Friedman S et al. (2020), Sodium ions allosterically modulate the M2 musc...

XB-ART-57155

Sci Rep 2020 Jul 07;101:11177. doi: 10.1038/s41598-020-68133-9.

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Sodium ions allosterically modulate the M2 muscarinic receptor.

Friedman S , Tauber M , Ben-Chaim Y .

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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.

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Species referenced: Xenopus
Genes referenced: gprc6a kcnj3

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	Figure 1. The effect of extracellular Na+ on the potency of the M2R. (a,b) 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 IACh, 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).
	Figure 2. Extracellular Na+ does not affect the basal GIRK current (IK). (a) Recording of IK from an oocyte that expresses the GIRK channel but not the M2R. Removing Na+ from the extracellular solution did not affect IK. (b) Collected results from 10 oocytes. Each two circles connected with a dashed line represent the amplitude of IK 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), IK was measured by subtracting the current at 24 mM K+ solution in the presence of 1 mM Ba2+ from the current in its absence. (c) Current–voltage relationship of IK at 72 mM Na+ solution (black circles) and in Na+ free solution (red circles). Results are given as mean (± SEM) from 11 oocytes.
	Figure 3. The effect of extracellular Na+ on the dissociation of ACh from the M2R. (a) The basic experimental protocol. The oocyte was clamped at − 80 mV and IACh was evoked by application of 1 µM ACh in 24 mM K+ solution. Following washout of the ACh with ACh-free solution the current declined and the time constant of the decay was fitted by a standard exponential equation (red dashed line). (b) Representative recordings from one oocyte in 72 mM Na+ solution (black) and in Na+ free solution (red). The currents shown are normalized to enable comparison of the kinetics of the current decline. (c) Collected results from 11 oocytes. Each two circles connected with a dashed line represent the time constant of the decay of IACh following ACh washout from one oocyte at 72 mM Na+ (black) and at Na+ free solution (red). The order of recordings was selected randomly.
	Figure 4. Mutation of Asp69 abolished the Na+ dependence of the M2R. (a) Dose response curves assembled from oocytes expressing the Asp69ASn mutant at 72 mM Na+ solution (black circles) and in Na+ free solution (red circles). The number of repetitions for each ACh concentration varied between 7 and 21 from 4 batches of oocytes. The solid black and red lines were generated by fitting a 3 parameters equation to the data (see “Methods”). (b) Representative recordings from one oocyte in 72 mM Na+ solution (black) and in Na+ free solution (red). The currents shown are normalized to enable comparison of the kinetics of the current decline. (c) Collected results from 10 oocytes. Each two circles connected with a dashed line represent the time constant of the decay of IACh following ACh washout from one oocyte at 72 mM Na+ (black) and at Na+ free solution (red). The order of recordings was selected randomly. The two conditions are not significantly different (p = 0.45).
	Figure 5. The effect of extracellular Na+ on the voltage-dependence of the potency of ACh toward the M2R. (a) Representative recordings from one oocyte at − 80 mV (left) and at + 40 mV (right) at Na+ free solution. (b,c) Dose response curves assembled from 5 experiments at 72 mM Na+ solution (b) and in Na+ free solution (c). For each oocyte, IACh was measured at − 80 mV (black circles) and at + 40 mV (red circles) at either Na+ concentration, and the response of each concentration was normalized to the response evoked by 10 µM ACh at the same holding potential. Each point represents mean (± SEM) from 8–30 oocytes. The solid black and red lines were generated by fitting a 3 parameters equation to the data (see “Methods”).

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