Gi/o-coupled muscarinic receptors co-localize with GIRK channel for efficient channel activation

G protein-gated inwardly rectifying K+ (GIRK) channel regulates cellular excitability upon activation of Gi/o-coupled receptors. In Gi/o-coupled muscarinic M2R, the intracellular third loop (i3) is known as a key domain for Gi/o coupling, because replacement of i3 of Gq-coupled muscarinic M1R with that of M2R enables the chimeric receptor (MC9) to activate the GIRK channel. In the present study, we showed that MC9, but not M1R, co-localizes with the GIRK channel and Gαi1 by Förster resonance energy transfer (FRET) analysis. When M1R was forced to stay adjacent to the channel through ligation with short linkers, M1R activated the GIRK channel. FRET analysis further suggested that the efficacy of channel activation is correlated with the linker length between M1R and the GIRK channel. The results show that co-localization is an important factor for activating the GIRK channel. In contrast, for MC9 and M2R, the GIRK channel was activated even when they were connected by long linkers, suggesting the formation of a molecular complex even in the absence of a linker. We also observed that replacement of 13 amino acid residues at the N-terminal end of i3 of MC9 with those of M1R impaired the co-localization with the GIRK channel as well as channel activation. These results show that localization of the receptor near the GIRK channel is a key factor in efficiently activating the channel and that the N-terminal end of i3 of M2R plays an important role in co-localization.


Introduction
G protein-gated inwardly rectifying potassium (GIRK) channel is a key protein that regulates cellular excitability and is activated by interacting with free Gβγ released from the heterotrimeric Gαβγ complex upon the activation of G protein-coupled receptors (GPCRs) [1]. The conformational rearrangement between the GIRK channel and Gβγ has been observed upon the receptor activation on the living cell membrane by Förster or bioluminescence resonance energy transfer analyses (FRET or BRET, respectively) [2,3] and the GIRK-Gβγ complex formation was demonstrated by structural analyses [4]. The GIRK channel is also known to bind to Gα i and Gα o in their resting and active forms [5][6][7][8], suggesting that Gβγ released from Gα i or Gα o immediately interacts with and activates the GIRK channel. In addition, several Gi/ocoupled receptors (Gi/o-Rs) have been reported to localize near Gi/o by FRET and BRET analyses [9][10][11]. Therefore, Gi/o-Rs, Gi/o and the GIRK channel have been suggested to form a ternary pre-signaling complex to effectively activate the GIRK channel.
Gi/o-Rs activate the GIRK channel through releasing Gβγ from pertussis toxin (PTX)-sensitive Gα i/o [1,12]. When the receptors are highly expressed, the GIRK channel is activated by receptors that stimulate PTX-resistant G protein, such as the Gq-coupled muscarinic receptors (M 1 R and M 3 R) or Gs-coupled adrenergic receptors (β1-AR and β2-AR) [13,14]. When surface expression of M 1 R is very high, the chance of M 1 R-Gi/o coupling may increase [13] and/ or the distance between M 1 R and GIRK channel may be shortened, which could allow Gβγ released from Gq to activate the channel. Interestingly, Gs-coupled β2-AR has been reported to functionally and physically interact with GIRK channels [15,16]. In addition, the rate of GIRK channel activation was thought to correlate with the co-localization of Gi/o-Rs with the channel [17]. We thought that the distance between GPCR and the GIRK channel is a critical factor for effective channel activation.
In this study, we first showed by FRET analyses that a chimeric receptor of M 1 R and M 2 R (MC9) [18], which activates the GIRK channel [19], locates adjacent to the channel and Gα i1 in transfected HEK293T cells. Next, we examined whether Gq-coupled M 1 R activates the GIRK channel when these molecules are forced to stay close to each other by ligating them with short linkers. The key amino acid residues in complex formation for activating the GIRK channel were further investigated by analyzing the changes in co-localization of MC9 mutants with the GIRK channel.

Electrophysiology
Macroscopic membrane currents were recorded from cells expressing fluorescent constructs by the whole cell patch clamp technique using Axopatch 200B amplifiers, Digidata1332A, and pClamp 9 software (Molecular Devices, San Jose, CA, USA). After establishing the whole cell configuration, the cell was held at -80 mV and ramp pulses (from -120 to +40 mV for 400 s) were applied every 5 s. The bath solution was composed of the following: 140 mM NaCl, 4 mM KCl, 1 mM CaCl 2 , 0.3 mM MgCl 2 , 10 mM HEPES (pH 7.4 adjusted with NaOH). The internal solution was composed of: 130 mM KCl, 5 mM Na 2 -ATP, 3 mM EGTA, 0.1 mM CaCl 2 , 10 mM HEPES, 4 mM MgCl 2 , 0.3 mM GTP-Li 3 (pH 7.3 adjusted with KOH). An agonist of muscarinic receptors, oxotremorine M (oxo-M), was applied by using fast perfusion system (VC-77SP, Warner Instruments, Hamden, CT, USA) as previously described [26]. To record the GIRK channel current, Na + ions in the bath solution were substituted with K + to enhance the driving force of K + and oxo-M was applied in 140 mM K + bath solution. The average of the holding current amplitude for 100 ms before applying the ramp pulse was measured and then normalized to the cell capacitance to calculate the current density (I). The basal current density (I 0 ) before application of oxo-M (10 μM) was subtracted from the maximum value (I max ) after agonist application to evaluate the agonist-induced current density (ΔI max ). To analyze the decay of the GIRK current, the ratio of the agonist-induced current density before washout of the agonist (ΔI last ) to (ΔI max ) was calculated. We also analyzed the onset of GIRK channel activation, with the application of oxo-M for 5 s controlled by the combination of Clampex9 and VC-77SP. The time to half-maximum current amplitude (t 1/2 ) was then measured for each trace.

Imaging and FRET analysis
Fluorescent images were obtained from cells expressing FP-fused constructs using a total internal reflection fluorescence (TIRF) microscope (Olympus, Tokyo, Japan) as previously described [25]. MetaFluor imaging software (Molecular Devices, Sunnyvale, CA, USA) was used to control the excitation of CFP and YFP and to acquire images. For FRET analysis, five images were acquired before and after acceptor bleaching, which was performed by excitation of YFP with a 515-nm laser line for 1 min (emission intensity of YFP was decreased by less than 5% in the bleaching procedure). Averaged emission intensities of CFP (I CFP ) and YFP (I YFP ) before and after acceptor bleaching were measured in each cell by subtracting their background intensities and were then normalized to the cell size. To calculate FRET efficiency, the increase in I CFP after acceptor bleaching was normalized to the total I CFP after bleaching in each cell. To make the CFP emission images, five CFP images before and after acceptor bleaching were averaged and their subtraction was performed by using MetaMorph software (ver 6, Molecular Devices).
To evaluate the activation of Gqi5 by MC9 constructs, the intensity of CFP-PH (I CFP-PH ) was measured under TIRF illumination. Cells expressing the fluorescent constructs were continuously perfused with bath solution by gravity at a rate of approximately 3 mL/min, and various concentrations of oxo-M were applied by changing the perfusion solution. The baseline I CFP-PH was the averages of I CFP-PH measured from 13 images before oxo-M application. The maximal decrease in I CFP-PH upon application of oxo-M (ΔI CFP-PH ) was normalized to the baseline I CFP-PH in each cell.

Statistical analysis
All data are expressed as the means ± S.E., with n indicating the number of data. Statistical significance between two groups was examined by unpaired Student's t-test and that between more than two groups was tested by one-way analysis of variance (ANOVA) followed by Tukey's test. Values of p 0.05 were considered statistically significant ( Ã :0.01<p 0.05, ÃÃ :0.001<p 0.01, ÃÃÃ :p 0.001, n.s.:p>0.05). Pearson correlation coefficients were calculated using Microsoft Excel2013.

i3 of M 2 R is a key structure in activating and staying adjacent to the GIRK channel
Application of an agonist oxo-M increased the amplitude of the inward current density in cells transfected with the GIRK channel and Gi/o-coupled M 2 R, but not with the channel and Gqcoupled M 1 R (Fig 1A and 1B, left and center panels). The amplitude of the agonist-induced GIRK channel current density (ΔI max ) was decreased to almost null by treating the cells with PTX (300 ng/mL) (Fig 1C left panels), showing that the effect of M 2 R is mediated by the activation of Gi/o. Replacement of the i3 of M 1 R with that of M 2 R (M 2 R-i3) enabled the chimeric receptor (MC9) to activate the GIRK channel, as previously reported [19]. The effect of MC9 was inhibited by PTX treatment (Fig 1C right panels) and the speed of current increase induced by the MC9 activation did not differ from that by M 2 R (S1 Fig), indicating that M 2 R-i3 is a critical structure for Gi/o coupling and GIRK channel activation. We then examined whether or not MC9 locates adjacent to the GIRK channel by analyzing FRET from GIRK1/ 2-CFP to receptor-YFP. In HEK293T cells expressing these constructs, acceptor bleaching resulted in the increases in intensities of CFP (I CFP ) (Fig 2A). Interestingly, the FRET efficiency from GIRK1/2-CFP to MC9-YFP was larger than that to M 1 R-YFP (Fig 2B left panels). As the surface expression level of the FP-tagged constructs were not different (S1 Table), MC9 but not M 1 R was suggested to co-localize with the GIRK channel. The FRET efficiency did not change by application of oxo-M (Fig 2B), suggesting that activation of receptor does not change the co-localization. We also analyzed the FRET from Gα i1 -CFP to receptor-YFP in HEK293T cells transfected together with Gβ 1 and Gγ 2 . The FRET efficiency from Gα i1 -CFP to MC9-YFP was higher than that to M 1 R-YFP both in the absence and presence of the agonist (Fig 2B bottom bars). The results support pre-coupling of MC9 with Gα i1 and show that precoupling is not changed by MC9 activation. The interaction between GIRK1/2 and Gα i1 showed higher FRET efficiency than that between GIRK1/2 and Gα q (Fig 2B, right panel), which is consistent with previous reports [5][6][7][8]. As a negative control for the FRET analysis, we chose a member of an anion-transporter family, prestin [24], since prestin is not expected to selectively interact with the GIRK channel and G protein. FRET values from CFP tagged constructs to prestin-YFP were similar to those to M 1 R-YFP (Fig 2), suggesting that M 1 R does not specifically interact with GIRK channel and Gα i1 . Taken together, MC9, GIRK, and Gi/o are adjacent to each other and may form a molecular complex.

Distance between receptor and GIRK1/2 channel is a key determinant for channel activation
The FRET analysis raised a question about the functional significance of co-localization. To address this question, Gq-coupled M 1 R was forced to stay adjacent to the GIRK channel by connecting them with a short linker composed of 34 glycine-rich amino acid (a.a.) residues ( Fig 3A). Application of oxo-M (10 μM) elicited a rapid increase in the amplitude of the inward current in cells transfected with M 1 R-YFP-34-GIRK1/2 (Fig 3B red lines). This effect remained after PTX treatment ( Fig 3C, Table 1), suggesting that Gβγ released from PTX-resistant Gα q activated the channel. The amplitude of the GIRK channel current decreased by approximately 30% at 25 s after application of oxo-M, which may have resulted from negative regulation induced by Gq signaling [27]. When the linker length was elongated, the GIRK channel was not activated; ΔI max was decreased in accordance with the increase in the linker length of the Cells were held at -80 mV and the ramp pulse (-120 to 40 mV for 400 ms) was applied every 5 s. The black bars on the traces indicate the timing of agonist application. Basal and maximal current amplitude was measured at the holding potential before (a) and during (b, red) oxo-M application. (B) Expanded traces corresponding to "a" (black lines) and "b" (red lines) are shown in middle panels. The ramp pulse protocol is shown above the expanded trace. The agonist-induced current densities at a holding potential of -80 mV (ΔI max ) was measured. (C) Summary of ΔI max is shown as bars. Numbers of data are shown in parentheses. ÃÃÃ :p 0.001, n.s.:p>0.05.
https://doi.org/10.1371/journal.pone.0204447.g001 M 1 R tandem constructs (Fig 3D open circles). In contrast, the application of oxo-M evoked a rapid increase in the inward current, even when MC9-YFP was connected to GIRK1/2 by a long linker of 535 a.a. residues (Fig 3E and 3F). The response was totally mediated by PTX-sensitive Gi/o ( Fig 3G, Table 1). Similar results were observed when the linker length was 100 or 265 a.a. (Fig 3H, Table 1), indicating that the effect of MC9 does not change by the linker length. ΔI max in the MC9 tandem constructs were 30% smaller than that in non-linker combination, but the difference was not statistically significant ( Fig 3H). The ligation of the receptor and the GIRK channel may have a slight inhibitory effect on the expression and/or activation of the GIRK channel. These results suggest that MC9, but not M 1 R, is in proximity of the GIRK channel, even when the linker is long, presumably by forming a molecular complex.
Next, the FRET efficiency between GIRK1/2-CFP and M 1 R-or MC9-YFP was measured for each tandem construct. FRET efficiency in the M 1 R tandem construct was decreased in accordance with the increase in the linker length (Fig 4A open diamonds), whereas that in the MC9 construct was not changed by the change of the linker length in the presence or absence of the agonist (Fig 4B triangles). For M 1 R, FRET efficiency was well-correlated with ΔI max ( Fig  4C, Pearson correlation coefficient is 0.98). The results suggest that receptor-GIRK signaling tightly depends on their distance. For MC9, the FRET efficiency and ΔI max did not change when the linker length was changed (Fig 4C, Pearson correlation coefficient is 0.53). These results support that MC9, but not M 1 R, forms a complex with the GIRK channel for efficient channel activation.

M 2 R forms a complex with GIRK channel
M 2 R-i3 is the key structure in complex formation with the GIRK channel and Gi/o (Figs 1 and  2). Thus, the results of the M 2 R tandem constructs connected to the GIRK channel by various linker residues were expected to be similar to those observed in MC9 constructs (Figs 3H and 4B). As expected, ΔI max as well as FRET efficiency were not changed with the increases in the linker length (Fig 5A and 5B) and ΔI max was not positively correlated with FRET efficiency (Fig 5C, Pearson correlation coefficient is -0.79). GIRK current increase induced by M 2 R was   completely inhibited by the PTX treatment ( Table 2), indicating that the effects were mediated by the activation of Gi/o in the M 2 R tandem constructs. The FRET efficiency from GIRK to M 2 R was smaller than that to MC9, which may be due to the difference in the C-terminal tail of the receptor at which YFP is tagged. Gi/o was suggested to be located in proximity to M 2 R as well as the GIRK channel; the FRET efficiency from Gα i1 -CFP to M 2 R-YFP was 2.8 ± 0.5 (n = 12, p = 0.001 compared to FRET from Gα i1 -CFP to prestin-YFP in Fig 2B). Taken together, these results indicate that M 2 R, Gi/o, and the GIRK channel form a molecular complex.

Dual roles of M 2 R-i3 in the activation of and co-localization with GIRK
We then evaluated the residues in M 2 R-i3 responsible for activation of and co-localization with the GIRK channel and activation of Gi/o. It was previously reported that most of the long M 2 R-i3 is not necessary for Gi/o coupling [28]. Indeed, the replacement of the long chain in the middle of M 2 R-i3 (K221-P379) with SGGGS did not decrease ΔI max and the FRET efficiency (ΔI max = 75.1 ± 12.5 pA/pF, n = 10; FRET = 5.5 ± 0.9%, n = 15, cf Figs 1 and 2). Thus, the proximal N-and distal C-terminal residues of M 2 R-i3 may be critical regions; the charged residues at the proximal N-terminal end of M 2 R-i3 as well as Val and Thr residues at the distal C-terminal end have been suggested to play important roles in Gi/o coupling [19,28,29]. Residues at the N-terminal end were replaced with those of M 1 R (MC9A-YFP and MC9B-YFP, Fig 6A) and also double mutations (V387A/T388A) were introduced at the C-terminal end. The emission intensities of YFP (I YFP ) fused to these constructs under TIRF illumination did not differ (I YFP in S2 and S3 Tables), indicating that their surface expression levels were similar. We first measured ΔI max and FRET from GIRK1/2-CFP to chimeric and mutant receptor-YFP. Although it was not statistically significant, ΔI max in VT/AA mutants was 30% smaller than that in MC9-YFP (Fig 6B), possibly due to the impairment of the activation of Gi/o. Interestingly, ΔI max in MC9B-YFP was significantly smaller than that in MC9-YFP (Fig 6B). Similarly, the FRET efficiency from GIRK1/2-CFP to MC9B-YFP, but not to MC9-VT/AA-YFP, was smaller than that to MC9-YFP (Fig 6C). ΔI max and FRET values appeared to be correlated in MC9-YFP chimeric constructs (Fig 6D): Pearson correlation coefficient is 0.79 (+VT/AA) and 0.97 (-VT/AA). Next, we examined the effect of the chimeric and double mutation on the Gi/o activation by using Gα qi5 , which is activated by Gi/o-Rs and stimulates Gq-phospholipase C (PLC) signaling [22]. As the last five a.a. residues of Gα qi5 are derived from Gα i1 and include the ADP-ribosylation site, Gα qi5 is inhibited by PTX. The activity of Gα qi5 signaling was monitored as the decrease in fluorescent intensity of the CFP-tagged PH domain (CFP-PH) under TIRF illumination (Fig 6E upper panel). Upon PIP 2 hydrolysis, CFP-PH translocates from the membrane into the cytosol, where TIRF illumination cannot reach [23,26], resulting in a decrease in the fluorescent intensity of CFP-PH (I CFP-PH ). Indeed, activation of MC9-YFP constructs decreased I CFP-PH in the presence of Gα qi5 (Fig 6E, lower left open circles). The amplitude of the decrease in I CFP-PH (ΔI CFP-PH ) was almost null when cells were treated with PTX ( Fig 6E filled circles and Table 3), suggesting that ΔI CFP-PH may reflect the efficacy of the receptor to activate Gi/o. As previously reported in studies of M 2 R [28,29], VT/AA attenuated Gα qi5 activation: ΔI CFP-PH in the VT/AA mutant was smaller than that in MC9-YFP (Fig 6F). In contrast, ΔI CFP-PH was not changed by chimeric mutations of MC9A and MC9B (Fig 6F). These results suggest a possibility that all MC9 constructs are equally not co-localized with Gα qi5 and/or PLC. Another possibility is that there is a difference in the extent of co-localization with Gα qi5 between the MC9, MC9A and MC9B, and that the difference of the co-localization does not change the extent of PLC activation. If this is the case, co-localization might not be critical for the PLC activation, highlighting the significance of co-localization for the GIRK channel activation. We also analyzed FRET from Gα i1 -CFP to MC9B-YFP and found that colocalization with Gα i1 appeared to be attenuated, but the difference was not significant (S3 Table). Taken together, at least the 13 a.a. residues at the N-terminal end of M 2 R-i3 play

Coupling properties of MC9B and MC9-VT/AA
The oxo-M concentration dependence of MC9B-YFP and MC9-VT/AA-YFP was then investigated. Examination of the concentration-ΔI CFP-PH relationship (Fig 7A) revealed that EC 50 did not differ between MC9 and MC9B. In the case of VT/AA mutant, 100 μM oxo-M was not the saturating concentration which made the estimation of EC 50 impossible and support that VT/ AA mutations impaired the Gα qi5 activation. The concentration-ΔI max of MC9-VT/AA-YFP showed that EC 50 of the mutant was 5-fold larger than that of MC9-YFP (Fig 7B). The lower affinity may represent impaired Gi/o activation. In the case of MC9B, the EC 50 was 2-fold larger than that of MC9 (Fig 7B filled circles). These results indicate that the coupling efficacy between MC9B and the GIRK channel was reduced. As the speed of the GIRK channel activation is another index of signaling efficacy, we analyzed the activation speed by using a motordriven fast-perfusion system to control agonist application. The time to reach the half-maximal current (t 1/2 ) upon activation of MC9B was longer than that of MC9 (Fig 7C), consistent with the results showing that GIRK and MC9B are distant from each other [17]. Taken together, the N-and C-terminal ends of M 2 R-i3 were shown to have different roles in receptor-GIRK signaling.

Discussion
We showed that M 2 R-i3 enables the chimeric MC9 to locate adjacent to Gα i1 and the GIRK channel, that Gq-coupled M 1 R activates the GIRK channel when it is in proximity to the Co-localization of GIRK channel and muscarinic receptors

Interaction between Gi/o-coupled muscarinic receptors, GIRK and Gi/o
Localization of MC9 or M 2 R in proximity to the GIRK channel was shown by FRET analysis and experiments using tandem constructs (Figs 2-5). These results were consistent with those of the previous FRET study of metabotropic receptor GABA B R, Gi/o, and GIRK [10]. As suggested for other Gi/o-Rs [9][10][11], MC9 and M 2 R are likely to be pre-coupled with Gi/o (Fig 2B). Because Gα i/o binds to the GIRK channel either in GTP-bound form or GDP-bound form [5][6][7][8]30], the Gi/o-GIRK coupling is thought to play important roles in the ternary complex formation. In fact, replacement of the helical domain of Gα q with that of Gα i was reported to enable the chimeric G protein to interact with the GIRK channel and the Gq-coupled M 1 R to activate the channel in cells treated with PTX [30]. Therefore, M 2 R and MC9 were suggested to localize near the GIRK channel by pre-coupling with Gi/o. In the case of MC9 chimeric mutants, MC9B showed significant impairment of the activation of and co-localization with the GIRK channel (Fig 6). Although it was not statistically significant in MC9B, the co-localization with Gα i1 was attenuated (S3 Table). The lack of statistical significance (MC9B-YFP vs MC9-YFP, S3 Table) was possibly due to the small FRET efficiency between Gα i1 -CFP and YFP tagged constructs relative to the background level. We think that the proximal N-terminal residues of M 2 R-i3 is a key region for the pre-coupling of M 2 R and MC9 with Gi/o. However, another possibility that the proximal N-terminal residues may play some roles in direct interaction with the GIRK channel cannot be excluded. FRET efficiency between MC9, Gi/o, and the GIRK channel was not changed by receptor activation (Figs 2B and 4B). These results were different from those of previous FRET studies, in which agonist-induced increases in the FRET efficiency was shown between Gq-Rs and Gα q [25,26] or between adenosine receptor A1R and Gi1 [31]. Similar FRET increases have been reported between adrenergic receptors (α2A-AR and β1-AR) and G protein [32,33]. As revealed in a recent single-molecule imaging study of G proteins and α2A-AR or β1-AR, receptors and G proteins dynamically associate with each other [34]. These reports supported the notion that the activated receptors associate with and activate G protein, which is detected as agonist-induced increases in FRET between receptor and G protein. In contrast, agonistinduced FRET increases were not detected in several Gi/o-Rs, which were suggested to precouple with Gi/o [9][10][11]. These reports are consistent with the results showing that pre-coupling is not changed by receptor activation. Interestingly, an agonist-induced decrease in FRET was observed between the opioid receptor and Gα i1 [9]. Therefore, the receptor-G protein interaction may differ depending on the receptor type.

Difference in efficacy of GIRK channel activation between M 1 R and MC9
M 1 R activates the GIRK channel only when it is in proximity of the channel by linking them with a short linker (Fig 3), but ΔI max upon activation of M 1 R was lower than that of MC9 ( Fig  3D and 3H). As FRET efficiency did not differ between the M 1 R and MC9 constructs ligated with the GIRK channel by a short linker (Fig 4A and 4B), the relative distance from M 1 R to the channel is likely to be similar to that from MC9. The weak effect of M 1 R on the GIRK channel activation may be because of the low abundance of the endogenous Gα q subunit, as (p 0.001 vs MC9-YFP). (C) Activation kinetics of GIRK channel current upon oxo-M application. Traces represent the GIRK channel current recorded from cells expressing MC9-YFP constructs and GIRK1/2. The application of oxo-M (10 μM) was controlled by a rapid perfusion system. Bars in right represent the half time to maximum (t 1/2 ). Numbers of experiments are indicated in parentheses. Ã :0.01<p 0.05, n.s.:p > 0.05. https://doi.org/10.1371/journal.pone.0204447.g007 co-expression of Gα s markedly increased the GIRK channel current amplitude for β2-AR [16]. We thus co-expressed Gα q with M 1 R-YFP-100-GIRK1/2, but co-expression failed to increase the amplitude; ΔI max was 6.4 ± 4.5 pA/pF (n = 3). This may be because of the inhibitory effects of Gα q on the GIRK channel [27,[35][36][37]. In fact, co-expression of Gα q with M 2 R-YFP-100-GIRK1/2 inhibited the effect of M 2 R on the GIRK channel; ΔI max was 3.6 ± 2.5 pA/pF (+Gα q , n = 4) and 78.2 ± 15.9 pA/pF (-Gα q , n = 5, P = 0.002). Therefore, the weak effect of M 1 R on the GIRK channel activation can be explained by the inhibitory effects of Gα q on the GIRK channel.

Localization in proximity is a key factor for activating GIRK channel
In MC9-YFP, the EC 50 of GIRK channel activation was 4-fold lower than that of Gα qi5 activation (Fig 7). This can be interpreted as the functional role of receptor-GIRK channel co-localization; localization of MC9 in the proximity to the GIRK channel enhances the efficacy of receptor-GIRK channel signaling through increasing a probability for MC9 to associate with complex of Gi/o and GIRK channel (Fig 2C Gα i1 vs. Gα q ). Interestingly, in the case of MC9B which impaired co-localization with the GIRK channel (Fig 6C), the difference of EC 50 between ΔI CFP-PH and ΔI max was attenuated. The EC 50 of GIRK channel activation was 2-fold lower than that of Gα qi5 activation (Fig 7A and 7B), which may be results of the decreases in a probability for MC9B to associate with Gi/o-GIRK channel complex. In contrast, mutations at the C-terminal end of M 2 R-i3 (VT/AA mutation) impaired the activation of Gα qi5 (Figs 6F and 7A), consistently with previous reports in which VT/AA mutation of M 2 R disrupted the Gi/o activation [27,28]. As for the GIRK channel activation, the double mutation did not abolish ΔI max but shifted the concentration-ΔI max relationship rightwards ( Fig 7B). As colocalization with the GIRK channel was not affected (Fig 6C), impairment of Gi/o activation in the double mutant may be compensated by co-localization, which enhances receptor-GIRK signaling.
In conclusion, we showed that the Gi/o-coupled muscarinic receptors co-localize with the GIRK channel and Gα i1 , which is mediated by at least 13 a.a. residues at the N-terminal end of M 2 R-i3, and that co-localization is another determinant of the efficacy of channel activation.