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26985.Full.Pdf 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.
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