Research Articles: Cellular/Molecular Heat but not mechanical hypersensitivity depends on voltage-gated CaV2.2 calcium channel activity in peripheral axon terminals innervating skin https://doi.org/10.1523/JNEUROSCI.0195-21.2021 Cite as: J. Neurosci 2021; 10.1523/JNEUROSCI.0195-21.2021 Received: 26 January 2021 Revised: 19 June 2021 Accepted: 19 July 2021 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2021 DuBreuil et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 Title: Heat but not mechanical hypersensitivity depends on voltage-gated CaV2.2 calcium 2 channel activity in peripheral axon terminals innervating skin 3 Abbreviated title: Peripheral CaV2.2 channels control heat hypersensitivity 4 Daniel M DuBreuil*#, Eduardo Javier Lopez Soto*, Simon Daste, Remy Meir, Daniel Li, Brian J 5 Wainger#, Alexander Fleischmann & Diane Lipscombe. Carney Institute for Brain Science and 6 the Department of Neuroscience, Providence, RI 02912, USA. #Departments of Neurology and 7 Anesthesia, Critical Care & Pain Medicine, Massachusetts General Hospital, Harvard Medical 8 School, Boston, MA 02114, USA. 9 *Authors contributed equally to this manuscript 10 11 Corresponding author: [email protected] 12 Number of pages: Including references and figure legends = 46 13 Number of figures: 7; Number of Tables: 1 14 Words in Abstract: 140; Introduction: 640; Words in Discussion: 1436 15 Conflict of Interest: B.J.W. is on the Scientific Advisory Board for Quralis; conflict of interest was 16 reviewed and managed by MGH and Partners Healthcare. No other conflicts of interest to 17 declare. 18 Acknowledgements: The authors thank Sylvia Denome for expert technical assistance. This 19 work was funded by grants NS055251 (DL), F31NS093818 (DD), T32MH020068 (DD), 20 NS062443 (DD), Warren Alpert Fellowship Award (EJLS), K99NS116123 (EJLS), DP2- 21 NS106664 (BJW), New York Stem Cell Foundation Robertson Investigator (BJW), and Robert J 22 and Nancy D Carney Institute for Brain Science (AF). 23 [1] 24 ABSTRACT 25 Voltage-gated CaV2.2 calcium channels are expressed in nociceptors at presynaptic terminals, 26 soma, and axons. CaV2.2 channel inhibitors applied to the spinal cord relieve pain in humans 27 and rodents, especially during pathological pain, but a biological function of nociceptor CaV2.2 28 channels in processing of nociception, outside presynaptic terminals in the spinal cord, is 29 underappreciated. Here, we demonstrate that functional CaV2.2 channels in peripheral axons 30 innervating skin are required for capsaicin-induced heat hypersensitivity in male and female 31 mice. We show that CaV2.2 channels in TRPV1-nociceptor endings are activated by capsaicin- 32 induced depolarization and contribute to increased intracellular calcium. Capsaicin induces 33 hypersensitivity of both thermal nociceptors and mechanoreceptors, but only heat 34 hypersensitivity depends on peripheral CaV2.2 channel activity, and especially a cell type- 35 specific CaV2.2 splice isoform. CaV2.2 channels at peripheral nerve endings might be important 36 therapeutic targets to mitigate certain forms of chronic pain. 37 38 SIGNIFICANCE STATEMENT (120 words maximum) 39 It is generally assumed that nociceptor termini in spinal cord dorsal horn are the functionally 40 significant sites of CaV2.2 channel in control of transmitter release, and the transmission of 41 sensory information from the periphery to central sites. We show that peripheral CaV2.2 42 channels are essential for the classic, heat hypersensitivity response to develop in skin following 43 capsaicin exposure. This function of CaV2.2 is highly selective for heat, but not mechanical 44 hypersensitivity induced by capsaicin exposure, and is not a property of closely related CaV2.1 45 channels. Our findings suggest that interrupting CaV2.2-dependent calcium entry in skin might 46 reduce heat hypersensitivity that develops after noxious heat exposure, and may limit the 47 degree of heat hypersensitivity associated with certain other forms of pain. [2] 48 INTRODUCTION 49 Voltage-gated calcium channels, CaV2.2 (N-type current) and CaV2.1 (P-type current), are the 50 primary sources of calcium that controls neurotransmitter release from nociceptor presynaptic 51 termini in the spinal cord dorsal horn (Diaz and Dickenson, 1997; Heinke et al., 2004; Motin and 52 Adams, 2008; Jayamanne et al., 2013). Opioids act through -opioid receptors and inhibit the 53 gating of presynaptic Cav2.2 channels in the spinal cord dorsal horn and decrease synaptic 54 transmission (Horvath et al., 2001; Andrade et al., 2010). CaV2.2 channels in nociceptors are 55 also targets of non-opioid analgesics including the selective CaV2.2 channel inhibitor - 56 conotoxin MVIIA (-CgTx MVIIA, Prialt®, Ziconotide, or SNX-111) (Miljanich and 57 Ramachandran, 1995; Bowersox et al., 1997; Miljanich, 2004; Patel et al., 2018). Intrathecal - 58 CgTx MVIIA mitigates otherwise intractable pain in human, and it is effective in animal models 59 of inflammatory and nerve injury-induced pain while having less effect on acute pain responses 60 (Miljanich and Ramachandran, 1995; Bowersox et al., 1997; Miljanich, 2004; Bannister et al., 61 2017). However, clinical use of CaV2.2 channel inhibitors is limited because of side effects due 62 to inhibition of CaV2.2 channels in brain and sympathetic nervous system (Miljanich, 2004). A 63 CaV2.2 channel inhibitor with greater selectivity against chronic pain would have significant 64 value. 65 CaV2.2 channels are expressed throughout nociceptors at presynaptic terminals, soma, and 66 peripheral nerves but their biological significance at sites beyond a well-accepted role at 67 presynaptic terminals is not well understood. Nociceptor free nerve endings in skin release 68 inflammatory signaling molecules, including neuropeptides calcitonin gene-related peptide 69 (CGRP), ATP and Substance P, that contribute to inflammatory hypersensitivity to non-noxious 70 and noxious stimuli (Louis et al., 1989; Costigan and Woolf, 2000; Cook and McCleskey, 2002; 71 Scholz and Woolf, 2002; Costigan et al., 2009). The signaling molecules that control release of [3] 72 pro-inflammatory molecules are not well characterized although TRPA1 receptors have been 73 proposed to play a role in peptide release (Lin King et al., 2019). A role for CaV channels is often 74 assumed, but direct evidence for their involvement is lacking (Chiu et al., 2012). Interestingly, 75 White and Cousins showed >20 years ago (White and Cousins, 1998), that daily intradermal 76 inhibition of CaV2.2, but not CaV2.1, channels attenuate mechanical hyperalgesia in a peripheral 77 nerve injury model, but this finding has not been pursued. More recently, Bean, Woolf and 78 colleagues (Lee et al., 2019) showed that external application of an inhibitor of both CaV2.2 and 79 NaV1.7 / 1.8 channels (CNCB-2) attenuates thermal hyperalgesia and mechanical 80 hyperalgesia/allodynia induced in postoperative and inflammatory pain models (Lee et al., 81 2019), but these studies did not directly address the contributions of CaV2.2 separate from NaV 82 channels. 83 CaV2.2 channel 1 subunits are encoded by the Cacna1b gene which generates splice isoforms 84 with different functional characteristics across tissue and cell-type, developmental age, and 85 disease state (Lipscombe, 2005; Lipscombe et al., 2013; Lipscombe and Lopez Soto, 2019). 86 Trpv1-lineage nociceptors express a unique CaV2.2 splice isoform, CaV2.2-e37a, whereas most 87 other neurons only express CaV2.2-e37b (Bell et al., 2004; Altier et al., 2007; Jiang et al., 2013; 88 Lopez Soto et al., 2019), and CaV2.2-e37a channels are more sensitive to modulation by 89 morphine compared with CaV2.2-e37b (Bell et al., 2004; Castiglioni et al., 2006; Altier et al., 90 2007; Raingo et al., 2007; Andrade et al., 2010; Marangoudakis et al., 2012; Jiang et al., 2013; 91 Macabuag and Dolphin, 2015). CaV2.2-e37a channels appear to be particularly critical for some 92 forms of hyperalgesia, although CaV2.2 splice isoforms contribute equally to acute nociception 93 (Altier et al., 2007; Andrade et al., 2010). 94 Here, we present functional evidence that CaV2.2 channels in Trpv1-lineage nociceptors are at 95 peripheral nerve endings in skin in addition to their accepted role at central synapses. At central 96 Trpv1-lineage nociceptor termini CaV2.2 channels contribute to the accurate transmission of [4] 97 information about acute noxious stimuli. Whereas, and most significantly, we show that in the 98 periphery, CaV2.2 channels and in particular CaV2.2-e37a isoforms, are necessary for capsaicin- 99 induced heat hypersensitivity. 100 MATERIALS AND METHODS 101 All mice used in this work, except for mice used for in culture calcium imaging, were bred at 102 Brown University and all protocols and procedures were approved by the Brown University 103 Institutional Animal Care and Use Committee. For in vitro calcium imaging, mice were bred at 104 Massachusetts General Hospital and all protocols