M2R. To do so, we wished to examine whether the removal of [Na + ] o affects the voltage-dependence of the M2R. We constructed dose-response curves at two holding potentials, − 80 mV and + 40 mV, as described before 19 . The oocyte was voltage clamp to either − 80 mV or + 40 mV (The order of holding potentials was selected randomly) and the response to three ACh concentrations was measured. Then the oocyte was washed for 10 min in ND96 solution and the experiment was repeated at the other holding potential. Because of the long duration required for the recording at the two holding potential, the dose response relationship was measured for each oocyte either in Na + free solution or in 72 mM Na + , and was compared to the dose-response curves obtained from oocytes from the same batches at the other [Na + ] o . Figure 5a shows representative recording from one oocyte at − 80 mV (left) and at + 40 mV (right) at Na + free solution. Dose response curves constructed from recordings obtained from 5 experiments at 72 mM Na + and at Na + free solution are shown in Fig. 5b,c, respectively. The results show that although the potency of ACh toward the M2R is decreased in low [Na + ] o , it remained voltage-dependent to a similar extent; depolarization increased the EC 50 3.8 fold at 72 mM Na + and 4.6-fold at Na + free solution.

Discussion
Na + modulates the affinity and activity of many GPCRs, and the functional role of this phenomenon, as well as its pharmacological potential, is only now begin to be revealed 39 . Recent advances in the understanding of the structure of class A GPCRs led to new insights into the structural mechanism by which Na + allosterically modulate these receptors 4,5 . This mechanism involves the binding of Na + to a conserved binding site in the transmembrane helices and a collapse of this site following activation of the receptor. This structural mechanism was suggested to account for the observed effect of Na + on the affinity of several GPCRs toward their agonists 4 . Although theoretical studies propose that muscarinic receptors may also be modulated by Na +16,17 , to the best of our knowledge, this has never been demonstrated experimentally for the M2R.
Here, we provide experimental support for this suggestion. We show that the potency of ACh toward the M2R, and specifically its dissociation from the M2R, is modulated by [Na + ] o . Interestingly, our results suggest that Na + increases the affinity of ACh toward the M2R. These results differ from the current notion in the field, where a negative allosteric effect of Na + on agonist binding was reported [16][17][18] . In particular, two studies with muscarinic receptors suggest the Na + increases the binding affinity of muscarinic antagonist and decreases the binding affinity of muscarinic agonists 40,41 . The first study showed that binding affinity of oxotremorine to cardiac muscarinic receptors is reduced when the tissue was incubated in high Na + concentration 40 . The reason for this difference from our results is not known. It is possible that it is due to the different experimental conditions employed in the two studies. For example, our study used expression system to investigate the M2R in isolation from other muscarinic receptors while Rosenberg et al. studied whole cardiac tissue, where although the M2R is the main muscarinic subtypes, other muscarinic receptor subtypes are expressed [42][43][44] and may affect the results. In addition, Rosenberg et al., as well as most other studies that addressed this question, incubated lysed membranes at different Na + conditions, thus allowing the interaction of Na + from both sides of the membrane. It was suggested that intracellular Na + may also haves an effect on the binding properties of GPCRs 45 . Therefore, comparison of the data obtained in such experiments to our data, where only extracellular Na + was modified, may not be adequate.
The second, more recent, study 41 has reported a mutation of serine residue in position 110 of the M2R with an arginine and suggested that this arginine residue mimics the stabilizing role of the Na + . This mutant exhibits Scientific RepoRtS | (2020) 10:11177 | https://doi.org/10.1038/s41598-020-68133-9 www.nature.com/scientificreports/ increased affinity to two M2R antagonists and reduced the affinity toward the muscarinic agonist iperoxo. These results led to the suggestion that this mutation, and thus, perhaps, also Na + , stabilizes the conformation of the whole receptor through tighter inter-helical interactions and therefore increases its affinity toward antagonist and increases it toward agonist. However, their data did not find an effect of Na + alone on the affinity of iperoxo toward the M2R. The source for the inconsistency with our results is not known at this time. It is possible that behavior of the synthetic, high affinity, agonist iperoxo is different from that of ACh. In addition, similar consideration as described above regarding the different interaction site of Na + may also apply here. www.nature.com/scientificreports/ Recent simulations 17,18 suggested that Na + that binds to a specific binding site translocate upon changes in membrane potential, and that these movements may give rise to the gating charges observed for M1R and M2R [26][27][28][29] . Other study, however, showed that three tyrosine residues that form a lid above the ligand binding site also contribute to the gating charge 29 . Mutating these tyrosine residues to phenylalanine significantly reduced the measured charge that moves in response to depolarization, although it did not completely abolish it. It is possible that both these mechanisms underlie the measured charge movement, although it is worth noting that charge movement associated currents were recorded in the absence of intracellular and extracellular Na + (but in the presence of NMDG, which may also bind the same binding site). The current study demonstrated (Fig. 5), that the potency of ACh toward the M2R is voltage-dependent even in the absence of extracellular Na + . This suggests that the allosteric effect of Na + and the voltage-dependence of the M2R do not share similar mechanism. This conclusion is consistent with recent studies that show that the M2R retain its voltage-sensitivity even when the main Na + interacting residue, Asp69, was mutated 29,46 . Thus, further research is needed in order to fully elucidate the mechanism of voltage-sensitivity of GPCRs. www.nature.com/scientificreports/

Methods
Preparation of cRNA and oocytes. cDNA plasmids of the two subunits of the G-protein activated inward rectifying K + channel (GIRK) (GIRK1 and GIRK2) and the M2R were linearized with the appropriate restriction enzymes. The Asp69Asn mutant was prepared using Quick-Change II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) 26,29 . The linearized plasmids were transcribed in vitro using a standard procedure 30 . Xenopus laevis oocytes were isolated and incubated in NDE96 solution composed of ND96 (in mM, 96 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 5 Hepes, pH adjusted to 7.5 with NaOH), with the addition of 2.5 mM Na + pyruvate, 100 U/ml penicillin and 100 mg/ml streptomycin 47 . A day after their isolation, the oocytes were injected with the following cRNAs: M2R (200 pg/oocyte) GIRK1 and GIRK2 (200 pg/oocyte for each) and Gαi3.
Materials were purchased from Sigma Israel (Rehovot, Israel).

Current measurements.
The currents were measured 4-7 days after cRNA injection and were recorded using two electrode voltage clamp amplifier 19  Data analysis. The dose response curves were fitted by the following equation: where Y is the normalized response, X is the concentration of ACh and EC 50 is the ACh concentration that gives the half-maximal response. The time constant of the decay of I ACh was extracted by fitting a single exponential to the decay of the current. We began the fit after the current declined to 80% of its maximal level, as described 36 . Statistical evaluation. Statistical analysis was done using Prism Graph pad software. Significance was evaluated by Student's two tailed paired (Figs. 2b, 3c,4c) or unpaired (Fig. 1d) t test.
Estimating the difference between the EC 50 values was done be the extra-sum-of-squares F test.
Ethics statement. All experimental procedures used in this study were performed in accordance with relevant guidelines and regulations and were approved by the Hebrew University's Animal Care and Use Committee (Ethical approval number NS-11-12909-3).
Received: 6 April 2020; Accepted: 15 June 2020 Scientific RepoRtS | (2020) 10:11177 | https://doi.org/10.1038/s41598-020-68133-9 www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/4.0/.