Regulating quantal size of neurotransmitter release through a GPCR voltage sensor

Quanfeng Zhanga,1, Bing Liua,1, Yinglin Lia,1, Lili Yina, Muhammad Younusa, Xiaohan Jianga, Zhaohan Lina, Xiaoxuan Suna, Rong Huanga, Bin Liua, Qihui Wua, Feipeng Zhua, and Zhuan Zhoua,2

aState Key Laboratory of Membrane Biology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine and Peking-Tsinghua Center for Life Sciences and PKU-IDG/McGovern Institute for Brain Research, Peking University, 100871 Beijing, China

Edited by Robert H. Edwards, University of California, San Francisco, CA, and approved September 11, 2020 (received for review March 25, 2020) Current models emphasize that membrane voltage (Vm) depolarization- ATP is the ligand of two families of purinergic receptors, P2Xs induced Ca2+ influx triggers the fusion of vesicles to the plasma and P2Ys. P2Xs are ion channels and P2Ys are GPCRs, in- membrane. In sympathetic adrenal chromaffin cells, activation of cluding Gq (P2Y1, 2, 4, 6, 11) and Gi types (P2Y12, 13, 14) (27, 28). a variety of G protein coupled receptors (GPCRs) can inhibit quantal Among the P2Ys, P2Y1 exists in most tissues, including epithelial size (QS) through the direct interaction of G protein Giβγ subunits and endothelial cells, platelets, and immune cells (27, 29), and with exocytosis fusion proteins. Here we report that, independently P2Y12 is strongly expressed on platelets, where it plays funda- from Ca2+, Vm (action potential) per se regulates the amount of mental roles in their activation and aggregation (28, 30, 31), as catecholamine released from each vesicle, the QS. The Vm regula- well as in microglia (32, 33), smooth muscle cells (34), and tion of QS was through ATP-activated GPCR-P2Y12 receptors. D76 chromaffin cells (35, 36). P2Y12 is a major target for drugs and D127 in P2Y12 were the voltage-sensing sites. Finally, we against cardiovascular diseases (thrombosis, stroke, and myo- revealed the relevance of the Vm dependence of QS for tuning cardial infarction) and currently, the top-selling drugs targeting autoinhibition and target cell functions. Together, membrane P2Y12 (clopidogrel and cangrelor) are used for antithrombotic voltage per se increases the quantal size of dense-core vesicle therapy (37–39). However, the subtype(s) of P2Ys that mediate → → βγ → release of catecholamine via Vm P2Y12(D76/D127) Gi AIQS remains unknown in ACCs. QS → myocyte contractility, offering a universal Vm-GPCR signal- To estimate the effects of GPCR-Giβγ on single-vesicle fusion, ing pathway for its functions in the nervous system and other

we used highly sensitive electrochemical microcarbon fiber NEUROSCIENCE systems containing GPCRs. electrodes (CFEs, 7-μm diameter) to measure the vesicle con- tents released from single fusion pores in ACCs (2, 12, 13, 19). membrane potential | GPCR/P2Y12 | dense core vesicle | quantal size | For real-time imaging of vesicle fusion events, total internal re- chromaffin cell flection fluorescence (TIRF) microscope imaging in neuropep- tide Y (NPY)-pHluorin transfected ACCs allows the distinction ccording to the classical Ca2+ hypothesis of presynaptic of kiss-and-run from full-fusion-like single-vesicle fusion modes Atransmission and neuroendocrine secretion, a presynaptic (12, 13, 40). To determine the sites responsible for specific action potential activates Ca2+ influx which triggers Ca2+-de- functions in a GPCR, reconstitution of the GPCR in a reporting pendent quantal (all or none) vesicular release of neurotrans- system and its functional assay are needed (38, 41–43). mitter/hormone (1). In 1996, we reported the example of Ca2+- In the present study, by using CFEs to measure QS and/or the dependent subquantal release, in which only part of the vesicular fusion mode of single-vesicle release events, TIRF live imaging content of a native transmitter (catecholamine) is released dur- ing a transient fusion event (fusion pore flickers, or kiss and run), Significance leading to a smaller quantal size (QS) in sympathetic adrenal chromaffin cells (ACCs) (2). Subquantal kiss-and-run release The amount of neurotransmitter release triggered by Ca2+ from was subsequently confirmed in ACCs (3, 4), other endocrine cells a presynaptic single vesicle (quantal size, QS) is fundamental – (5, 6), glia (7, 8), and neurons (9 11). In 2005, the regulator of and determines the strength of synaptic transmission. The subquantal kiss-and-run release was found: G protein-coupled present study provides compelling evidence that the QS of βγ receptor (GPCR)-dependent Gi reduces QS (7). In addition, catecholamine is increased by membrane depolarization per se endogenous dynamin-1 limits expansion of the vesicle fusion 2+ (bypassing Ca ) via the voltage-sensitive ATP-GPCR/P2Y12 in pore and maintains all Ca2+-induced exocytotic release via the sympathetic chromaffin cells. D76-P2Y12 and D127-P2Y12 are kiss-and-run/subquantal release mode under physiological con- revealed as the voltage-sensing sites by introducing two GPCR ditions in ACCs (12). More recently, the mechanism underlying reporting systems. We also establish the physiology relevance subquantal catecholamine release has been revealed to consist of of voltage dependence of catecholamine QS, offering a sig-

the joint kiss-and-run fusion pore and matrix binding (13). naling pathway—Vm → P2Y12(D76/D127) → Giβγ → QS → The molecular machine that gates the fusion pore is known to myocyte contractility—for diverse functions in the nervous involve the SNARE complex and other regulators (14, 15), in- system and other systems containing GPCRs. cluding synaptotagmin (16), dynamin (12, 17–19), microdomain 2+ Ca (7, 11, 20), and GPCR-dependent Giβγ (12, 19, 21–23). Author contributions: Z.Z. designed research; Q.Z., Bing Liu, Y.L., L.Y., M.Y., X.J., Z.L., X.S., Giβγ is downstream of Gi-GPCR activation because only Gi is R.H., Bin Liu, and Q.W. performed research; Q.Z., Bing Liu, Y.L., M.Y., and Z.Z. analyzed sufficiently abundant to allow Giβγ to function (24). There are data; and Q.Z., F.Z., and Z.Z. wrote the paper. many Gi-GPCRs, including those activated by the native trans- The authors declare no competing interest. mitters acetylcholine (25, 26), somatostatin, and ATP (or ADP). This article is a PNAS Direct Submission. ATP inhibition of QS (termed AIQS here) is induced via the Published under the PNAS license. GPCR-Gi-βγ pathway in ACCs (19). Its physiological relevance 1Q.Z., Bing Liu, and Y.L. contributed equally to this work. lies in the fact that ATP release (13) evoked from a neighboring 2To whom correspondence may be addressed. Email: [email protected]. cell directly leads to AIQS (19). AIQS is downstream of the This article contains supporting information online at https://www.pnas.org/lookup/suppl/ ligand-GPCR-Gi-βγ signaling pathway, which is initiated by a doi:10.1073/pnas.2005274117/-/DCSupplemental. native ligand that regulates the GPCR. First published October 12, 2020.

www.pnas.org/cgi/doi/10.1073/pnas.2005274117 PNAS | October 27, 2020 | vol. 117 | no. 43 | 26985–26995 Downloaded by guest on September 27, 2021 to confirm single-vesicle fusion modes, and two complementary broad-spectrum antagonist of P2X purinoceptors (48, 49), did GPCR reporting systems (a high-throughput assay of Gi-α-IP3- not block the depolarization effect (Fig. 2C). Thus, depolariza- 2+ [Ca ]i for screening sites and a precision assay of Gi-βγ-GIRK tion relieves AIQS via Gi-P2Y(s). Strikingly, ARC66096 (10 μM, current for validation) to determine the voltage-sensing sites in a specific antagonist of P2Y12) (50, 51) blocked the effect of the P2Y-GPCR, we discovered that P2Y12 mediates AIQS, depolarization on AIQS (Fig. 2D). These pharmacological ex- which is disinhibited by depolarization (membrane voltage [Vm]) periments suggested that P2Y12 is a candidate for the via two voltage-sensing sites (D76 and D127) in native depolarization-induced relief of AIQS. neuroendocrine chromaffin cells. To confirm the pharmacological findings, we first performed reverse transcriptional PCR (RT-PCR) of rat adrenal medulla Results and found four Gq-coupled receptors (P2Y1, P2Y2, P2Y4, and Depolarization Per Se Relieves the Intrinsic AIQS. We have previ- P2Y6) and three Gi-coupled receptors (P2Y12, P2Y13, and ously demonstrated that subquantal catecholamine release oc- P2Y14)(SI Appendix, Fig. S5A and Table S2). Second, following curs in ACCs (2), and the QS is regulated by GPCR-Gi-βγ the pharmacological results (Fig. 2), we designed and validated 2+ following elevation of the intracellular Ca concentration two knockdown (KD) shRNAs targeting P2Y12 in rat ACCs (SI 2+ [Ca ]i by caffeine (20 mM) (19). When caffeine was applied for Appendix, Fig. S5 C–F) and found that P2Y12 was the major 10 s, a burst of amperometric spikes—representing quantal cat- receptor mediating AIQS. Western blot analysis of HEK293A echolamine release from single large dense-core vesicles—was cells and immunofluorescent staining of native chromaffin cells evoked in ACCs (Fig. 1A). Indeed, ATP inhibited the QS revealed that P2Y12 was knocked down (∼80% reduction) by the (Fig. 1A, see also refs. 13, 19), or the AIQS phenotype was shRNAs and could be fully rescued (Fig. 3 A and B). Depolar- produced by the smaller QS (Fig. 1A, see also ref. 19). Surpris- ization disinhibited the AIQS in control scrambled (Fig. 3C and ingly, when a cell was depolarized by high KCl (70 mM), the SI Appendix, Fig. S6A), but not P2Y12-KD (shRNA 1 or 2) ACCs AIQS phenotype was abolished (Fig. 1A). The effect of Vm on (Fig. 3 D and E and SI Appendix, Fig. S6 B and C), while over- GPCR-Gi-βγ-based AIQS was fully reversible (Fig. 1A) and in- expressing shRNA-resistant P2Y12 fully recovered the Vm de- 2+ dependent of [Ca ]i (Fig. 1B). The statistics of this Vm- pendence of AIQS (Fig. 3F and SI Appendix, Fig. S6D). Thus, dependent AIQS are shown in Fig. 1 D and E and SI Appen- P2Y12 is essential for voltage-dependent AIQS in rat ACCs. 2+ dix, Fig. S1. ACCs were bathed in 0 Ca solution containing Finally, using P2Y12-knockout (KO) mice (SI Appendix, Fig. 1 mM EGTA during caffeine stimulation to ensure no Ca2+ S5B), the depolarization-sensitive AIQS was present only in 2+ influx during depolarization. [Ca ]i measurements using Fura-2 wild-type (WT) but not P2Y12-KO cells (Fig. 3G). Taken to- 2+ showed that the caffeine-induced [Ca ]i elevation was inde- gether, P2Y12 is not only the ATP receptor for AIQS, but is also pendent of depolarization (Fig. 1 B and F). The depolarization responsible for its Vm dependence. from −70 mV to −20 mV by 70 mM KCl (but not caffeine or ATP) was confirmed by current-clamp recordings (Fig. 1 C and Depolarization Regulates P2Y12-Gi Signals via the Voltage-Sensing F). Note that ATP was required for the Vm-dependent AIQS Sites D76 and D127 in Reconstituted Systems. Based on the find- phenotype and in the absence of ATP, depolarization had no ing that P2Y12 mediated the Vm dependence of AIQS in ACCs – SI Appendix – effect on the QS (SI Appendix, Figs. S1 and S14). Furthermore, (Figs. 1 3 and , Figs. S1 S6), we proposed that P2Y12 the QS did not change with repetitive caffeine stimulation (SI activity is voltage dependent and P2Y12 contains voltage-sensing Appendix, Fig. S2). sites. To test this hypothesis at the molecular level, we designed a α 2+ To ensure that the relief of the inhibitory effect of ATP is due P2Y12 reporting system via the P2Y12-Gi- -IP3-[Ca ]i signaling to membrane depolarization per se, we repeated the experiments pathway (Fig. 4A), which is a high-throughput assay for multiple 2+ B shown in Fig. 1, but replacing 70 mM KCl depolarization by cells per experiment with [Ca ]i imaging (Fig. 4 ) to assess the C whole-cell voltage clamp. Similar to 70 mM KCl depolarization voltage-dependence of P2Y12 in reporting cells (Fig. 4 ). The 2+ α (Fig. 1), whole-cell depolarization from −70 mV to 0 mV abol- Ca indicator GCaMP3, P2Y12, and the G i3q chimera were ished the AIQS (SI Appendix, Fig. S3). Thus, the AIQS pheno- co-overexpressed in HeLa cells and the Gαi3q chimera results in type is produced by membrane depolarization per se. coupling with Gαi-coupled receptors but signaling through the α 2+ In addition to CFE recordings, single-vesicle exocytosis can be G q-mediated PLC-IP3-[Ca ]i mobilization pathway (42, 52, imaged live by TIRF, which measures the dynamics of single 53) (Fig. 4 A and B and SI Appendix, Fig. S7 A, B, and E and see large dense-core vesicle release of pHluorin-tagged NPY in also Materials and Methods). Membrane depolarization by neurons (40, 44) and ACCs (13, 45). The Ca2+-dependent 70 mM KCl (SI Appendix, Fig. S7H) inhibited the increased 2+ full-fusion-like mode, corresponding to full quantal release, and [Ca ]i induced by 2MesADP (P2Y12 agonist) (27) in the P2Y12- the kiss-and-run mode, corresponding to subquantal release (13) reconstituted reporting cells (Fig. 4 C and E and Movies S5–S7), were identified by TIRF imaging (SI Appendix, Fig. S4 A and B confirming that Vm modulates P2Y12 function: depolarization α and Movies S1–S4, see also refs. 13, 40, 45). Consistent with the decreases P2Y12 activation as measured by the G i pathway. CFE recordings, TIRF imaging revealed that ATP shifted the Using this high-throughput imaging assay, we screened for fusion mode from full-fusion-like to kiss-and-run (full-fusion-like voltage-sensing sites in P2Y12 and found two amino acid sites: ratio changed from 68 to 33%), while this AIQS-like effect was D76 and D127 (Fig. 4 C–E and SI Appendix, Fig. S10A) (54). largely abolished by depolarization (full-fusion-like ratio Either D76N or D127N mutation abolished the Vm dependence E SI Appendix changed from 33 to 62%, Fig. 1G and SI Appendix, Fig. S4). of P2Y12 (Fig. 4 and , Fig. S8), indicating that these are the voltage-sensing sites of P2Y12. P2Y12 Is the ATP Receptor for Voltage-Dependent AIQS. Since AIQS Next, to further analyze and quantify the voltage-sensitive sites was present in ACCs (Fig. 1, see also ref. 19), we aimed to of P2Y12, we designed another reporting system for P2Y12 acti- identify the associated receptor. Because AIQS is probably vation based on the P2Y12-Gi-βγ-GIRK signaling pathway. In produced by an unknown subtype of P2Y receptor coupled to Gi this precision assay, we used patch clamp to evaluate the voltage in ACCs (19), we first identified candidates in pharmacological dependence of P2Y12 (Fig. 4 F–I and SI Appendix, Fig. S9, see experiments. When ACCs were treated with ATP (100 μM), also refs. 31, 41, 43). P2Y12 and GIRK1/4 channels together were 70 mM KCl depolarization disinhibited AIQS and increased QS coexpressed in HEK293A cells. Following ADP stimulation, the (Fig. 2A). In contrast, suramin (100 μM), a broad-spectrum an- activation of P2Y12 was detected by the whole-cell GIRK current tagonist of P2 purinoceptors (46, 47), fully removed the effect of IGIRK (Fig. 4 F and G and SI Appendix, Fig. S9). To assess the depolarization on AIQS (Fig. 2B). However, PPADS (50 μM), a voltage dependence of P2Y12, we calculated the ratio of IGIRK at

26986 | www.pnas.org/cgi/doi/10.1073/pnas.2005274117 Zhang et al. Downloaded by guest on September 27, 2021 NEUROSCIENCE

Fig. 1. ATP inhibition of quantal size (AIQS) of vesicular catecholamine release is attenuated by membrane depolarization voltage (Vm). (A) Basic charac- teristics of voltage-dependent AIQS. (Upper) cartoon of Vm-AIQS with steps 1 through 3. (Lower) ATP (100 μM) inhibits the QS of amperometric spikes in- duced by caffeine (Caf; 20 mM for 10 s) and this is reversed by 70 mM KCl (70K)-induced depolarization (dashed boxes show averaged quantal events). Each cell was exposed four times to caffeine-containing solutions. The reversible Vm-AIQS contains four steps (1, 2, 3, and 2′): (step 1) Ca2+ is released from the ER

store by caffeine and triggers vesicular catecholamine release events recorded as spikes in amperometric current (Iamp) recordings; (step 2) ATP activates P2Y receptors and reduces QS; (step 3) membrane depolarization (by 70K) removes the inhibitory effect of ATP; and finally (step 2′) AIQS recovers after removing 2+ 2+ depolarization. (B) Intracellular Ca [Ca ]i (F340/F380) elevation measured by Fura2/AM, corresponding to A.(C) Membrane potential recorded by current clamp, corresponding to A. Caffeine with or without ATP does not change the membrane potential, while 70 mM KCl depolarizes it by ∼50 mV. (D) Cu- mulative distribution of QS corresponding to the four steps (1, 2, 3, and 2′) of Fig. 1A (198 spikes for group 1, 157 spikes for group 2, 193 spikes for group 3, and 166 spikes for group 2′. The four groups are matched measurements from n = 15 cells. For groups 2 vs. 3, ***P < 0.001, K-S test). (E) Quantitative analysis of single-vesicle release events (quanta) from cells of Fig. 1D. Analyzed spikes meet the 5-SD noise threshold criterion. ATP significantly reduces QS, while depolarization by 70 mM KCl completely removes the inhibitory effect of ATP. When the 70 mM KCl is washed out, QS is inhibited by ATP again (the four groups are matched measures from n = 15 cells, paired Friedman test, post hoc Dunn’s multiple comparisons test). (F, Left) Depolarization does not affect the 2+ 2+ caffeine-induced [Ca ]i elevation in Ca -free bath (n = 14 cells, Friedman test, post hoc Dunn’s multiple comparisons test). (F, Right) KCl (70 mM) depolarizes Vm by ∼50 mV (from –70 mV to –20 mV, n = 5 cells, paired Student’s t test). (G) Confirmation of voltage-dependent AIQS by another orthogonal assay of TIRFM imaging. Statistics of TIRF imaging data (SI Appendix, Fig. S4) confirm that the probability ratio [FFL ratio = N(FFL)/N(FFL + KAR), where N(FFL) is the number of full-fusion-like (FFL) events and N(FFL + KAR) is the total number of FFL and kiss-and-run (KAR) events]. Similar to the QS index from CFE recordings in E (multiple treatments were applied to the same cells), the corresponding index (FFL ratio) from TIRFM imaging is also increased by depolarization (70K) (n = 9 cells, Friedman test, post hoc Dunn’s multiple comparisons test, further details in SI Appendix, Fig. S4 and Materials and Methods). A–C show different cells with the same time scale. Data are presented as the mean ± SEM. (E–G)*P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant.

Zhang et al. PNAS | October 27, 2020 | vol. 117 | no. 43 | 26987 Downloaded by guest on September 27, 2021 D127E, abolished the voltage dependence of γ(Vm) (Fig. 4 J and K and SI Appendix,Fig.S10B–F). These results from the precision assay confirmed that not only the sites but also the negative charges (either D or E) of both D76 and D127 were essential for the Vm dependence of P2Y12.Taken together, D76 and D127 are the voltage-sensing sites of P2Y12.

Validation of P2Y12 Vm Sensors for AIQS in Chromaffin Cells. Next, we examined the effects of the mutations D76N and D127N on the Vm dependence of AIQS (Vm-AIQS) in native ACC cells. First, comparing WT and KD cells (P2Y12-shRNA-KD, Fig. 3 A–F), the rescue by shRNA1-resistant WT restored both AIQS and Vm-AIQS in ACCs (Fig. 5A). However, replacing the shRNA1- resistant WT by either shRNA1-resistant P2Y12-D76N or -D127N, the Vm-AIQS effect (but not AIQS itself) was abol- ished (Fig. 5 B–D). Thus, both D76 and D127 are voltage-sensing sites of P2Y12 responsible for the Vm-AIQS effect (Fig. 5E).

Physiological Relevance of the Vm Dependence of AIQS. Next, we investigated whether the Vm dependence of AIQS exists in ad- renal slice ACCs (Fig. 6A). CFEs were used to record the am- perometric current (Iamp) representing catecholamine overflow from all nearby quantal release from slice ACCs bathed in Ca2+- free solution containing 10 mM EGTA after stimulation (Fig. 6 A and B). ATP reversibly inhibited the caffeine-induced Iamp (Fig. 6B). Importantly, corresponding to the Vm-AIQS in cultured ACCs (Fig. 1A), the Vm dependence of the ATP- inhibited Iamp induced by caffeine was preserved in adrenal sli- ces, as 70 mM KCl depolarization disinhibited this Iamp (Fig. 6B). That this voltage-dependent phenomenon is inde- pendent of Ca2+ channels was demonstrated by using the 2+ Ca channel blocker CdCl2 (SI Appendix,Fig.S12).. Similar to the Vm-dependent AIQS in cultured ACCs (Fig. 3G), the Vm dependence of ATP-inhibited Iamp in adrenal slices (Vm-AIQS in situ) was also abolished by P2Y12-KO (Fig. 6C). Although depolarization in these slice experiments was by 70 mM KCl, the Vm-AIQS in situ likely remains with physi- ological stimulation because, when depolarization by 70 mM KCl (Figs. 1–3) was replaced by whole-cell depolarization with either action potentials (SI Appendix,Fig.S13)at physiological frequencies (55, 56) or Vm pulses (SI Appendix, — Fig. 2. P2Y12 is the ATP receptor for voltage-dependent AIQS Fig. S3), the Vm dependence of AIQS remained in pharmacological evidence. (A) AIQS is regulated by voltage. Statistically, cultured ACCs. normalized QS (QS normalized by averaged QS in control bath, black) is To provide proof of concept for the physiological impact of the significantly increased by 70 mM KCl (70K) depolarization (red) in ACCs treated with 100 μM ATP (n = 17 cells, **P < 0.01, paired Student’s t test). Vm-AIQS effect via catecholamine release from adrenal slices, we Inset, averaged quantal spikes without (black solid line) and with (red determined whether the caffeine-induced release could affect dashed line) 70 mM KCl depolarization. (B) Suramin (100 μM, a broad- cardiac myocytes, the major peripheral targets of sympathetic spectrum P2XR and P2YR blocker) removes the effect of 70 mM KCl depo- ACCs (57, 58). We collected the fluid (30 μL/gland) from adrenal larization on AIQS (n = 13 cells, P = 0.49, paired Student’s t test). (C) PPADS slices (two slices per gland) incubated in either control (0 mM (50 μM, a P2XR-specific blocker) does not remove the effect of 70 mM KCl Ca2+ +100μM ATP + 20 mM caffeine) or depolarization depolarization on AIQS (n = 8 cells, *P < 0.05, paired Student’s t test). (D) (control + 70 mM KCl) solution. Compared to the control solu- ARC66096 (10 μM, a P2Y12 blocker) removes the 70 mM KCl-depolarization = = ’ tion, the depolarization solution should contain more catechol- effect on AIQS (n 19 cells, P 0.74, paired Student s t test). Data are amine produced by the Vm-AIQS effect. These stock solutions presented as the mean ± SEM. *P < 0.05, **P < 0.01; NS, not significant. were diluted 10 times into working solutions (2 mM Ca2+ and 2 mM caffeine) and applied to beating myocytes (Fig. 6D). In the paired ADP doses 10 μM (middle dose) and 100 μM (satu- contrast to the control solution, the depolarization solution in- D–F ration dose): γ(Vm) = ΔI /ΔI = I (10 μM)/I (100 creased the contractility of myocytes (Fig. 6 ), implying po- 10 100 GIRK GIRK – μM), where Vm is the holding potential (SI Appendix, Fig. S11B). tential regulation of cardiac excitation contraction coupling by Importantly, γ(Vm) was larger at −100 mV than at −40 mV, Vm-AIQS. Finally, in addition to the ligand-GPCR pair of ATP-P2Y , indicating stronger P2Y activation and greater ADP-P2Y 12 12 12 somatostatin-GPCR is also known to inhibit QS in ACCs (13, binding affinity with hyperpolarization (Fig. 4 H–J). To deter- 19). We found that, like ATP-P2Y12, somatostatin-SSTR (so- mine whether the negatively charged D76 and D127 were critical matostatin receptor) (59) had a similar phenotype of Vm de- for the Vm dependence, in addition to D76N and D127N pendence (Figs. 1–6 and SI Appendix, Fig. S15), indicating that (negative-to-neutral charge), we made D76E and D127E muta- the Vm dependence of GPCR-Gi signaling is likely a general tions (negative-to-negative charge). In contrast to native P2Y12 phenomenon in native mammalian cells. In principle, the pres- (Fig. 4 H–J), the mutation D76N or D127N, but not D76E or ence of two types of Gi-GPCRs (P2Y12 and SSTR) allows the

26988 | www.pnas.org/cgi/doi/10.1073/pnas.2005274117 Zhang et al. Downloaded by guest on September 27, 2021 NEUROSCIENCE

Fig. 3. P2Y12 is the ATP receptor for voltage-dependent AIQS—genetic evidence. (A) Western blots and statistics of P2Y12 expression in HEK293A cells transfected with scrambled, P2Y12-KD shRNA1, P2Y12-KD shRNA2, and P2Y12-rescue plasmids (five independent experiments, one-way ANOVA, post hoc Tukey’smultiple comparisons test). (B) Immunostaining and statistics of P2Y12 expression (scrambled, 25 cells; shRNA1, 26 cells; shRNA2, 10 cells; rescue, 17 cells, one-way ANOVA, post hoc Tukey’s multiple comparisons test). (Scale bar, 5 μm.) (C, Left)TypicalCFEIamp traces of ATP (100 μM)-induced AIQS from scrambled cells with or without 70 mM KCl (70K) depolarization. (C, Right) Statistics of the effect of depolarization on QS in 26 scrambled cells (**P < 0.01, Wilcoxon test). Inset, averaged quantal

spikes without (black solid line) and with (red dashed line) 70 mM KCl depolarization (Vm). (D and E) Statistics of averaged quantal events and AIQS in 21 P2Y12- KD-shRNA1 cells (D, P = 0.64, Wilcoxon-test) and 19 P2Y12-KD shRNA2 cells (E, P = 0.59, Wilcoxon test) with or without 70 mM KCl depolarization (Vm). (F) Statistics of AIQS in 30 P2Y12-rescued cells (**P < 0.01, Wilcoxon test) with or without 70 mM KCl depolarization (Vm). (G, Upper Left) 70 mM KCl depolarization-induced AIQS is intact in a WT cell. (G, Upper Right) Cumulative distribution curve of QS (n = 14 cells, four groups [1, 2, 3, and 2′] of quantal events [167, 141, 140, and 125]). Forgroups2vs.3,***P < 0.001, K-S test and statistics of Vm-AIQS in 14 WT cells (Friedman test, post hoc Dunn’s multiple comparisons test). (G, Lower Left)70mM

KCl depolarization-induced AIQS is eliminated in a P2Y12-KO cell. (G, Lower Right) Cumulative distribution curve of QS (n = 12 cells; groups 1, 2, 3, and 2′ of 136, 120, 118, and 101 events). For groups 2 vs. 3, NS, not significant, K-S test and statistics of Vm-AIQS in 12 P2Y12-KO cells (Friedman test, post hoc Dunn’smultiple comparisons test). Data are presented as the mean ± SEM. (C–G)**P < 0.01, ***P < 0.001; NS, not significant.

cell to be regulated by both neurotransmitters (ATP and so- Discussion matostatin) arising from other vesicles or cells. On the other Neurotransmitter release from presynaptic cells induced by ac- hand, a single depolarization can shut down the Vm-dependent tion potentials is fundamental, and neurotransmission depends autoinhibition of both Gi-GPCRs. on QS. In the present work we found that, after Ca2+ as the

Zhang et al. PNAS | October 27, 2020 | vol. 117 | no. 43 | 26989 Downloaded by guest on September 27, 2021 coregulator to trigger vesicle fusion, depolarization—in addition to activating voltage-gated channels—per se acts as coregulator of GPCR to determine the QS. The P2Y12-mediated Vm de- pendence of QS has broad physiological relevance, providing the entire pathway [Vm → P2Y12 (D76/D127) → Giβγ → QS → secretion] the ability to modulate the hormone level in circu- lating plasma for targeted cells throughout the body (including cardiac myocytes, Fig. 6G). The major finding of the present work was that Vm depolar- ization relieved AIQS (ATP inhibition of QS) via ATP- dependent GPCR (P2Y)-Giβγ in rodent ACCs. This was sup- ported by the findings that in cultured ACCs: 1) depolarization per se relieved the AIQS, and increased the QS of caffeine- induced secretion by ∼200% (Fig. 1 A–F and SI Appendix, Figs. S1–S3), a characteristic termed Vm-AIQS. 2) TIRF imag- ing confirmed that depolarization shifted the vesicle fusion mode from kiss-and-run to full fusion and increased the proportion of full-fusion events from 33 to 62% (Fig. 1G and SI Appendix, Fig. S4 and Movies S1–S4), which confirmed Vm-AIQS. 3) Vm- AIQS also persisted in the more physiological adrenal slice (Fig. 6A–C). 4) Like ATP-P2Y, another native ligand-GPCR pair, somatostatin and its GPCR, possessed a Vm-AIQS-like phenotype with somatostatin replacing ATP (SI Appendix, Fig. S15), indicating that Vm-GPCR-QS signaling extends beyond GPCR-P2Y12, and could be a modulatory mechanism shared by many GPCRs and cell types. Regarding the molecular identity and mechanisms of Vm- AIQS, we found that P2Y12 and its voltage-sensing sites are re- sponsible because RT-PCR showed that P2Y12 was present in rat and mouse ACCs (SI Appendix, Fig. S5 A and B); using phar- macological methods, Vm-AIQS was blocked by suramin (an antagonist of P2Ys and P2Xs) and ARC66096 (an antagonist of P2Y12) (Fig. 2 B and D), but not by PPADS (an antagonist of P2Xs) (Fig. 2C); using genetic approaches, the phenotype was largely blocked either by P2Y12-KO (Fig. 3G) or KD by two P2Y12-shRNAs (Fig. 3 A–E), and was rescued by the corre- sponding shRNA-resistant P2Y12 (Fig. 3F and SI Appendix, Fig. S6); using assays of P2Y12 function in two complementary re- constitution systems (Fig. 4 and SI Appendix, Figs. S7 and S9), P2Y12-D76 and -D127 were identified as the Vm-sensing sites, Fig. 4. Dissecting the voltage-sensing sites of P2Y12 by two reconstituted GPCR- because mutagenesis of either D76N or D127N abolished the α 2+ signaling assays. (A) Cartoon of the P2Y12-Gi- -IP3-[Ca ]i assay. HeLa cells were Vm dependence of P2Y12 while their basic P2Y12-Gi signals α transfected with the plasmids P2Y12,GCaMP3(cytosol),andGi3q. The P2Y12- remained (Fig. 4 and SI Appendix, Figs. S8 and S10); and over- specific agonist 2MesADP (10 μM) activates the P2Y12-Gαi3q-PLC-IP3 pathway to expressing either D76N or D127N in ACCs silenced by P2Y12- induce Ca2+ release from the ER (SI Appendix,Fig.S7). (B) Confocal image of 2+ shRNA#1 abolished the Vm dependence, while the control WT- multiple HeLa cells transfected with P2Y12,Gαi3q, and GCaMP3 plasmids. (C)[Ca ]i P2Y12 did not (Fig. 5). Taken together, D76 and D127 are the (ΔF/F0) triggered by 2MesADP with or without depolarization. Overlapped gray 2+ Vm-sensing sites in P2Y responsible for Vm-AIQS. [Ca ]i traces from many cells in the same dish; black/red traces show averaged 12 [Ca2+]. Depolarization (70 mM KCl, 70K) reversibly decreases the averaged ampli- Regarding physiological relevance, the Vm-AIQS occurred in i 2+ tude of ΔF/F0 spikes. (D)P2Y12 purinergic receptor model based on the crystal the quantal release evoked by increasing cytosolic Ca through structure (PDB accession no. 4PXZ) (54). Arrows indicate the positions of the two either the caffeine-sensitive endoplasmic reticulum (ER) store 2+ 2+ candidate Vm-sensing residues D76 and D127. (E) Statistics of evoked [Ca ]i signals. (Fig. 1A) or by whole-cell dialysis with high Ca (SI Appendix, 2+ Δ 2+ Compared with the WT (C), Vm depolarization reduced [Ca ]i ( F/F0)ofP2Y12-WT, Fig. S13). In principle, the quantal release evoked by Ca influx but not D76N or D127N mutation. For WT, n = 108 cells; for the D76N mutation, through ion channels (19) or other internal Ca2+ stores (60, 61) n = 45 cells; for the D127N mutation, n = 76 cells (SI Appendix,Fig.S8). Friedman ’ would also be regulated by the Vm-dependent GPCRs. The Vm- test, post hoc Dunn s multiple comparisons test. (F) Cartoon of the P2Y12-Gi- – βγ-GIRK current assay (SI Appendix,Fig.S9). (G) Whole-cell recordings of GIRK AIQS was present not only in cultured ACCs (Figs. 1 3, and 5 and SI Appendix, Figs. S1, S3, S4, S6, and S13), but also in fresh current (IGIRK) induced by 140 mM KCl (140 K) in HEK293A cells coexpressing GIRK1/ A–C 4andP2Y12 (ΔI10 and ΔI100 were IGIRK induced by 10 and 100 μM ADP). Note that adrenal slices (Fig. 6 ). In WT but not P2Y12-KO slices, IGIRK is larger at 100 μMthanat10μMADP(seealsoSI Appendix,Fig.S9E). (H)IGIRK AIQS was preserved as ATP-inhibited Iamp (the sum of release evoked by 10 μMor100μM ADP measured at Vm = −40 mV (Left)or−100 mV from all nearby cells), which is proportional to the total cate- Δ Δ γ = Δ Δ (Right). The IGIRK ratio ( I10/ I100) was defined as (Vm) I10/ I100. Statistics are cholamine release from the adrenal medulla into the blood. shown in J (WT). (I)Theγ(Vm)-Vm curve showing that γ(Vm) is voltage dependent. Thus, the Vm dependence of AI-QS (or AI-Iamp) could sub- γ γ − − (J) Statistics of (Vm). For WT-P2Y12, (Vm) is potentiated at 100 mV versus 40 stantially regulate the catecholamine level in the circulation and − γ = − γ = = < mV. At 40 mV, (Vm) 0.55; at 100 mV, (Vm) 0.79, (n 29 cells, ***P 0.001, thus all of its peripheral targets (57, 58), including myocyte paired Student’s t test). With the D76N or D127N mutation, the Vm-dependence of D–F – γ(Vm) is abolished (n = 15 cells for D76N; n = 18 cells for D127N, see also SI Ap- contractility (Fig. 6 ). Excitation contraction coupling was enhanced by the fluid from adrenal slices exposed to 70 mM KCl, pendix,Fig.S10, paired Student’s t test). (K) Summary of the effects of P2Y12 mutations on the ratio γ(Vm) (SI Appendix,Fig.S10). Data are presented as the which depolarizes ACCs, disinhibits P2Y12, and increases the median with interquartile range (E)orthemean± SEM (J). *P < 0.05, **P < 0.01, catecholamine level in solution. This provides proof of concept, ***P < 0.001; NS, not significant. that the effect of Vm-AIQS can impact the heart and other

26990 | www.pnas.org/cgi/doi/10.1073/pnas.2005274117 Zhang et al. Downloaded by guest on September 27, 2021 NEUROSCIENCE

Fig. 5. The P2Y12-D76 and -D127 sites are voltage sensors in chromaffin cells. (A, Left) The depolarization dependence of AIQS is rescued by P2Y12*, which is a functional P2Y12 resistant to shRNA1. (A, Right) Statistics of QS (n = 11 cells, Friedman test, post hoc Dunn’s multiple comparisons test). Dashed boxes show averaged quantal events. (B and C, Left) The Vm dependence of AIQS cannot be rescued by either D76N-P2Y12 (B) or D127N-P2Y12 (C). (B and C, Right) Statistics of QS (B, n = 17 cells, one-way ANOVA, post hoc Tukey’s multiple comparisons test; C, n = 7 cells, Friedman test, post hoc Dunn’s multiple com-

parisons test). (D) Summary of the P2Y12 Vm dependence of AIQS. (E) Model of voltage-sensitive P2Y12 regulation of quantal release in native ACCs. ATP is released from chromaffin vesicles and activates autoinhibitory P2Y12, which inhibits quantal catecholamine release. Meanwhile, depolarization deactivates P2Y12 via the D76 and D127 sites, and thus relieves the inhibitory effect of ATP. Data are the mean ± SEM. (A–C)*P < 0.05, **P < 0.01; NS, not significant.

peripheral functions through regulating the blood catecholamine HEK293A cells coexpressing P2Y12 and GIRK1/4 ion channels, level. which reported P2Y12 function as GIRK currents for Vm-GPCR The discovery of the Vm sensors D76 and D127 was made analysis (Fig. 4 F–K and SI Appendix, Figs. S9–S11, see also refs. possible by using two complementary GPCR reporting systems: a 41, 43). In principle, these two complementary assays can be 2+ high-throughput [Ca ]i-imaging assay (Gαi3q) and a high- used to study the Vm dependence and/or other functions of precision patch-clamp assay (Gβγ-GIRK) (Fig. 4 and SI Appen- P2Y12 (and other GPCRs, with slight modifications). 2+ dix, Figs. S7–S11). Our GPCR-Gαi3q-[Ca ]i assay used HeLa In contrast to the present findings of Vm-dependent QS in cells coexpressing P2Y12, the Gαi3q chimera, and GCaMP3, rodent neuroendocrine chromaffin cells, previously Parnas and 2+ which reported the function of P2Y12 by cytosolic [Ca ]i using Parnas have found the Vm-sensing sites responsible for M2- fluorescence imaging for screening Vm-P2Y12 sites (Fig. 4 A–E mediated GIRK currents in oocytes and provided pharmaco- and SI Appendix, Figs. S7 and S8). This assay is superior to logical evidence supporting a Vm-dependent M2 effect on pe- previous assays (42, 52, 53) for its higher throughput using real- ripheral excitatory postsynaptic currents (26). Mahaut-Smith and time imaging. Our high-precision patch-clamp assay consisted of colleagues found that Vm-dependent binding between ADP and

Zhang et al. PNAS | October 27, 2020 | vol. 117 | no. 43 | 26991 Downloaded by guest on September 27, 2021 Fig. 6. Potential relevance of voltage-dependent GPCR-P2Y12 mediation of catecholamine release. (A) Image of a freshly prepared mouse adrenal slice 150 μm thick. (A, Inset) Enlargement showing a recording CFE (Φ 7 μm) in the slice with visible individual ACCs. (B, Left) In an adrenal slice bathed in 0 Ca2+

(containing 10 mM EGTA) and ATP (100 μM), the ATP-inhibited catecholamine overflow (Iamp, amperometric current) evoked by caffeine (20 mM for 40 s) is fully removed by 70 mM KCl (70K) depolarization in WT ACCs. (B, Right) Statistics of WT (n = 9 slices from four mice, Friedman test, post hoc Dunn’s multiple

comparisons test). (C)AsinB, except the Vm dependence of Iamp is abolished by P2Y12-KO. (C, Right) Statistics of KO (n = 9 slices from four mice, one-way ANOVA, post hoc Tukey’s multiple comparisons test). (D, Upper) Cartoon of the protocol for the catecholamine fluid collection from adrenal slices treated with 0 Ca + ATP + caffeine (control fluid) or 0Ca + 70K + ATP + caffeine (70K fluid). The fluids were puffed onto myocytes through the patch pipette (10 to 12 μm in diameter) to test their effects. (D, Lower) Setup to record excitation–contraction coupling in a myocyte. (E) Diagram of how catecholamine fluid was used to potentiate myocyte contractility (see F for details). (F) Quantification of ΔL2/ΔL1, where ΔL1 and ΔL2 represent the contraction length of myocytes before and after application of depolarization fluid (0Ca + 70K + ATP + caffeine) versus control fluid (0Ca + ATP + caffeine) (n = 20 myocytes for control fluid, and n = 22 myocytes for depolarization fluid, unpaired Student’s t test). (G) Model of the Vm-AIQS signal pathway to a target cell. The Vm dependence of QS

implies a broad physiological relevance by providing the entire Vm-P2Y12 pathway in vivo. Data are presented as the mean ± SEM. (B, C, and F)*P < 0.05; NS, not significant.

2+ P2Y1 modulates [Ca ]i in nonexcitable blood megakaryocytes, the Vm dependence of GPCRs (M2 or P2Y1) by genetic with pharmacological evidence supporting Vm-dependent P2Y1 knockdown and rescue in native cells. In the present study, we (62). These earlier reports, however, lacked crucial evidence of demonstrated not only the phenotypes of the Vm dependence of

26992 | www.pnas.org/cgi/doi/10.1073/pnas.2005274117 Zhang et al. Downloaded by guest on September 27, 2021 catecholamine release via P2Y12-AIQS (Figs. 1–3) and the mo- Cell Culture and Transfection. Rat adrenal chromaffin cells (ACCs) were pre- lecular mechanisms (Fig. 4), but also crucial validation using pared as described previously (19, 67) and mouse ACCs were prepared sim- genetic KO, KD, and rescue (Figs. 3 and 5). Particularly, we not ilarly with minor modifications. Briefly, the adrenal glands were isolated only identified the Vm-sensing sites of P2Y but also confirmed from anesthetized animals (10% chloral hydrate), cut into pieces and, after 12 removing the cortex, incubated in papain solution for 40 min at 37 °C. The them in native ACCs (Figs. 2–5). These provide an example of pieces were then triturated gently through a 200-μL pipette tip. After cen- physiological Vm per se having a direct physiological impact on trifugation, cells were quickly plated on coverslips precoated with 0.1% quantal vesicle release in both cultured (Figs. 1–3 and 5 and SI poly-L-lysine, incubated at 37 °C under 5% CO2, and used within 24 to 96 h. Appendix, Figs. S1, S3, S4, S6, and S13) and slice ACCs ACCs were transfected for genetic manipulations using a 10-μL Neon elec- + (Fig. 6 A–C). The sodium ion (Na ) is known to allosterically troporation system (Invitrogen, MPK1096) according to the manufacturer’s modulate GPCR activation by binding the highly conserved TM2 instructions (68). aspartate residue in family A GPCRs (63–65). The present work HEK293A cells were cultured in Dulbecco’s modified Eagle’s medium demonstrated that the aspartate residues of TM2 (D76) and (DMEM) supplemented with 10% fetal bovine serum (FBS) and transfected with VigoFect (Vigorous Biotechnology Beijing Co.) using the following TM3 (D127) are the voltage sensors of P2Y12 and regulate exocytosis in native cells and slices (Figs. 4–6), establishing the plasmids (per 3.5-cm diameter dish): For gene-silencing experiments, shRNAs μ μ essential physiological relevance of GPCR voltage dependence, (2 g) and their target P2Y12 (1 g) were delivered into cells and Western probably by influencing the interaction between the Na+ pocket blotting analysis was performed 4 d later. To measure GIRK currents, rat P2Y12 (1 μg) and a bicistronic plasmid expressing the GIRK1 and GIRK4 of D76 and the DRY motif of D127 (66). subunits (1 μg) were transfected and after 24 h of expression, the HEK293A Since the structure and function of GPCRs are well conserved in cells were placed on sterile, poly-L-lysine–coated glass coverslips and the all tissues, in addition to P2Y12 regulating catecholamine release, GIRK current was recorded. future work is needed to determine: 1) the Vm dependence of other HeLa cells were cultured in DMEM supplemented with 10% FBS and

phenotypes (i.e., event number) by P2Y12,orotherGPCRsand transfected with P2Y12,Gαi3q, and GCaMP3 (1 μg each) for 36 h using physiological functions, including released vesicle cargos of synaptic Lipo2000 (Invitrogen) before Ca2+-imaging experiments. The coexpression neurotransmitters, neurotrophins (nerve growth factor, insulin), rate of the three plasmids in cells was ∼85%. inflammatory factors (NF-κB, substance P), and other hormones (5- HT, dopamine); and 2) whether Vm-GPCR-QS also exists in the Adrenal Slice Preparation. Adrenal slices were prepared as described previ- central nervous system, other nonneuronal systems, and other ani- ously (13, 69). Briefly, adrenal glands were removed from adult mice (2 to 4 mals including humans. mo old) and immediately placed in ice-cold cutting solution containing (in The present work establishes a signal pathway linking excitation- mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose (pH 7.4, saturated with 95% O and 5% CO ). Then, a single gland

2 2 NEUROSCIENCE quantal size (QS), Vm → GPCR-P2Y12 → Giβγ → fusion pore → G without fat was glued with 3% agarose to the stage of a vibratome (Leica VT QS (Fig. 6 ), which coexists with the canonical pathway linking 1200S) and cut into 150-μm slices. The slices were incubated for at least 2+ excitation-quantal release, Vm → Ca influx → quantal release in 30 min at 37 °C in cutting solution and then kept at room temperature sympathetic chromaffin cells. As Vm and GPCRs are present in all before recordings. Slices were used within 12 h after cutting. neuronal and nonneuronal cells, the regulation of GPCR and its downstream signals (i.e., vesicle release) by Vm could affect phys- Electrophysiology. An EPC10/2 amplifier with Patchmaster software (HEKA iological/pathological functions beyond adrenal chromaffin cells and Elektronik) was used to obtain whole-cell patch-clamp recordings as de- sympathetic nervous system. scribed previously (40). The normal extracellular buffer was composed of (in 2+ mM): 145 NaCl, 2 CaCl2, 2.8 KCl, 1 MgCl2, 10 Hepes (pH 7.4). For the Ca -free Materials and Methods solution (0Ca), 2 mM Ca2+ was replaced by 1 mM EGTA for cultured chro- maffin cells or 10 mM EGTA for adrenal slices. Patch pipettes were filled with Animals and Chemicals. The P2Y12-KO mice on a C57BL/6 background were intracellular solution containing (in mM): 100 K+-aspartate, 40 KCl, 5 NaCl, 7 gifts from Junling Liu, Shanghai Jiaotong University, Shanghai, China, and MgCl , 10 EGTA, 0.025 GTP, 5 Na+-ATP, 20 Hepes (pH 7.2). For GIRK current maintained in the Animal Center of Peking University. Sprague-Dawley rats 2 recordings, a high-K+ buffer was used as the extracellular solution (as above, (adult, 150 g) were from Beijing Vital River Laboratory Animal Technology + + Co., Ltd. All procedures and animal care were approved by the Institutional but containing 140 mM K and 2.4 mM Na ) (43). GIRK currents were Animal Care and Use Committee of Peking University (Beijing, China) and measured in the whole-cell configuration as inward currents (holding po- − − – 2+ the Association for Assessment and Accreditation of Laboratory Animal tential: 40 mV, 70 mV, or 100 mV) (43). For pure Ca current recording, μ Care. Adult mice (C57BL/6 strain, 1 to 4 mo old, both sexes) were used for all 2 mM CaCl2 was increased to 10 mM CaCl2 and 1 M tetrodotoxin was added experiments. Mice and rats were housed under a 12-h light/dark cycle with to the extracellular solution. The intracellular pipette solution contained (in food and water. The details of all animals and chemicals are in SI Appendix, mM) 153 CsCl, 1 MgCl2, 10 H-Hepes, and 4 Mg-ATP, pH 7.2. During experi- Table S1. ments, patched cells were continuously superfused with extracellular buffer or agonist-containing solution. Igor software (WaveMetrics) was used for all offline data analyses. All experiments were performed at room temperature Plasmids. The full-length rat P2Y12 receptor (P2Y12, NM_022800) was subcl- unless otherwise indicated. oned into pIRES2-EGFP (Clontech) or p3XFLAG-CMV (Sigma). The P2Y12 site mutations D76N, D127N, D76E, and D127E were produced by PCR using a μ QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies). The Electrochemistry. Highly sensitive, low-noise, 7- m carbon fiber electrodes (ProCFE, Dagan) were used for the electrochemical monitoring of quantal nucleotide target sequences GCA GTA AAT CGA ACT TCA TCA (P2Y12- release of catecholamines from single ACCs as described previously (2, 19). shRNA1) and GCT TCG TTC CCT TCC ACT TTG (P2Y12-shRNA2) were chosen to Quantal events were analyzed as described previously (2, 19). Stimulation silence the expression of P2Y12. A random sequence (TTC TCC GAA CGT GTC ACG T) that was predicted to target no genes in human, rat, and mouse cells solutions (caffeine or other drugs) were delivered by a perfusion system < served as a negative control (scrambled) (Guangzhou RiboBio Co., Ltd). (Yibo) with a fast exchange time ( 100 ms). Annealed double-stranded oligonucleotides encoding the target sequences were inserted into the vector pRNAT-H1.1-RFP/GFP to generate plasmids TIRF Imaging. TIRF images were captured on an inverted microscope with a × expressing shRNAs against P2Y12. An RNAi (P2Y12-shRNA1)-resistant form of 100 TIRF objective lens (numerical aperture, 1.45; Olympus IX-81) at an rat P2Y12 for the rescue experiments was generated by introducing the exposure time of 50 ms using an Andor electron-multiplying charge-coupled following silent mutations: GTA GCA AGT CAA ATT TTA. All constructs were device with Andor iQ software. The temperature was kept at ∼35 °C throughout verified by DNA sequencing. The bidirectional expression vector pBI-CMV1 (a all TIRF experiments using a laboratory-made heater. Kiss-and-run and full- kind gift from D. E. Logothetis, Virginia Commonwealth University, Rich- fusion-like events were defined as follows: in kiss-and-run events, the fluores- mond, VA) was used to simultaneously and constitutively express GIRK1 and cence signal was restricted to the center of the release site; in full-fusion-like GIRK4. The Gαi3q was a kind gift from Xiao Yu, Shandong University, events, the fluorescence signal diffused to the surroundings of the release site Shandong, China. The GCaMP3 was from Addgene Co. The NPY-pHluorin (SI Appendix,Fig.S4B and see also refs. 8, 13, 40). Exocytotic events were plasmid was constructed from NPY-Venus (a kind gift from Nikita Gamper, stimulated by puffing 20 mM caffeine and analyzed using ImageJ (NIH) as University of Leeds, Leeds, UK). described previously (13).

Zhang et al. PNAS | October 27, 2020 | vol. 117 | no. 43 | 26993 Downloaded by guest on September 27, 2021 Ca2+ Imaging. For Ca2+ imaging using Fura-2, isolated chromaffin cells were were IRDye 800CW goat anti-rabbit IgG (LIC-926-32211, LI-COR Biosciences), incubated in a bath solution containing 5 μM Fura-2/AM (Molecular Probes) and IRDye 680CW goat anti-mouse IgG (LIC-926-32220, LI-COR Biosciences). 2+ for 15 min at 37 °C. The [Ca ]i was measured by dual-wavelength ratio- metric fluorometry. The Fura-2 was excited by light alternating between Immunofluorescence. Cells were prefixed in 4% paraformaldehyde for 15 min, 340 nm and 380 nm using a monochromatic system (TILL Photonics), and the washed three times with PBS, then permeabilized with 0.3% Triton in PBS for emission fluorescence was measured using a cooled charge-coupled device. 3 min. After the cells were incubated with 2% BSA for 1 h, they were in- 2+ The [Ca ]i was calculated from the ratio of F340 to F380, and the sampling cubated with the primary antibody rabbit anti-P2Y (Anaspec, AS-55043A) 2+ 12 frequency was 1 Hz, triggered by the HEKA amplifier. The Ca fluorescence overnight at 4 °C. Then they were washed with 2% BSA in PBS, and incu- signals were analyzed using Igor software (Wavemetrix) (19). bated with the secondary antibody (Alexa Fluor 594 goat anti-rabbit IgG, For Ca2+-imaging using GCaMP3, HeLa cells (4th to 12th generation after A11037, Invitrogen). After that, the cells were mounted on coverslips im- recovery from freezing) were transfected with three plasmids: P2Y ,Gαi3q, 12 mersed in 50% glycerol. Fluorescence images were acquired on a confocal and GCaMP3 (1 μg each) for ∼36 h using Lipo2000. The P2Y12-specific agonist 2+ microscope (Zeiss 710) and analyzed with ImageJ. 2MesADP (10 μM) was puffed onto the cells to trigger [Ca ]i oscillations (typically one to three spikes, SI Appendix, Fig. S7E). All of the cells were from three to six batches; 10 to 20 cells per batch for each condition (control Reverse Transcription PCR. Total RNA was extracted using the TRIzol reagent ’ or mutations) were imaged for statistics. Considering that the peak of the (Invitrogen) according to the manufacturer s instructions and mRNA was 2+ reverse transcribed with the Transgen kit (AU311). The forward and reverse [Ca ]i oscillation represents the strength of P2Y12-Gαi3q signaling, the 2+ oligonucleotide primers we have used are listed in SI Appendix, Table S2. amplitude of [Ca ]i in a cell was defined as the maximum peak during the 2+ 90-s stimulation (SI Appendix, Fig. S7E). [Ca ]i was measured by ΔF/F0, where F0 is basal fluorescence before 2MesADP stimulation and ΔFisthe Statistics. All experiments were replicated at least three times. Data are 2+ 2+ ± “ ” fluorescence change during a [Ca ]i spike. The [Ca ]i signals were captured shown as the mean SEM or the median with interquartile range. n on an inverted confocal microscope (Zeiss 710) using a 488-nm laser and the represents the number of independent experiments as reported in the fig- light emitted from GCaMP3 was recorded at 500 to 540 nm. Time series ure legends. All data were tested for normality prior to choosing a proper videos (512 × 512 pixels) were acquired at 1 Hz under a 40× oil objective lens statistical test. If the data passed the normality test, the paired Student’s (Zeiss). Data were analyzed with ImageJ. t test was applied for comparison between two matched groups and one- way ANOVA followed by Tukey’s multiple comparisons test was applied Excitation–Contraction Coupling in Myocytes. Myocytes were isolated from when multiple groups were compared with one variable. If the data did not adult C57 mice (1.5 to 2 mo old, weight ∼25 g) (70–72). A bipolar electrode pass normality, the Wilcoxon matched-pairs signed rank test (Wilcoxon test) (laboratory made) was used to evoke contractions; a brief, 50-V, 5-ms pulse was applied for comparison between two matched groups and the Friedman (Nihon Kohden, Electronic Stimulator SEN-3201, Isolator SS-102J) was ap- test followed by Dunn’s multiple comparisons test was used for multiple plied for pacemaking. The contractions evoked before and after drug matched groups. The Kolmogorov–Smirnov (K-S) test was applied for cu- treatments were imaged under an inverted IX-81 Olympus microscope, and mulative distribution comparison between two groups. All tests were con- analyzed with ImageJ. ducted using Prism V7.0 (GraphPad Software, Inc.) and SPSS 20.0 (Statistical Package for the Social Sciences). Statistical tests were two-tailed and the Western Blot. Samples were lysed with lysate buffer containing 20 mM Hepes level of significance was set at P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001). at pH 7.4, 100 mM KCl, 2 mM EDTA, 1% Nonidet P-40, 1 mM phenyl- methanesulfonyl fluoride, and 2% proteinase inhibitor (539134, Calbio- Data Availability. All study data are included in the article and supporting chem). The homogenate was centrifuged at 15,000 × g for 30 min and the information. supernatant was collected and boiled in sodium dodecyl sulfate– polyacrylamide gel electrophoresis buffer. Proteins were electrophoresed and transferred to nitrocellulose filter membranes. Each membrane was ACKNOWLEDGMENTS. We thank Drs. Junling Liu (Shanghai Jiaotong Uni- versity) for the P2Y -KO mice, Shiqiang Wang and Peace Cheng (Peking blocked by incubation for 1 h with PBS containing 0.1% Tween-20 (vol/vol), 12 University) for providing myocytes, Xiao Yu (Shandong University) for Gαi3q, and 5% nonfat dried milk (wt/vol). After washing with 0.1% Tween-20 Diomedes E. Logothetis (Northeastern University) and Hailin Zhang (Hebei containing PBS (PBST), the blots were incubated with primary antibodies Medical University) for GIRK, and Xiaoke Chen (Stanford University) and Iain at 4 °C overnight in PBST containing 2% bovine serum albumin (BSA). Sec- C. Bruce (Peking University) for reading the manuscript. This work was sup- ondary antibodies were then applied at room temperature and left for 1 h. ported by the National Natural Science Foundation of China (31930061, Blots were scanned with an Odyssey infrared imaging system (LI-COR Bio- 31761133016, 21790394, 31171026, 31330024, 31327901, 31521062, and sciences) and quantified with ImageJ. The primary antibodies were as fol- 21790390), the National Key Research and Development Program of China lows: rabbit anti-P2Y12 (Anaspec, AS-55043A), mouse anti-flag (F1804, (2016YFA0500401), and the National Basic Research Program of China Sigma), and mouse anti–β-actin (A5316, Sigma); the secondary antibodies (2012CB518006).

1. J. Del Castillo, B. Katz, Quantal components of the end-plate potential. J. Physiol. 124, 12. Q. Wu et al., Dynamin 1 restrains vesicular release to a subquantal mode in mam- 560–573 (1954). malian adrenal chromaffin cells. J. Neurosci. 39, 199–211 (2019). 2. Z. Zhou, S. Misler, R. H. Chow, Rapid fluctuations in transmitter release from single 13. Q. Zhang et al., Differential co-release of two neurotransmitters from a vesicle fusion vesicles in bovine adrenal chromaffin cells. Biophys. J. 70, 1543–1552 (1996). pore in mammalian adrenal chromaffin cells. Neuron 102, 173−183.e4 (2019). 3. A. Albillos et al., The exocytotic event in chromaffin cells revealed by patch amper- 14. T. C. Sudhof, J. E. Rothman, Membrane fusion: Grappling with SNARE and SM pro- ometry. Nature 389, 509–512 (1997). teins. Science 323, 474–477 (2009). 4. E. Ales et al., High calcium concentrations shift the mode of exocytosis to the 15. J. Rettig, E. Neher, Emerging roles of presynaptic proteins in Ca++-triggered exocy- kiss-and-run mechanism. Nat. Cell Biol. 1,40–44 (1999). tosis. Science 298, 781–785 (2002). 5. M. Braun et al., Corelease and differential exit via the fusion pore of GABA, serotonin, 16. M. Segovia et al., Push-and-pull regulation of the fusion pore by synaptotagmin-7. and ATP from LDCV in rat pancreatic beta cells. J. Gen. Physiol. 129, 221–231 (2007). Proc. Natl. Acad. Sci. U.S.A. 107, 19032–19037 (2010). 6. N. Takahashi, T. Kishimoto, T. Nemoto, T. Kadowaki, H. Kasai, Fusion pore dynamics 17. P. Samasilp, S. A. Chan, C. Smith, Activity-dependent fusion pore expansion regulated and insulin granule exocytosis in the pancreatic islet. Science 297, 1349–1352 (2002). by a calcineurin-dependent dynamin-syndapin pathway in mouse adrenal chromaffin 7. X. Chen, L. Wang, Y. Zhou, L. H. Zheng, Z. Zhou, “Kiss-and-run” glutamate secretion in cells. J. Neurosci. 32, 10438–10447 (2012). cultured and freshly isolated rat hippocampal astrocytes. J. Neurosci. 25, 9236–9243 18. A. Anantharam et al., A new role for the dynamin GTPase in the regulation of fusion (2005). pore expansion. Mol. Biol. Cell 22, 1907–1918 (2011). 8. D. N. Bowser, B. S. Khakh, Two forms of single-vesicle astrocyte exocytosis imaged 19. X. K. Chen et al., Activation of GPCRs modulates quantal size in chromaffin cells with total internal reflection fluorescence microscopy. Proc. Natl. Acad. Sci. U.S.A. 104, through G(betagamma) and PKC. Nat. Neurosci. 8, 1160–1168 (2005). 4212–4217 (2007). 20. R. H. Chow, J. Klingauf, C. Heinemann, R. S. Zucker, E. Neher, Mechanisms deter- 9. Q. Zhang, Y. Li, R. W. Tsien, The dynamic control of kiss-and-run and vesicular reuse mining the time course of secretion in neuroendocrine cells. Neuron 16, 369–376 probed with single nanoparticles. Science 323, 1448–1453 (2009). (1996). 10. L. He, X. S. Wu, R. Mohan, L. G. Wu, Two modes of fusion pore opening revealed by 21. Z. Zurawski et al., Gbetagamma directly modulates vesicle fusion by competing with cell-attached recordings at a synapse. Nature 444, 102–105 (2006). synaptotagmin for binding to neuronal SNARE proteins embedded in membranes. 11. Y. Wang et al., Ligand- and voltage-gated Ca2+ channels differentially regulate the J. Biol. Chem. 292, 12165–12177 (2017). mode of vesicular neuropeptide release in mammalian sensory neurons. Sci. Signal. 22. T. Blackmer et al., G protein betagamma directly regulates SNARE protein fusion 10, eaal1683 (2017). machinery for secretory granule exocytosis. Nat. Neurosci. 8, 421–425 (2005).

26994 | www.pnas.org/cgi/doi/10.1073/pnas.2005274117 Zhang et al. Downloaded by guest on September 27, 2021 23. K. M. Betke, C. A. Wells, H. E. Hamm, GPCR mediated regulation of synaptic trans- 49. G. J. McLaren et al., Investigation of the actions of PPADS, a novel P2x-purinoceptor mission. Prog. Neurobiol. 96, 304–321 (2012). antagonist, in the Guinea-pig isolated vas deferens. Br. J. Pharmacol. 111, 913–917 24. G. Kadamur, E. M. Ross, Mammalian phospholipase C. Annu. Rev. Physiol. 75, 127–154 (1994). (2013). 50. R. G. Humphries, W. Tomlinson, A. H. Ingall, P. A. Cage, P. Leff, FPL 66096: A novel, 25. Z. Zhou, S. Misler, Action potential-induced quantal secretion of catecholamines from highly potent and selective antagonist at human platelet P2T-purinoceptors. Br. rat adrenal chromaffin cells. J. Biol. Chem. 270, 3498–3505 (1995). J. Pharmacol. 113, 1057–1063 (1994). 26. I. Parnas, H. Parnas, Control of neurotransmitter release: From Ca2+ to voltage de- 51. J. Simon et al., Activity of adenosine diphosphates and triphosphates on a P2Y(T) pendent G-protein coupled receptors. Pflugers Arch. 460, 975–990 (2010). -type receptor in brain capillary endothelial cells. Br. J. Pharmacol. 132, 173–182 27. M. P. Abbracchio et al., International union of pharmacology LVIII: Update on the P2Y (2001). G protein-coupled nucleotide receptors: From molecular mechanisms and patho- 52. J. Bohnekamp, T. Schoneberg, Cell adhesion receptor GPR133 couples to Gs protein. physiology to therapy. Pharmacol. Rev. 58, 281–341 (2006). J. Biol. Chem. 286, 41912–41916 (2011). 28. M. Idzko, D. Ferrari, H. K. Eltzschig, Nucleotide signalling during inflammation. Na- 53. Q. X. Hu et al., Constitutive Galphai coupling activity of very large G protein-coupled ture 509, 310–317 (2014). receptor 1 (VLGR1) and its regulation by PDZD7 protein. J. Biol. Chem. 289, 29. G. Burnstock, Purine and pyrimidine receptors. Cell. Mol. Life Sci. 64, 1471–1483 24215–24225 (2014). (2007). 54. J. Zhang et al., Agonist-bound structure of the human P2Y12 receptor. Nature 509, 30. M. Cattaneo et al., Molecular bases of defective signal transduction in the platelet 119–122 (2014). P2Y12 receptor of a patient with congenital bleeding. Proc. Natl. Acad. Sci. U.S.A. 100, 55. A. M. de Diego, L. Gandia, A. G. Garcia, A physiological view of the central and pe- 1978–1983 (2003). ripheral mechanisms that regulate the release of catecholamines at the adrenal 31. G. Hollopeter et al., Identification of the platelet ADP receptor targeted by antith- medulla. Acta Physiol. (Oxf.) 192, 287–301 (2008). rombotic drugs. Nature 409, 202–207 (2001). 56. M. E. Holman, H. A. Coleman, M. A. Tonta, H. C. Parkington, Synaptic transmission 32. S. E. Haynes et al., The P2Y12 receptor regulates microglial activation by extracellular from splanchnic nerves to the adrenal medulla of Guinea-pigs. J. Physiol. 478, 115–124 nucleotides. Nat. Neurosci. 9, 1512–1519 (2006). (1994). 33. H. Tozaki-Saitoh et al., P2Y12 receptors in spinal microglia are required for neuro- 57. H. Q. Yang et al., beta2-adrenergic stimulation compartmentalizes beta1 signaling pathic pain after peripheral nerve injury. J. Neurosci. 28, 4949–4956 (2008). into nanoscale local domains by targeting the C-terminus of beta1-adrenoceptors. 34. A. K. Wihlborg et al., ADP receptor P2Y12 is expressed in vascular smooth muscle cells Circ. Res. 124, 1350–1359 (2019). and stimulates contraction in human blood vessels. Arterioscler. Thromb. Vasc. Biol. 58. R. Huang et al., Ca(2+)-independent but voltage-dependent quantal catecholamine 24, 1810–1815 (2004). secretion (CiVDS) in the mammalian sympathetic nervous system. Proc. Natl. Acad. Sci. 35. G. Burnstock, G. E. Knight, Cellular distribution and functions of P2 receptor subtypes U.S.A. 116, 20201–20209 (2019). in different systems. Int. Rev. Cytol. 1240,3 –304 (2004). 59. B. Kailey et al., SSTR2 is the functionally dominant somatostatin receptor in human 36. S. J. Ennion, A. D. Powell, E. P. Seward, Identification of the P2Y(12) receptor in nu- pancreatic beta- and alpha-cells. Am. J. Physiol. Endocrinol. Metab. 303, E1107–E1116 cleotide inhibition of exocytosis from bovine chromaffin cells. Mol. Pharmacol. 66, (2012). 601–611 (2004). 60. M. L. Mundorf, K. P. Troyer, S. E. Hochstetler, J. A. Near, R. M. Wightman, Vesicular 37. H. K. Eltzschig, M. V. Sitkovsky, S. C. Robson, Purinergic signaling during inflamma- Ca(2+) participates in the catalysis of exocytosis. J. Biol. Chem. 275, 9136–9142 (2000). tion. N. Engl. J. Med. 367, 2322–2333 (2012). 61. C. L. Haynes, L. A. Buhler, R. M. Wightman, Vesicular Ca2+-induced secretion pro- 38. K. Zhang et al., Structure of the human P2Y12 receptor in complex with an antith- moted by intracellular pH-gradient disruption. Biophys. Chem. 123,20–24 (2006).

rombotic drug. Nature 509, 115–118 (2014). 62. I. S. Gurung, J. Martinez-Pinna, M. P. Mahaut-Smith, Novel consequences of voltage- NEUROSCIENCE 39. I. von Kugelgen, K. Hoffmann, Pharmacology and structure of P2Y receptors. Neu- dependence to G-protein-coupled P2Y(1) receptors. Br. J. Pharmacol. 154, 882–889 ropharmacology 104,50–61 (2016). (2008). 40. Z. Chai et al., Ca V 2.2 gates calcium-independent but voltage-dependent secretion in 63. V. Katritch et al., Allosteric sodium in class A GPCR signaling. Trends Biochem. Sci. 39, mammalian sensory neurons. Neuron 96, 1317–1326.e4 (2017). 233–244 (2014). 41. Y. Ben-Chaim, O. Tour, N. Dascal, I. Parnas, H. Parnas, The M-2 muscarinic G-protein- 64. C. Zhang et al., High-resolution crystal structure of human protease-activated re- coupled receptor is voltage-sensitive. J. Biol. Chem. 278, 22482–22491 (2003). ceptor 1. Nature 492, 387–392 (2012). 42. B. R. Conklin, Z. Farfel, K. D. Lustig, D. Julius, H. R. Bourne, Substitution of three amino 65. W. Liu et al., Structural basis for allosteric regulation of GPCRs by sodium ions. Science acids switches receptor specificity of Gq alpha to that of Gi alpha. Nature 363, 337, 232–236 (2012). 274–276 (1993). 66. A. J. Venkatakrishnan et al., Molecular signatures of G-protein-coupled receptors. 43. A. Rinne, A. Birk, M. Buenemann, Voltage regulates adrenergic receptor function. Nature 494, 185–194 (2013). Proc. Natl. Acad. Sci. U.S.A. 110, 1536–1541 (2013). 67. K. Duan, X. Yu, C. Zhang, Z. Zhou, Control of secretion by temporal patterns of action 44. N. Matsuda et al., Differential activity-dependent secretion of brain-derived neuro- potentials in adrenal chromaffin cells. J. Neurosci. 23, 11235–11243 (2003). trophic factor from axon and dendrite. J. Neurosci. 29, 14185–14198 (2009). 68. C. Wang et al., Synaptotagmin-11 inhibits clathrin-mediated and bulk endocytosis. 45. D. Perrais, I. C. Kleppe, J. W. Taraska, W. Almers, Recapture after exocytosis causes EMBO Rep. 17,47–63 (2016). differential retention of protein in granules of bovine chromaffin cells. J. Physiol. 560, 69. X. W. Chen et al., DTNBP1, a schizophrenia susceptibility gene, affects kinetics of 413–428 (2004). transmitter release. J. Cell Biol. 181, 791–801 (2008). 46. P. M. Dunn, A. G. Blakeley, Suramin: A reversible P2-purinoceptor antagonist in the 70. W. Shang et al., Cyclophilin D regulates mitochondrial flashes and metabolism in mouse vas deferens. Br. J. Pharmacol. 93, 243–245 (1988). cardiac myocytes. J. Mol. Cell. Cardiol. 91,63–71 (2016). 47. C. H. Hoyle, G. E. Knight, G. Burnstock, Suramin antagonizes responses to P2- 71. M. Xu et al., Intermolecular failure of L-type Ca2+ channel and ryanodine receptor purinoceptor agonists and purinergic nerve stimulation in the Guinea-pig urinary signaling in hypertrophy. PLoS Biol. 5, e21 (2007). bladder and taenia coli. Br. J. Pharmacol. 99, 617–621 (1990). 72. Z. Zhou, D. M. Bers, Ca2+ influx via the L-type Ca2+ channel during tail current and 48. A. U. Ziganshin et al., Selective antagonism by PPADS at P2X-purinoceptors in rabbit above current reversal potential in ferret ventricular myocytes. J. Physiol. 523, isolated blood vessels. Br. J. Pharmacol. 111, 923–929 (1994). 57–66 (2000).

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