King’s Research Portal DOI: 10.1113/JP278211 Document Version Peer reviewed version Link to publication record in King's Research Portal Citation for published version (APA): Lainez, S., Tsantoulas, C., Biel, M., & McNaughton, P. A. (2019). HCN3 ion channels - roles in sensory neuronal excitability and pain: HCN3 ion channels and pain. The Journal of Physiology, 597(17), 4661-4675. https://doi.org/10.1113/JP278211 Citing this paper Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections. 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Oct. 2021 DOI: 10.1113/JP278211 HCN3 ion channels: roles in sensory neuronal excitability and pain Sergio Lainez1,*, Christoforos Tsantoulas1,3,*, Martin Biel2 and Peter A McNaughton1,3. Running title: HCN3 ion channels and pain 1Wolfson Centre for Age-Related Research, King´s College London, Guy´s Campus, London SE1 1UL, UK. 2Center for Integrated Protein Science (CIPS-M) and Center for Drug Research, Department of Pharmacy, Ludwig-Maximilians-Universität Munchen, Munich, Germany. 3Correspondence should be addressed to Peter McNaughton, email: [email protected], or to Christoforos Tsantoulas, email [email protected]. * These authors contributed equally to this paper. Keywords: HCN3, Ion channel, Neuron, Sensory, Nociception, Pain Key points: HCN ion channels conducting the Ih current control the frequency of firing in peripheral sensory neurons signalling pain Previous studies have demonstrated a major role for the HCN2 subunit in chronic pain but a potential involvement of HCN3 in pain has not been investigated HCN3 was found to be widely expressed in all classes of sensory neurons (small, medium, large) where it contributes to Ih HCN3 deletion increased the firing rate of medium, but not small, sensory neurons This is an Accepted Article that has been peer-reviewed and approved for publication in the The Journal of Physiology, but has yet to undergo copy-editing and proof correction. Please cite this article as an 'Accepted Article'; doi: 10.1113/JP278211. This article is protected by copyright. All rights reserved. Pain sensitivity both acutely and following neuropathic injury was largely unaffected by HCN3 deletion, with the exception of a small decrease of mechanical hyperalgesia in response to a pinprick We conclude that HCN3 plays little role in either acute or chronic pain sensation Summary HCN ion channels govern the firing rate of action potentials in the pacemaker region of the heart and in pain-sensitive (nociceptive) nerve fibres. Intracellular cAMP promotes activation of the HCN4 and HCN2 isoforms, while HCN1 and HCN3 are relatively insensitive to cAMP. HCN2 modulates action potential firing rate in nociceptive neurons and plays a critical role in all modes of inflammatory and neuropathic pain, but the role of HCN3 in nociceptive excitability and pain is less studied. Using antibody staining, we found that HCN3 is expressed in all classes of somatosensory neurons. In small nociceptive neurons, genetic deletion of HCN2 abolished the voltage shift of the Ih current carried by HCN isoforms following cAMP elevation, while the voltage shift was retained following deletion of HCN3, consistent with the sensitivity of HCN2 but not HCN3 to cAMP. Deletion of HCN3 had little effect on the evoked firing frequency in small neurons, but enhanced the firing of medium-sized neurons, showing that HCN3 makes a significant contribution to the input resistance only in medium-sized neurons. Genetic deletion of HCN3 had no effect on acute thresholds to heat or mechanical stimuli in vivo, and did not affect inflammatory pain measured with the formalin test. Nerve-injured HCN3 KO mice exhibited similar levels of mechanical allodynia and thermal hyperalgesia to WT mice, but reduced mechanical hyperalgesia in response to a pinprick. These results show that HCN3 makes some contribution to excitability, particularly in medium-sized neurons, but has no major influence on acute or neuropathic pain processing. Key points HCN ion channels generate an inward current that can regulate action potential firing in somatosensory nerve fibres and can play an important role in pain sensation. The HCN1 isoform plays a limited role only in cold sensation following nerve injury. HCN2, on the other hand, is a key regulator of excitability in nociceptive nerve fibres, and controls the perception of inflammatory and neuropathic pain, but has no influence on acute pain sensation. Here we examine a potential role for the HCN3 isoform in neuronal excitability and pain. HCN3 is widely expressed in somatosensory neurons, and contributes to the regulation of firing of action potentials in medium-sized neurons, amongst which many have a nociceptive function. Genetic deletion of HCN3, however, had little impact on acute pain sensation, on inflammatory pain, nor on pain following nerve injury (neuropathic pain). We conclude that HCN3 does not play an important role in pain sensation. This article is protected by copyright. All rights reserved. 2 Introduction Ion channels belonging to the Hyperpolarization activated, Cyclic Nucleotide-gated ion channels family (HCN ion channels) have recently attracted interest as drivers of chronic pain (Chaplan et al., 2003; Brown et al., 2004; Luo et al., 2007; Momin et al., 2008; Emery et al., 2011; Acosta et al., 2012; Weng et al., 2012; Noh et al., 2014; Young et al., 2014; Tsantoulas et al., 2016; Tsantoulas et al., 2017). Genetic deletion or pharmacological block of HCN2 abolishes thermal hyperalgesia in inflammatory pain, and both thermal and mechanical hyperalgesia in neuropathic pain (Emery et al., 2011; Emery et al., 2012; Young et al., 2014; Tsantoulas et al., 2017). Genetic deletion or pharmacological inhibition of HCN1, on the other hand, has more limited effects on either inflammatory or neuropathic pain, although it does provide partial analgesia in some modalities of neuropathic pain, including in cold hypersensitivity triggered by oxaliplatin- or nerve injury-induced neuropathies (Momin et al., 2008; Tibbs et al., 2013; Resta et al., 2018). In the present study we sought to determine whether HCN3 ion channels play a role in acute, inflammatory or neuropathic pain. The HCN family comprises four members (HCN1-4) that carry an inward current activated by hyperpolarization from the membrane resting potential. In both the heart and the nervous system the membrane current carried by HCN ion channels has a pace-making role, because the channels carry an inward current and are activated by membrane hyperpolarization following an action potential, leading to rebound membrane depolarization and initiation of a further action potential (Biel et al., 2009; DiFrancesco, 2010). A significant difference between members of the HCN family is that the voltage-dependence of activation of HCN2 and HCN4 is shifted in the positive direction on the voltage axis by direct cAMP binding to the C-terminus, but while both HCN1 and HCN3 contain a cAMP-binding domain, their voltage- dependence is largely insensitive to elevations of cAMP (Santoro et al., 1998; Ludwig et al., 1999; Mistrik et al., 2005). Thus the inward current carried by HCN2 and HCN4 can be enhanced by physiologically important transmitters which act to elevate intracellular cAMP levels, such as adrenaline in the heart (DiFrancesco, 2010) or prostaglandin E2 in nociceptive neurons (Emery et al., 2012). Expression of HCN1 and HCN4 underlies the pacemaker current (If) in the pacemaker region of the rodent heart (Baruscotti et al., 2011; Fenske et al., 2013), although species differences have also been noted (Thollon et al., 2007; Stillitano et al., 2008). In neurons, the main mediators of the hyperpolarisation-activated current, Ih, are HCN1, 2 and 3 (Momin et al., 2008). HCN3 has been shown to be expressed in rat DRG neurons by RT-PCR, in situ hybridisation and immunocytochemistry (Chaplan et al., 2003; Kouranova et al., 2008; Cho et al., 2009), although in mice, by contrast, mRNA for HCN3 has been reported to be expressed at low levels (Moosmang et al., 2001; Schnorr et al., 2014). No overt phenotype is seen in HCN3-/- mice, which are viable and healthy throughout development from embryonic to adult stages (Fenske et al., 2011). No impairment in cardiac function is seen in HCN3-/- mice, and pacemaker activity is normal, although there is a change in the repolarization rate of the epicardial ventricular action potential, leading to an increase of both the T-wave amplitude and the QT duration in the ECG at low heart rates (Fenske et al., 2011). Recent work has also shown some changes in the fear responses of HCN3-/- mice (Stieglitz et al., 2017). This article is protected by copyright. All rights reserved. 3 To investigate a possible role for HCN3 in pain, we examined in vitro electrophysiological and expression data in sensory neurons from HCN3 and HCN2-deficient mice.