Ben Chaim Y , Bochnik S , Parnas I , Parnas H .

	Figure 2. Measurement of τdec fro the decline of the GIRK current.(A) The functional assay used. Binding of ACh to the m2R leads to G-protein activation. βγ subunits of the activated G-protein then bind to the GIRK channel and open it, inducing IACh. Upon ligand washout, ACh unbinds from the receptor and IACh deactivates. (B) 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 (1). Following washout of the ACh with ACh-free solution the current declines (2). The kinetics of the washout, as measured by the disappearance of a colored solution from the bath, is shown as red symbols and line (see text for details) (C) τdec depends on the affinity of the agonist to the receptor. IACh decline after wash out faster than GIRK current evoked by OXO. Representative recordings from one oocyte are shown. The currents shown here and in Figs. 3A and 5B, C are normalized to enable comparison of the kinetics of the current decline. Inset collected data from 14 oocytes. The two bars are significantly different (p<0.0001).
	Figure 3. Voltage sensitivity of τdec.(A) The decline of the current evoked by ACh at different holding potentials following washout (beginning at time 0) with agonist free solution. (B) Average± SEM of τdec at different holding potentials from 8 to 18 oocytes for each holding potential. (C) The dependency of RL in membrane potential, from experimental data taken from [6] (empty symbols) and from the data shown at (B) (filled symbols).
	Figure 4. The mechanism proposed to shift receptor between two affinity states.(A) A schematic description. RH is coupled to a G-protein. Depolarization induces charge movement in a putative voltage sensor which induces conformational change in L3. This change reduces the probability of the GPCR to couple to its G-protein and that in turn shifts the GPCR into RL. Hyperpolarization reverses the process and the GPCR shifts back to RH (dashed arrow). (B) The proposed mechanism for voltage sensitivity in the m2R in the context of the ternary complex model [23]. The GPCR can reside in either RH (bound to G-protein, (R−Gp)H)) or in RL. Depolarization reduces the likelihood of the receptor to be in RH state (red arrow). When coupled to the G protein, the GPCR acquires koffH (low value of koff). When uncoupled to the G protein the GPCR acquires koffL (high value of koff).
	Figure 5. The mechanism of voltage dependent shift in koff.(A) Effect of PTX treatment on dissociation of [3H]ACh from m2R-expressed oocytes. Left, PTX uncouples the G-protein from the GPCR. This causes reduction of the binding affinity and abolishment of the voltage sensitivity of the affinity and abolish the voltage sensitivity of the binding (Middle, taken with permission from [7]). Right, Dissociation of [3H]ACh from m2R-expressed oocytes after different washout times, at two membrane potentials, with and without PTX treatment. The data is average ±SEM of 10 and 12 oocytes. The specific binding after PTX treatment was not significant for all data points. As seen, after PTX treatment the voltage dependence of τdis was abolished. (B) τdec is voltage insensitive in m2R- ELALL expressed oocytes. Replacement of 5 residues in the N-terminal of L3 (Left) abolished the voltage sensitivity of the binding affinity (Middle,taken with permission from [6]) Right, Measurements of τdec at −60 mV and +40 mV from the same oocyte. Inset. Collected data from 17 (−60 mV) and 23 (+40 mV) oocytes. The bars are not significantly different; p = 0.74. (C) τdec is voltage insensitive in m2R-Y426A expressed oocytes. Left, Side view representation of the m2R based on [36] highlighting residue Y426 (red) in the ligand binding site. A ligand bound to the binding site is shown as a blue sphere. Middle, Dose-Response relation of the Y426A mutant exhibits low affinity and lack of voltage sensitivity. Taken with permission from [8]. Right, τdec at −80 mV and +40 mV measured from the same oocyte. Inset, Collected data obtained with m2R-Y426A expressed oocytes. Each bar represents the mean± SEM of 11 (−80 mV) and 6 (+40 mV) oocytes. The bars are not significantly different (p = 0.67).
	Figure 1. The dissociation rate of [3H]ACh from m2R-expressed oocytes is voltage dependent.(A, top) The measurement procedure (see Materials and Methods and [7]). (A, bottom) An example of data obtained from one experiment at resting membrane potential. Results after 2, 5 and 7 sec washout are shown (n = 9, 8 and 12 oocyte, respectively) (B) An example from one experiment at two membrane potentials as depicted. The data was normalized as follows: The binding of [3H]ACh was first measured after 2 sec washout in agonist free solution (initial sampling), and then after 2 and 5 sec of additional wash. (C) Collected data from a total of 9 experiments at resting potential (−88 mV) and under depolarization (+5 mV) fitted by an exponential decay. Each data point was normalized to the binding of the initial sampling and represents the average ± SEM of 16–59 oocytes (The SEM values at +5 mV are smaller than the symbols). Inset, results of binding measurements of [3H]ACh to m2R-expressed oocyte (Taken with permission from [7]). (D) Binding of [3H]ACh to m1R- expressed oocytes after different washout times, at two membrane potentials. Each data point was normalized to the binding of the initial sampling and represents the average ±SEM of 7 to 57 oocytes. Inset, results of binding measurements of [3H]ACh to m1R-expressed oocyte (Taken with permission from [7]).

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