Ben-Chaim Y et al. (2019), The coupling of the M2 muscarinic receptor to i...

XB-ART-56438

PLoS One 2019 Oct 31;1410:e0224367. doi: 10.1371/journal.pone.0224367.

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The coupling of the M2 muscarinic receptor to its G protein is voltage dependent.

Ben-Chaim Y , Broide C , Parnas H .

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G protein coupled receptors (GPCRs) participate in the majority of signal transduction processes in the body. Specifically, the binding of an external agonist promotes coupling of the GPCR to its G protein and this, in turn, induces downstream signaling. Recently, it was shown that agonist binding to the M2 muscarinic receptor (M2R) and to other GPCRs is voltage dependent. Here we examine, whether the coupling of the M2R to its G protein is also voltage-dependent. We first show, in Xenopus oocytes, that the activity of the M2R in the absence of agonist (constitutive activity) can be used to report the coupling. We then show that the coupling is, by itself, voltage dependent. This novel finding is of physiological importance, as it shows that the actual signal transduction, whose first step is the coupling of the GPCR to its cognate G protein, is voltage dependent.

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Species referenced: Xenopus laevis
Genes referenced: gprc6a kcnj3

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	Fig 1. Measurement of constitutive activity in the M2R, using the atropine procedure.(A) The effect of 100 μM atropine on oocytes expressing both the M2R and GIRK channel. (top) A representative recording from one oocyte. (bottom) Collected data from 26 oocytes. Each two data points connected with a line represent IKS (black) and IKB (red), from one oocyte. The averages of IKS and IKB are denoted by the horizontal lines. The ratio IKS/ IKB is given in the box. (B) The effect of 100 μM atropine on oocytes expressing only the GIRK channel. (top) A representative recording from one oocyte. (bottom) Data from 5 oocytes. Each two data points connected with a line represent IK from one oocyte before (black) and after (red) the addition of atropine. The averages of IK before and after the addition of atropine are denoted by the horizontal lines.
	Fig 2. Measurement of constitutive activity in the M2R, using two groups of oocytes.(A) group 1: oocytes expressing both the M2R and the GIRK channel. (top) Representative recording. (bottom) Collected data from 61 oocytes. Here and in B, each data point depicts IK measured from one oocyte, and the average IK is denoted by the horizontal line. (B) group 2: oocytes expressing only the GIRK channel. (top) Representative recording. (bottom) Collected data from 61 oocytes. The ratio IKS/ IKB is given in the box.
	Fig 3. Constitutive activity in PTX treated oocytes.(top) Representative recording from a PTX-treated oocyte (solid line). Recording from control, PTX-untreated, oocyte is shown for comparison as dashed line. The arrows indicate the addition of atropine. (bottom) Collected data from 20 PTX-treated oocytes. Each two data points connected with a line represent IK from one oocyte, before (black) and after (red) the addition of atropine. The averages of IK before and after the addition of atropine are denoted by the horizontal solid lines. The averages of IKS and IKB from control, PTX-untreated, oocytes are shown for comparison as horizontal dashed lines. The ratio IKS/ IKB from PTX-treated oocytes, is given in the box.
	Fig 4. The voltage dependence of the constitutive activity of the M2R.(A) The experimental protocol (upper part) and the IK produced at each holding potential (lower part). IK was calculated by subtracting the current amplitude in ND96 from that in 24 mM K+ in the same holding potential. An example of recording in one holding potential (-80 mV) is shown in green. (B) IKS (black) and IKB (red) measured at various holding potentials. Each data point represents mean ±SEM from 69 oocytes. (C) The voltage dependence of the ratio IKS/IKB.
	Fig 5. Voltage dependence of the binding affinity and the constitutive activity of the wt M2R and the triple mutant.(A) Dose-response curves obtained from several experiments at -80 mV (black circles) and at +40 mV (red circles) using various concentrations of ACh from wt M2R. Each data point represents the mean ± SEM, n = 5–8; taken from [18]. (B) IKS (black) and IKB (red) measured at various holding potentials from wt M2R; taken from Fig 4B. (C) Dose-response curves from the triple mutant at -80 mV (black circles) and at +40 mV (red circles). Each data point represents the mean ± SEM, n = 6–8. (D) IKS (black circles) and IKB (red circles) measured at various holding potentials. Each data point represents mean ±SEM from 21 oocytes.

References [+] :

Barchad-Avitzur, A Novel Voltage Sensor in the Orthosteric Binding Site of the M2 Muscarinic Receptor. 2016, Pubmed, Xenbase