Berlin S , Artzy E , Handklo-Jamal R , Kahanovitch U , Parnas H , Dascal N , Yakubovich D .

             plasma membrane
             G-protein activated inward rectifier potassium channel activity

                bipolar disorder

	Figure 1 Increasing expression levels of M2R accelerates the activation of GIRK1/2. (A) – a representative GIRK1/2 activation experiment. Oocytes were injected with the following RNAs: GIRK1 and GIRK2 (2 ng/oocyte each) and 500 pg/oocyte M2R. Ievoked was elicited by 10 µM ACh. Inset- zoom-in on the activation phase of Ievoked (black plot) and a mono-exponential fit (red). (B) – kinetics of GIRK1/2 activation. Oocytes expressed a constant amount of GIRK1/2 (2 ng RNA/oocyte), with increasing levels of M2R, in the range 10-1000 pg/oocyte, and τact was determined by monoexponential fitting as shown in A (N=2-7 experiments, n= 13-25 cells).
	Figure 2 Estimating the surface density of M2R-YFP. All data are from one experiment. (A) - (left) Representative micrographs of oocytes (membrane at equator) expressing equal amounts of RNAs of YFP-GIRK1 and GIRK2, and 1 ng M2R (wt). Injected YFP-GIRK1 mRNA amounts are indicated. (right) Representative currents from oocytes from the same experiment (from the same groups as in left panels). Note the gradual increase in Ibasal and Itotal, reaching a plateau at 2 ng/oocyte of channel’s RNA. The current measurements in this experiment were done with a slower perfusion rate and were not included in the kinetic analysis. (B) – Representative micrographs of oocytes, injected with the indicated amounts of M2R-YFP, imaged at the same day and under identical settings as those in A. (C) – Itotal of YFP-GIRK1/GIRK2 channels as a function of channel’s RNA dose. (D) – calibration of the surface density of YFP. YFP-GIRK1 fluorescence (in arbitrary units, AU) is plotted versus channel density induced by the two lowest doses of GIRK RNA (<1 ng/oocyte), within the linear range of fluorescence-current relationship. Channel density was calculated from Itotal as explained in the text. The correlation between fluorescence and number of YFP-GIRK1 molecules is shown with superimposed linear regression line, extended to origin of coordinates. The regression equation was y=4x, i.e. one channel/µm2 corresponds to fluorescence intensity of 4 AU. Note that, since each channel has two YFP molecules, the calibration factor in this experiment is: 1 YFP molecule/µm2 = 2 AU. (E) –estimating the surface density of M2R-YFP, for RNA concentrations of 1, 2 and 5 ng/oocyte. YFP fluorescence, in AU, is shown on the left Y-axis. M2R-YFP surface density (right axis) was calculated using the calibration factor derived from YFP-GIRK1/GIRK2 measurements. The relationship between M2R-YFP RNA dose and M2R-YFP surface density was fitted with linear regression, extended to the origin of coordinates, in the form y=62.5x.
	Figure 3 Quantitative analysis of dose-dependency of τact and amplitude of Ievoked on plasma membrane density of M2R-CFP. (A)-Whole cell radioligand-labeling by quinuclidinyl benzilate (QNB*) shows that identical doses of injected RNA produce similar surface expression of M2R, M2R-CFP and M2R-YFP, for three different amounts of receptor RNAs. B-F, quantitative analysis of incremental expression of M2R-CFP reveals collision-coupling activation of YFP-GIRK1/GIRK2 channels. Oocytes were injected with constant mRNA amounts of YFP-GIRK1 and GIRK2 (1 ng each) but with increasing doses of M2R-CFP RNA. (B) – RNA dose-dependent increase in the surface levels of M2R-CFP. Representative micrographs of oocytes (left) expressing m2R-CFP and summary of expression (right). RNA amounts of M2R-CFP RNA, in ng/oocyte, are indicated in yellow. n=7-12 oocytes in each group. (C) – expression of M2R-CFP does not affect the surface level of YFP-GIRK1/GIRK2 except for the decrease at the highest dose of M2R-CFP, 25 ng/oocyte (**, p<0.01). Left panel shows representative micrographs of YFP-GIRK1/GIRK2 – expressing oocytes. Numbers within the images indicate the amounts of M2R-CFP RNA, in ng/oocyte. Right panel shows summary of expression data (n=7-12 oocytes in each group). (D) – representative normalized Ievoked, elicited by 10 µM ACh in oocytes injected with the indicated amounts of M2R-CFP RNA. For simplicity, only the initial (activation) phase of Ievoked is shown. (E, F) – τact is reduced (E) and Ievoked amplitude (F) is increased with increased surface density of M2R-CFP. AU, arbitrary units. τact and Ievoked data are from cells exemplified in B – D; n=5-12 in each group.
	Figure 4 Simulating activation kinetics and its dependence on M2R surface density. (A) – scheme of the G-protein cycle. (B) – graded contribution model of GIRK1/2 activation. Rate constants of reactions shown in A and B are summarized in Table 1. (C) –comparison of the experimentally observed and predicted ACh-evoked currents, with GIRK-Gβγ interaction parameters from two structural models, 4KFM and BS. (D) - representative analysis of the time course of GIRK1/2 activation. The experimental result (dotted black line) is from an oocyte injected with 0.5 ng M2R RNA. Superimposed are simulated currents according to graded contribution model of GIRK1/2 activation. The experimental parameters in this cell were: Ibasal = 15.5 µA, Itotal= 22.8 µA. Estimated channel density was 36 channels/µm2. Initial concentrations of Gα and Gβγ available to the channel are: for the case of 4KFM model 3.23 molecules Gαi/channel and 0.45 molecules Gβγ/channel; for the case of BS model, 3.24 molecules Gαi/channel and 0.46 molecules Gβγ/channel. Each plot represents the recorded or simulated current normalized to its maximum. (E) – mean τact values from all experiments with wild-type M2R, superimposed on data obtained from fitting of simulated time-courses according to different models. Each experimental point shows mean value of τact ± SEM from one experiment (n=3-7 oocytes). Simulated time courses were generated for the case described by Yakubovich et al. as “high density group”, i.e. Ibasal =13.36 µA, Itotal =17.2 µA, n=21 channels/µm2. Amounts of available Gα and Gβγ molecules per one channel, that are required to obtain the observed Ievoked, were calculated using the graded contribution model: with the BS structure-based parameters, 3.65 Gβγ and 0.39 Gα molecules/channel; with the for 4kfm structure-based parameters, 3.62 Gβγ and 0.38 Gα molecules/channel.
	Figure 5 Physical tethering of M2R and Gαi3 converts a collision coupling mechanism to a preformed-complex: experiment and simulation. (A) – Incremental expression of fused M2R-Gαi3C351G (PTX-insensitive) in the presence of coexpressed A-protomer of pertussis toxin (PTX; 0.2 ng RNA/oocyte) shows increase in Ievoked with growing amounts of injected RNA (black plot), but kinetics of activation remain unchanged (red plot). Right- Histogram of evoked currents (black) and τact (red) of GIRK1/2 coexpressed with M2R-wt. Result shown are from one experiment; number of cells (n) tested in each group are shown above experimental points or in the bar. No significant change in τact was found (one way ANOVA, P = 0.154). Spearman correlation coefficient calculated for analysis of evoked currents ~ 1, P = 0.0167. (B) – scheme of G-protein utilized to simulate GIRK1/2 activation by M2R-Gαi3C351G. Blue arrows and numbering denote reactions that are shared with M2R wt activation pathway, as described in Figure 4. Red arrows denote reactions present only in the current scheme. The numbering of reactions and the rate constants are the same as in Table 1. (C) – simulated Ieoked values obtained assuming a range of expression level of fused M2R-Gαi3C351G. (D) – summary of τact obtained from fitting time-course of activation of GIRK1/2 by range of M2R-Gαi3C351G densities with mono-exponential function. Three possible scenarios were simulated for analysis of M2R-Gαi3C351G experiments. Black bars; M2R-Gα concatemer is assumed to have same affinity to Gβγ as Gα, and no change in Gβγ concentration is assumed with concatemer expression. Red bars; M2R-Gα concatemer is assumed to have same affinity to Gβγ as Gα, and 1:1 increase in Gβγ concentration is assumed with concatemer expression. Green bars; M2R-Gα concatemer is assumed to have 10-fold lower affinity to Gβγ than Gα and no change in Gβγ concentration is assumed with concatemer expression. For simulation it was assumed that GIRK1/2 is expressed at levels similar to “intermediate density group” described in Yakubovich et al. (2015) i.e. under pre-expression conditions there are ~ 9.7 channels/µm2 and 3.5 Gβγ molecules/channel. It is also assumed that under PTX expression conditions most endogenous Gαi3 is ADP-ribosylated and subsequently degraded.
	Figure 6 Simulation of GIRK1/2 activation according to cooperative gating model. (A) – Simulated values of Ievoked obtained for a range of M2R densities. (B) – τact of mono-exponential fit of simulated Ievoked obtained for a range of M2R densities. For A and B, the rate constants were taken from Touhara et al. (1) and it was assumed that Gβγ =16 molecules/channel and Gα = 9 molecules/channel (red circles). (C) – Simulated Ievoked obtained from simulation with khydrolysis = 0.02 s-1 and a range of M2R densities. (D) - τact of mono-exponential fit of time-course from simulation of Ievoked with khydrolysis = 0.02 s-1 and a range of M2R densities. For calculations done in (C, D), khydrolysis was assumed to be 0.02 s-1 and Gβγ = 9 molecules/channel and Gα = 2 molecules/channel (blue circles).
	Supplemental Figure S1 | Kinetic measurements of time course of AMPA-R activation. (A) –configuration of the experimental chamber used for fast perfusion experiments. (B)– a representative record (black dots) of current evoked by 1 mM glutamate. The red dots represent mono-exponential fit of the activation time course of the current (τact=49.5 s).
	Supplemental Figure S2 | Linear relationship between Gβγ-activated YFP-GIRK1/GIRK2 current and total surface density is observed in the range 0-1 ng of YFP-GIRK1 RNA. (A) – representative confocal images of oocytes expressing YFP-GIRK1/GIRK2. Channels were expressed by injecting the indicated doses of RNA of YFP-GIRK1 and GIRK2 (1:1) and activated by coexpression of 5 ng Gβ and 1 ng Gγ RNA. (B) – correlation between total GIRK surface density and the Gβγ-dependent GIRK current, Iβγ. Iβγ is the total agonist-independent current in Gβγ-expressing oocytes. It was measured in 24 mM K+ solution by subtracting the non-GIRK currents remaining after inhibition of >95% GIRK activity by 5 mM Ba2+ (Rubinstein et al., 2007). Total surface density is reflected in YFP fluorescence levels, functional channel density is proportional to Iβγ. Correlation between Iβγ and fluorescence is linear for RNA doses of YFP-GIRK1/GIRK2 up to 1 ng/oocyte of each subunit. n = 6-12 for each point.
	Supplemental Figure S3 | Estimation of Gβγ and Gα densities for simulation with cooperative gating model. (A) – Ibasal currents rendered by different pairs of Gβγ and Gα densities selected for simulation. (B) – Itotal currents rendered by different pairs of Gβγ and Gα densities selected for simulation.Red bars indicate Gβγ and Gα densities used for calculations in Figures 6A, B. Blue bars indicate Gβγ and Gα densities used for calculations in Figures 6C, D. All calculations except the 9:2 Gβγ/Gα pair (blue bar) have been made for the condition of fast hydrolysis of GTP, khydrolysis=2 s-1. The 16:9 Gβγ/Gα pair was the lowest dose of G protein subunits per channel that reproduced Itotal under this condition. Lower doses of Gβγ/Gα produced lower Itotal. The 9:2 Gβγ/Gα pair was the lowest G protein subunits combination that reproduced Itotal under the condition of low GTP hydrolysis rate (khydrolysis=0.02 s-1).
	Figure 1. Increasing expression levels of M2R accelerates the activation of GIRK1/2. (A) – a representative GIRK1/2 activation experiment. Oocytes were injected with the following RNAs: GIRK1 and GIRK2 (2 ng/oocyte each) and 500 pg/oocyte M2R. Ievoked was elicited by 10 µM ACh. Inset- zoom-in on the activation phase of Ievoked (black plot) and a mono-exponential fit (red). (B) – kinetics of GIRK1/2 activation. Oocytes expressed a constant amount of GIRK1/2 (2 ng RNA/oocyte), with increasing levels of M2R, in the range 10-1000 pg/oocyte, and τact was determined by monoexponential fitting as shown in A (N=2-7 experiments, n= 13-25 cells).
	Figure 2. Estimating the surface density of M2R-YFP. All data are from one experiment. (A) - (left) Representative micrographs of oocytes (membrane at equator) expressing equal amounts of RNAs of YFP-GIRK1 and GIRK2, and 1 ng M2R (wt). Injected YFP-GIRK1 mRNA amounts are indicated. (right) Representative currents from oocytes from the same experiment (from the same groups as in left panels). Note the gradual increase in Ibasal and Itotal, reaching a plateau at 2 ng/oocyte of channel’s RNA. The current measurements in this experiment were done with a slower perfusion rate and were not included in the kinetic analysis. (B) – Representative micrographs of oocytes, injected with the indicated amounts of M2R-YFP, imaged at the same day and under identical settings as those in A. (C) – Itotal of YFP-GIRK1/GIRK2 channels as a function of channel’s RNA dose. (D) – calibration of the surface density of YFP. YFP-GIRK1 fluorescence (in arbitrary units, AU) is plotted versus channel density induced by the two lowest doses of GIRK RNA (<1 ng/oocyte), within the linear range of fluorescence-current relationship. Channel density was calculated from Itotal as explained in the text. The correlation between fluorescence and number of YFP-GIRK1 molecules is shown with superimposed linear regression line, extended to origin of coordinates. The regression equation was y=4x, i.e. one channel/µm2 corresponds to fluorescence intensity of 4 AU. Note that, since each channel has two YFP molecules, the calibration factor in this experiment is: 1 YFP molecule/µm2 = 2 AU. (E) –estimating the surface density of M2R-YFP, for RNA concentrations of 1, 2 and 5 ng/oocyte. YFP fluorescence, in AU, is shown on the left Y-axis. M2R-YFP surface density (right axis) was calculated using the calibration factor derived from YFP-GIRK1/GIRK2 measurements. The relationship between M2R-YFP RNA dose and M2R-YFP surface density was fitted with linear regression, extended to the origin of coordinates, in the form y=62.5x.
	Figure 3. Quantitative analysis of dose-dependency of τact and amplitude of Ievoked on plasma membrane density of M2R-CFP. (A)-Whole cell radioligand-labeling by quinuclidinyl benzilate (QNB*) shows that identical doses of injected RNA produce similar surface expression of M2R, M2R-CFP and M2R-YFP, for three different amounts of receptor RNAs. B-F, quantitative analysis of incremental expression of M2R-CFP reveals collision-coupling activation of YFP-GIRK1/GIRK2 channels. Oocytes were injected with constant mRNA amounts of YFP-GIRK1 and GIRK2 (1 ng each) but with increasing doses of M2R-CFP RNA. (B) – RNA dose-dependent increase in the surface levels of M2R-CFP. Representative micrographs of oocytes (left) expressing m2R-CFP and summary of expression (right). RNA amounts of M2R-CFP RNA, in ng/oocyte, are indicated in yellow. n=7-12 oocytes in each group. (C) – expression of M2R-CFP does not affect the surface level of YFP-GIRK1/GIRK2 except for the decrease at the highest dose of M2R-CFP, 25 ng/oocyte (**, p<0.01). Left panel shows representative micrographs of YFP-GIRK1/GIRK2 – expressing oocytes. Numbers within the images indicate the amounts of M2R-CFP RNA, in ng/oocyte. Right panel shows summary of expression data (n=7-12 oocytes in each group). (D) – representative normalized Ievoked, elicited by 10 µM ACh in oocytes injected with the indicated amounts of M2R-CFP RNA. For simplicity, only the initial (activation) phase of Ievoked is shown. (E, F) – τact is reduced (E) and Ievoked amplitude (F) is increased with increased surface density of M2R-CFP. AU, arbitrary units. τact and Ievoked data are from cells exemplified in B – D; n=5-12 in each group.
	Figure 4. Simulating activation kinetics and its dependence on M2R surface density. (A) – scheme of the G-protein cycle. (B) – graded contribution model of GIRK1/2 activation. Rate constants of reactions shown in A and B are summarized in Table 1. (C) –comparison of the experimentally observed and predicted ACh-evoked currents, with GIRK-Gβγ interaction parameters from two structural models, 4KFM and BS. (D) - representative analysis of the time course of GIRK1/2 activation. The experimental result (dotted black line) is from an oocyte injected with 0.5 ng M2R RNA. Superimposed are simulated currents according to graded contribution model of GIRK1/2 activation. The experimental parameters in this cell were: Ibasal = 15.5 µA, Itotal= 22.8 µA. Estimated channel density was 36 channels/µm2. Initial concentrations of Gα and Gβγ available to the channel are: for the case of 4KFM model 3.23 molecules Gαi/channel and 0.45 molecules Gβγ/channel; for the case of BS model, 3.24 molecules Gαi/channel and 0.46 molecules Gβγ/channel. Each plot represents the recorded or simulated current normalized to its maximum. (E) – mean τact values from all experiments with wild-type M2R, superimposed on data obtained from fitting of simulated time-courses according to different models. Each experimental point shows mean value of τact ± SEM from one experiment (n=3-7 oocytes). Simulated time courses were generated for the case described by Yakubovich et al. as “high density group”, i.e. Ibasal =13.36 µA, Itotal =17.2 µA, n=21 channels/µm2. Amounts of available Gα and Gβγ molecules per one channel, that are required to obtain the observed Ievoked, were calculated using the graded contribution model: with the BS structure-based parameters, 3.65 Gβγ and 0.39 Gα molecules/channel; with the for 4kfm structure-based parameters, 3.62 Gβγ and 0.38 Gα molecules/channel.
	Figure 5. Physical tethering of M2R and Gαi3 converts a collision coupling mechanism to a preformed-complex: experiment and simulation. (A) – Incremental expression of fused M2R-Gαi3C351G (PTX-insensitive) in the presence of coexpressed A-protomer of pertussis toxin (PTX; 0.2 ng RNA/oocyte) shows increase in Ievoked with growing amounts of injected RNA (black plot), but kinetics of activation remain unchanged (red plot). Right- Histogram of evoked currents (black) and τact (red) of GIRK1/2 coexpressed with M2R-wt. Result shown are from one experiment; number of cells (n) tested in each group are shown above experimental points or in the bar. No significant change in τact was found (one way ANOVA, P = 0.154). Spearman correlation coefficient calculated for analysis of evoked currents ~ 1, P = 0.0167. (B) – scheme of G-protein utilized to simulate GIRK1/2 activation by M2R-Gαi3C351G. Blue arrows and numbering denote reactions that are shared with M2R wt activation pathway, as described in Figure 4. Red arrows denote reactions present only in the current scheme. The numbering of reactions and the rate constants are the same as in Table 1. (C) – simulated Ieoked values obtained assuming a range of expression level of fused M2R-Gαi3C351G. (D) – summary of τact obtained from fitting time-course of activation of GIRK1/2 by range of M2R-Gαi3C351G densities with mono-exponential function. Three possible scenarios were simulated for analysis of M2R-Gαi3C351G experiments. Black bars; M2R-Gα concatemer is assumed to have same affinity to Gβγ as Gα, and no change in Gβγ concentration is assumed with concatemer expression. Red bars; M2R-Gα concatemer is assumed to have same affinity to Gβγ as Gα, and 1:1 increase in Gβγ concentration is assumed with concatemer expression. Green bars; M2R-Gα concatemer is assumed to have 10-fold lower affinity to Gβγ than Gα and no change in Gβγ concentration is assumed with concatemer expression. For simulation it was assumed that GIRK1/2 is expressed at levels similar to “intermediate density group” described in Yakubovich et al. (2015) i.e. under pre-expression conditions there are ~ 9.7 channels/µm2 and 3.5 Gβγ molecules/channel. It is also assumed that under PTX expression conditions most endogenous Gαi3 is ADP-ribosylated and subsequently degraded.
	Figure 6. Simulation of GIRK1/2 activation according to cooperative gating model. (A) – Simulated values of Ievoked obtained for a range of M2R densities. (B) – τact of mono-exponential fit of simulated Ievoked obtained for a range of M2R densities. For A and B, the rate constants were taken from Touhara et al. (1) and it was assumed that Gβγ =16 molecules/channel and Gα = 9 molecules/channel (red circles). (C) – Simulated Ievoked obtained from simulation with khydrolysis = 0.02 s-1 and a range of M2R densities. (D) - τact of mono-exponential fit of time-course from simulation of Ievoked with khydrolysis = 0.02 s-1 and a range of M2R densities. For calculations done in (C, D), khydrolysis was assumed to be 0.02 s-1 and Gβγ = 9 molecules/channel and Gα = 2 molecules/channel (blue circles).

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