GPCR voltage dependence controls neuronal plasticity and behavior

G-protein coupled receptors (GPCRs) play a paramount role in diverse brain functions. Almost 20 years ago, GPCR activity was shown to be regulated by membrane potential in vitro, but whether the voltage dependence of GPCRs contributes to neuronal coding and behavioral output under physiological conditions in vivo has never been demonstrated. Here we show that muscarinic GPCR mediated neuronal potentiation in vivo is voltage dependent. This voltage dependent potentiation is abolished in mutant animals expressing a voltage independent receptor. Depolarization alone, without a muscarinic agonist, results in a nicotinic ionotropic receptor potentiation that is mediated by muscarinic receptor voltage dependency. Finally, muscarinic receptor voltage independence causes a strong behavioral effect of increased odor habituation. Together, this study identifies a physiological role for the voltage dependency of GPCRs by demonstrating crucial involvement of GPCR voltage dependence in neuronal plasticity and behavior. Thus, this study suggests that GPCR voltage dependency plays a role in many diverse neuronal functions including learning and memory.


Supplemental Figure 2: mAChR-A Gq pathway is functional and voltage dependent
A. Dose-response curves for wt mAChR-A (left) and mAChR-A-KK (right) Gq activated Clcurrents in oocytes at -80 mV (black; n=10,11,6,26 and 7,10,10,28 for wt mAChR-A and mAChR-A-KK, respectively and in ascending order of ACh nM) and +40 mV (red; n=9,15,16,24 and n=6,7,7,20 for wt mAChR-A and mAChR-A-KK, respectively and in ascending order of ACh nM). As with the GIRK currents, mAChR-A-KK is in the high activity state as indicated by the overlap with the dose response curve of wt mAChR-A at +40 mV (blue line)

B.
Absolute current values of wt mAChR-A and mAChR-A-KK, for ACh evoked currents in oocytes at -80 mV (left, black; n=24 and 29, respectively) and +40 mV (right, red; n=13 and 29, respectively) elicited by a saturating level (10 4 nM) of ACh. No difference is observed between the different conditions, indicating that the receptor maximal activity is not impaired.
C. 2-photon functional imaging was performed using GH298-GAL and the genetically encoded Ca 2+ indicator UAS-GCaMP6f on the background of wt or mAChR-A-KK flies. To examine only Ca 2+ release from internal stores which results from the activation of the Gq pathway, nominal 0 Ca 2+ external solution with TTX was used. A single plane of the AL was imaged. Peak fluorescence signal for wt mAChR-A (left, n=11) and mAChR-A-KK (right, n=12) elicited by a saturating level of muscarine (10 5 nM) are presented. No significant difference is observed, indicating that the release of Ca 2+ from internal stores by the Gq pathway is not impaired. Each dot represent a single fly.
For all panels, error bars represent the standard error of the mean (SEM).

Supplemental Figure 3: mAChR-A-KK mutant does not affect β-arrestin signaling
A. Example of traces showing receptor desensitization by β-arrestin as measured from AChevoked GIRK currents decay. Weak desensitization of ACh-evoked GIRK currents is observed when the mAChR-A receptor was expressed alone (left), and strong desensitization when mAChR-A was co-expressed with β-arrestin and GRK3 (right).

B.
Averaged traces depicting the desensitization kinetics with β-arrestin and GRK3 in wt mAChR-A (black line; n=7; SEM is shaded area) and in mAChR-A-KK (blue; n=6; SEM is shaded area). The currents are normalized to the maximal current evoked by ACh at a given oocyte.
C. The mean desensitization values obtained from traces such as shown in panel A. No significant difference was observed for both conditions (i.e. with or without β-arrestin and GRK3) between wt mAChR-A (grey; n=7 and 12 respectively) and mAChR-A-KK (blue; n=6 and 8 respectively), (p = 0.013, Two sample two sided t-test). Error bars represent the SEM.

Supplemental Figure 4: Voltage step and lower stimulation frequency controls for PTP
A. Left, normalized EPSC before and after depolarization in wt flies (expressing wt mAChR-A) for a holding potential of -40 mV. Right, mean normalized EPSC obtained from the data presented on the left. Applying a voltage step of -40mV without the HFS protocol did not change iLN EPSC (n=7, Paired sample t-test).
B. Left, normalized EPSC before and after a lower frequency PTP (50 Hz) obtained in the mAChR-A-KK fly strain (expressing mAChR-A-KK) for holding potential of -60 mV. Right, mean normalized EPSC obtained from the data presented on the left. No potentiation was observed. GH298-GAL4 was used to drive UAS-GFP. Each line represents a single fly (n=9, Paired sample t-test).
For all panels, shaded error bands represent the standard error of the mean (SEM).

Supplemental Figure 5: Voltage pre-pulse increases mAChR-A responses
A depolarizing pre-pulse step (40 mV for 30 seconds) before the muscarinic pulse potentiated the response to muscarine in wt flies (left, no pre-pulse, n=9, pre-pulse, n=8) but not in the mAChR-A-KK fly strain (right, no pre-pulse, n=8, pre-pulse, n=8). Each dot represents a single fly (p(wt) = 0.0009, Two sample two sided t-test). B. Examples of flies walking trajectories inside the behavioral chamber and scheme of the habituation protocol. At the beginning of the experiment no odor is presented for one minute, then an odor is presented to the right side of the chamber (gray) for two minutes. This protocol is then repeated for with the odor presented to the left side of the chamber. Following this, the odor is presented in both sides for thirty minutes. Finally, the initial protocol is repeated. wt flies retain their odor avoidance after thirty minutes of odor exposure whereas mAChR-A-KK show reduced odor avoidance.

Supplemental Figure 7: Controls for behavioral habituation
A. The valence (positive values indicate attraction) flies assign to apple cider vinegar (ACV) before and after the odor habituation protocol (30 minute exposure). For wt flies, there was no change in odor valence, however, the mAChR-A-KK fly strain displayed a strong and significant reduction in odor attraction, indicating odor habituation. (wt before, n=39, wt after, n=29, mAChR-A-KK before n=33, mAChR-A-KK after n= 38, p(kk) = 0.0009, Two sample two sided t-test). Each dot represent a single fly.
B. The valence wt flies assign to ethyl butyrate before and after the odor habituation protocol (1 h exposure). A strong and significant reduction in odor avoidance was observed indicating odor habituation. (wt before, n=65, wt after, exposure n=66, p<10 -10 , Two sample two sided ttest). Each dot represent a single fly.
C. The valence mAChR-A-KK flies assign to ethyl butyrate before and after 30 minutes without exposure to the odor. No significant reduction in odor avoidance was observed. (mAChR-A-KK before, n=48, mAChR-A-KK, after 30 min n=45, Two sample t-test). Each dot represent a single fly.