ASICs and neuropeptides

Neuropharmacology xxx (2015) 1e6

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Invited review

ASICs and neuropeptides Jonathan S. Vick, Candice C. Askwith* The Department of Neuroscience, The Ohio State University Wexner Medical Center, United States

a r t i c l e i n f o

a b s t r a c t

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The acid sensing ion channels (ASICs) are proton-gated cation channels expressed throughout the nervous system. ASICs are activated during acidic pH fluctuations, and recent work suggests that they are involved in excitatory synaptic transmission. ASICs can also induce neuronal degeneration and death during pathological extracellular acidosis caused by ischemia, autoimmune inflammation, and traumatic injury. Many endogenous neuromodulators target ASICs to affect their biophysical characteristics and contributions to neuronal activity. One of the most unconventional types of modulation occurs with the interaction of ASICs and neuropeptides. Collectively, FMRFamide-related peptides and dynorphins potentiate ASIC activity by decreasing the proton-sensitivity of steady state desensitization independent of G protein-coupled receptor activation. By decreasing the proton-sensitivity of steady state desensitization, the FMRFamide-related peptides and dynorphins permit ASICs to remain active at more acidic basal pH. Unlike the dynorphins, some FMRFamide-related peptides also potentiate ASIC activity by slowing inactivation and increasing the sustained current. Through mechanistic studies, the modulation of ASICs by FMRFamide-related peptides and dynorphins appears to be through distinct interactions with the extracellular domain of ASICs. Dynorphins are expressed throughout the nervous system and can increase neuronal death during prolonged extracellular acidosis, suggesting that the interaction between dynorphins and ASICs may have important consequences for the prevention of neurological injury. The overlap in expression of FMRFamide-related peptides with ASICs in the dorsal horn of the spinal cord suggests that their interaction may have important consequences for the treatment of pain during injury and inflammation. This article is part of a Special Issue entitled ‘ASIC Channels’. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Acid sensing ion channel ASIC Neuropeptide Dynorphin FMRFamide Steady state desensitization

1. Introduction The acid sensing ion channels (ASICs) are proton-gated cation channels and members of the degenerin/epithelial sodium channel (DEG/ENaC) super family (Waldmann et al., 1997a). There are four ASIC genes (ACCN1-4) which encode six known subunit isoforms including ASIC1a and ASIC1b, ASIC2a and ASIC2b, ASIC3, and ASIC4 (Akopian et al., 2000; Babinski et al., 1999; Bassler et al., 2001; Chen et al., 1998; de Weille et al., 1998; Garcia-Anoveros et al., 1997; Grunder et al., 2000; Lingueglia et al., 1995; Price et al., 1996; Waldmann et al., 1996). Three subunits combine to form functional homomeric (i.e. ASIC1a) or heteromeric channels (i.e. ASIC1a/ ASIC2b), each with characteristic biophysical properties and tissue distributions (Jasti et al., 2007; Baron et al., 2002; Benson et al., * Corresponding author. 4066B Graves Hall, 333 West 10th Avenue, Department of Neuroscience, The Ohio State University, Columbus, OH 43210, United States. E-mail address: [email protected] (C.C. Askwith).

2002; Hesselager et al., 2004). ASICs are enriched in the dorsal root ganglia (DRG), olfactory bulbs, hippocampus, amygdala, cerebellum, and cerebral cortex (Waldmann and Lazdunski, 1998). Broadly speaking, ASIC1b and ASIC3 are found in sensory neurons while ASIC1a, ASIC2a, ASIC2b, and ASIC4 are found in both sensory and central neurons. In central neurons, ASICs are localized to the cell body, dendrites, and dendritic spines (Wemmie et al., 2002). Recent work shows that ASICs are activated during synaptic transmission (Kreple et al., 2014; Du et al., 2014). Specifically, acidic pH fluctuations in the synapse are due, at least in part, to proton release from synaptic vesicles within active regions of the brain (Du et al., 2014; DeVries, 2001; Magnotta et al., 2012). Furthermore, acidic pH fluctuations are a major form of neuromodulation in the retina (Wang et al., 2014). Thus, protons and ASICs represent a neurotransmitter system that functions in concert with more traditional neurotransmitters, such as glutamate, to mediate neuronal signaling.

http://dx.doi.org/10.1016/j.neuropharm.2014.12.012 0028-3908/© 2014 Elsevier Ltd. All rights reserved.

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Mice with disruptions in individual ASIC genes (ACCN1-3) are healthy, reproduce, and display no obvious signs of dysfunction (Wemmie et al., 2002; Price et al., 2000, 2001). Moreover, simultaneous disruption of ASIC1, ASIC2, and ASIC3 results in viable animals (Kang et al., 2012). ASIC knockout animals, however, do display particular abnormalities in behavioral and sensory transduction. In particular, disruption of ASIC1a, which eliminates proton-gated currents activated by a pH above 5 in central neurons, results in deficiencies in behaviors linked to fear, anxiety, panic, and depression (Ziemann et al., 2009; Coryell et al., 2009, 2007; Wemmie et al., 2004; Wemmie et al., 2003; Dwyer et al., 2009; Pidoplichko et al., 2014). Interestingly, disruption of ASIC2 has similar effects suggesting that both ASIC1a and ASIC2 are essential for proper function in the brain (Price et al., 2014). Similarly, the localization of ASICs to cutaneous nerve terminals and the involvement of ASICs in sensory transduction suggests that acidic pH fluctuations are also critical for normal sensory inputs (Price et al., 2000, 2001). No mutations in ACCN1-3 have yet been shown to be the cause of a human disease and no therapeutics have yet been proven to improve human heath by targeting ASICs. However, ASICs are involved in a number of pathophysiological conditions and thus represent novel therapeutic targets in the treatment of neurological injury. ASIC1a activation attenuates seizure duration and ASIC3 may contribute to migraine (Ziemann et al., 2008; Yan et al., 2011). ASIC1a also mediates neurodegeneration and death under pathological conditions that induce long lasting cerebral acidosis. In this way, ASIC1a contributes to neuronal injury in cerebral ischemia (Duan et al., 2011; Pignataro et al., 2007; Xiong et al., 2004; Yang et al., 2011), autoimmune inflammation (Arun et al., 2013; Friese et al., 2007; Vergo et al., 2011), and traumatic injury (Yin et al., 2013). ASIC1 and ASIC3 also contribute to hyperalgesia (Mazzuca et al., 2007; Walder et al., 2011, 2010; Ikeuchi et al., 2008; Diochot et al., 2012). Through the genetic deletion or use of inhibitors of ASICs, such as amiloride, venom peptides (i.e. psalmotoxin 1 (PcTx1) or mambalgins), the severity and overall presentation of these nervous system diseases in animal models can be alleviated (Diochot et al., 2012; Escoubas et al., 2000). ASICs generally produce transient currents that inactivate in the continued presence of acidic extracellular pH. The exact concentration of protons required to induce activation varies between subunits and is affected by endogenous neuromodulators and toxins. Under certain conditions, the inactivation of ASICs can be incomplete and allow them to produce sustained currents in the continued presence of acidic extracellular pH (Lingueglia et al., 1997; Waldmann et al., 1997b). When ASICs are exposed to mildly acidic extracellular pH, which itself is not sufficient for robust activation, they become desensitized at steady state (Babini et al., 2002). ASICs that have undergone steady state desensitization are refractory to further decreases in extracellular pH. Steady state desensitization allows ASICs to selectively respond to rapid and localized pH fluctuations. In addition, there is an intimate connection between extracellular calcium and ASIC gating. Higher concentrations of calcium attenuate activation and steady state desensitization as well as accelerate the recovery from inactivation. There is thought to be a calcium binding site, as yet unidentified, within the extracellular domain of ASICs that interferes with the protonation of residues linked to activation and steady state desensitization (Immke and McCleskey, 2001; Zhang et al., 2006). However, exactly where these residues reside within ASICs is the subject of debate. ASICs have several structural domains defined by their resemblance to the anatomy of a closed fist (Jasti et al., 2007). The large extracellular domain of the channel is partially responsible for the gating characteristics of ASICs including the proton sensitivity of activation, steady state desensitization, and

inactivation. The knuckle and finger domains project from the apical part of the channel and contain residues that are glycosylated (Jasti et al., 2007; Jing et al., 2012, 2011; Kadurin et al., 2008; Saugstad et al., 2004). The palm domain is composed of beta sheets and lines the central region of the channel. The thumb domain is exposed to the outside of the channel and is critical for the proton sensitivity of activation (Jasti et al., 2007; Sherwood et al., 2009). The wrist domain connects the extracellular to the transmembrane domains and is involved in transducing extracellular signals into changes in gating characteristics (Li et al., 2009; Passero et al., 2009; Tolino et al., 2011; Yang et al., 2009). The channel also contains a “beta-ball” domain which is critical for the conformational changes that accompany gating and is central to the knuckle, palm, and thumb domains (Bargeton and Kellenberger, 2010). One of the most highlighted regions in the extracellular domain is the acidic pocket, an area composed of the thumb, palm, and beta-ball domains of adjacent subunits. This region is integral for activation and steady state desensitization (Jasti et al., 2007; Sherwood et al., 2009; Li et al., 2009; Yang et al., 2009; Paukert et al., 2008). 2. ASICs and neuropeptides The DEG/ENaC super family includes FaNaC, a molluscan ion channel that is gated by the neuropeptide FMRFamide (Phe-MetArg-Phe-amide) and modulated by acidic pH (Lingueglia et al., 1995). FaNaC-like receptors have been identified in mollusks and hydra (Dürrnagel et al., 2010; Golubovic et al., 2007) but not in mammals. While FMRFamide is not produced in mammals, injection of FMRFamide into the brain can alter behavior and blood pressure in rodents (Hinuma et al., 2000; Muthal et al., 1997). Mammals do express FMRFamide-related peptides, including neuropeptide FF (NPFF), neuropeptide AF (NPAF), RFamide related peptide 1 and 3, (RFRP-1, RFRP-3), 26-RFamide, prolactin releasing peptide (PrP), and Kiss1 (Hinuma et al., 2000; Yang et al., 1985; Jiang et al., 2003; Hinuma et al., 1998; Lee et al., 1996). The pharmacology of the FMRFamide-related peptides in mammals is thought to arise from the activation of five distinct G proteincoupled receptors (Parhar et al., 2012). However, the FMRFamiderelated peptides also have effects that are independent of G protein-coupled receptor activation (Allard et al., 1989; Gayton, 1982; Kavaliers, 1987; Raffa, 1988; Raffa et al., 1986; Roumy and Zajac, 1998). ASICs, like FaNaC, are affected by the FMRFamiderelated peptides, but while FaNaC is activated by neuropeptides and modulated by acidic pH, ASICs are activated by acidic pH and modulated by neuropeptides (Askwith et al., 2000; Perry et al., 2001). Several aspects of ASIC current are affected by FMRFamide and the FMRFamide-related peptides (Fig. 1). Most notably, FMRFamide slows inactivation and induces a pH-dependent sustained current in ASIC1 and ASIC3 (Askwith et al., 2000). Moreover, the sustained current displays reduced ion selectivity compared to the transient peak current. The effect of FMRFamide is observed in Xenopus oocytes expressing different ASIC subunits, cultured cells transfected with different ASIC subunits, and native proton-gated currents from isolated DRG or central neurons (Askwith et al., 2000; Allen and Attwell, 2002). FMRFamide affects both homomeric ASIC1 and ASIC3 as well as heteromeric ASIC1b/ASIC3, ASIC1a/2a, ASIC1a/ 2b, and ASIC3/2a (Askwith et al., 2000; Catarsi et al., 2001; Chen et al., 2006; Sherwood and Askwith, 2008; Xie et al., 2003). With ASIC3/2a, FMRFamide slows inactivation and enhances the amplitude of the transient current (Catarsi et al., 2001). Homomeric ASIC2a or heteromeric ASIC2a/2b are unaffected by FMRFamide. In DRG neurons, an extensive complement of ASIC1, ASIC2, and ASIC3 are expressed (Benson et al., 2002). The increase in the

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Fig. 1. Amino acid sequences of the FMRFamide-related peptides and dynorphins. (A) The sequence of FMRFamide and human FMRFamide-related neuropeptides. The “RFamide” moiety is shown in red. (B) Amino acid sequences of the dynorphins. The smallest dynorphin peptide that modulates ASIC1a is highlighted in red.

sustained current by FMRFamide is dependent on ASIC3 in DRG neurons, as disruption of ASIC3 eliminates the effect of FMRFamide (Askwith et al., 2000; Deval et al., 2003; Xie et al., 2002). Furthermore, while loss of ASIC2 is without effect, loss of ASIC1 from mouse DRG neurons potentiates the effects of FMRFamide (Xie et al., 2003). These results indicate that ASIC3 preferentially mediates the increase in the sustained current observed with FMRFamide in DRG neurons. In central neurons that do not express ASIC3, FMRFamide slows inactivation and some sustained current is observed, however, the effect is less pronounced than that observed in DRG neurons (Allen and Attwell, 2002). FMRFamide also decreases the proton sensitivity of steady state desensitization of ASIC1b/ASIC3 (Chen et al., 2006) and ASIC1a (Sherwood and Askwith, 2008) such that more acidic pH is required to induce desensitization (Fig. 2). Endogenous FMRFamide-related peptides also modulate ASIC inactivation and induce sustained current. In particular, NPFF and RFRP-1 increase the sustained proton-gated currents in mouse DRG neurons. Similarly, the mammalian neuropeptides NPFF and QRFP decrease the proton sensitivity of steady state desensitization (Xie et al., 2003). Ultimately, the specific effect of a given FMRFamide-related peptide is dependent upon its primary sequence in combination with the ASIC subunit and species identity. Furthermore, while the concentration of FMRFamiderelated peptide needed to modulate ASICs is high, ASICs are localized to synapses and respond to acidic pH fluctuations within the synaptic cleft. The synaptic cleft is an area that sees a localized and highly concentrated accumulation of neuropeptide after neurotransmitter release and is an ideal location for the modulation of ASICs by the FMRFamide-related peptides. FMRFamide action generally requires the peptide to be applied before the extracellular solution is acidified. For example, if

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FMRFamide is present at pH 7.4 prior to a pH reduction sufficient for activation, there is an increase in the sustained current of ASIC1a and ASIC1b/ASIC3 (Fig. 2B). If FMRFamide is co-applied during the pH reduction, little change in the current kinetics occurs. Accordingly, the effect of FMRFamide remains regardless of whether or not it is continuously applied during the pH reduction (Askwith et al., 2000; Chen et al., 2006). These results suggest that FMRFamide initially binds to ASICs in the closed or inactivated state, presumably through a C-terminal arginine, as neutralization of this positive charge to valine attenuates the increase in the sustained protongated current in rat DRG neurons (Ostrovskaya et al., 2004). The effects of the FMRFamide-related peptides are also pH dependent, being most pronounced at pH 5, and eliminated by increasing extracellular calcium (Chen et al., 2006; Ostrovskaya et al., 2004). These results suggest that the FMRFamide-related peptides have their greatest effects when ASICs are in a protonated state and calcium is not bound. Although exactly how the FMRFamiderelated peptides modulate the gating of ASICs is not well understood, recent work suggests that the palm domain is essential for the effects of FMRFamide. In the inactivated state of ASIC1a, L415 in the region that links beta sheets 11 and 12 within the palm domain (b11eb12linker) hydrophobically interacts with I307 in a7 of the thumb domain, V367 in b10 of the palm domain, and L280 in b9 of the palm domain. The L415C and L280C mutations result in an increase in the sustained current of ASIC1a when treated with MTSET. The L280C mutation also potentiates the increase in the sustained and transient currents by the synthetic FMRFamide-related peptide, FRRFa, in ASIC1a. These results suggest that the FMRFamiderelated peptides affect the lower palm domain by interfering with the conformational changes necessary for inactivation (Frey et al., 2013). Despite this, not all of the FMRFamide-related peptides that interact with ASICs slow inactivation; some decrease the proton sensitivity of steady state desensitization. Thus, there are likely additional interactions between the FMRFamide-related peptides and ASICs that are as yet unknown. In addition to FMRFamide-related peptides, the dynorphin peptides modulate ASICs. Dynorphins are highly basic peptides expressed abundantly throughout the nervous system (Fig. 1). Specifically, the dynorphins are expressed in the amygdala and contribute to fear learning (Bilkei-Gorzo et al., 2012). Both big dynorphin and dynorphin A decrease the proton-sensitivity of steady state desensitization of ASIC1a and ASIC1b independent of opioid receptor activation (Fig. 2C). Big dynorphin also decreases the proton sensitivity of steady state desensitization of protongated currents in mouse cortical neurons. Big dynorphin is nearly 1000 times more potent than dynorphin A in its modulation of ASICs, while dynorphin B is without effect. Thus, like the FMRFamide-related peptides, their primary structures determine not only their potency but their specific modulatory effects. Like the

Fig. 2. FRRFamide, FMRFamide, and Dynorphin A Modulation of Human ASIC1a. Representative traces of human ASIC1a expressed in Xenopus oocytes showing the response to FRRFamide (A), FMRamide (B), or Dynorphin A(1-17) (C), Note that all three peptides inhibit steady-state desensitization of ASIC1a and allow activation after incubation with pH 6.8. Only FMRFamide and FRRFamide slow inactivation and induce a sustained current (red arrows). Dynorphin A, FRRFamide, and FMRFamide were applied for two minutes at pH 7.9(50e100 mM) and during the pH 6.8 conditioning solution indicated in gray boxes above the trace. The pH 5.0 test solution was applied for 30 s as indicated in white boxes above the trace.

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FMRFamide-related peptides, dynorphins are most effective when applied before the extracellular solution is acidified. However, unlike the FMRFamide-related peptides, big dynorphin does not slow inactivation and induce sustained currents. The dynorphins appear to only affect homomeric ASIC1 and heteromeric ASIC1a/2b channels, and the presence of ASIC3 subunits attenuates the effects of dynorphin (Sherwood and Askwith, 2009). PcTx1 interacts with the acidic pocket of the extracellular domain and the surrounding area to increase the proton-sensitivity of steady state desensitization of ASIC1a (Escoubas et al., 2000; Chen et al., 2005) and ASIC1a/2b (Sherwood et al., 2011). Interestingly, PcTx1 competes with big dynorphin in the modulation of steady state desensitization of ASIC1a, suggesting that they may share a binding site. Furthermore, PcTx1 can also affect pain as intrathecal injections of PcTx1 reduce mechanical and thermal hyperalgesia in mice (Mazzuca et al., 2007). The interactions between neuropeptides and ASICs could have a variety of physiological and pathological consequences. The FMRFamide related peptides are abundant within the spinal cord and both ASICs and the FMRFamide-related peptides localize to the dorsal horn of the spinal cord. In addition, both the FMRFamiderelated peptides and ASICs affect pain processing. As FMRFamide decreases the proton-sensitivity of steady state desensitization and slows inactivation, resulting in a sustained proton-gated current in DRG neurons, extracellular acidification and concomitant accumulation of FMRFamide-related peptides during injury and inflammation may contribute to the role of ASICs in hyperalgesia. Taken together, these studies suggest that the FMRFamide-related peptides preferentially interact with the extracellular domain of the inactivated or closed channel and potentiate ASIC activity by decreasing the proton-sensitivity of steady state desensitization and slowing inactivation (Waldmann et al., 1997a). Moreover, the dynorphin peptides preferentially interact near the acidic pocket in the extracellular domain of the channel and potentiate ASIC activity by decreasing the proton-sensitivity of steady state desensitization (Akopian et al., 2000). The importance of the interaction between FMRFamide-related peptides and ASICs in the brain is less clear. The FMRFamide-related peptides have predominantly been studied in the hypothalamus, a location where the function of ASICs remains unknown. Thus, although FMRFamide slows inactivation of ASICs in Purkinje neurons from the cerebellum, it is not clear how this effect may contribute to neuronal activity in the brain. On the other hand, dynorphins and ASICs are both localized to the amygdala and involved in fear and anxiety. The dynorphin peptides big dynorphin and dynorphin A decrease the proton-sensitivity of steady state desensitization of proton-gated currents in cortical neurons. This would allow ASICs to continue to respond to reductions in pH even after the basal pH has become modestly acidic. Moreover, big dynorphin enhances neuronal death following prolonged extracellular acidosis (Sherwood and Askwith, 2009). Interestingly, mutations in the dynorphin A peptide cause neurodegeneration in spinocerebellar ataxia 23 (SCA23) through non-opioid receptor dependent mechanisms (Bakalkin et al., 2010; Watanabe et al., 2012), indicating that inhibition of ASIC1a activity could provide a novel strategy to attenuate neurological injury in this genetic disease (Vig et al., 2014). Dynorphins can modulate NMDA receptors as well as ASICs suggesting dynorphins may interact with additional channels (Caudle and Mannes, 2000). Thus, understanding the interaction between ASICs and neuropeptides may have profound implications for the prevention of ASIC-mediated injuries in conditions of constitutive cerebral acidosis. New insights to promote the development of novel therapeutics that can limit ASIC activity though modulation of steady state desensitization and inactivation are an important first step in this process. In short, preventing the

interaction of the FMRFamide-related peptides with ASICs may prove useful in the treatment of hyperalgesia while preventing the interaction of the dynorphin peptides with ASICs may reduce the severity of neuronal injury by preventing neuronal death upon prolonged extracellular acidosis.

Acknowledgments This work was possible through an NIH R01 NINDS #NS062967. J. S. Vick was supported from a P30 NINDS Core Grant #NS045758. The authors acknowledge Thomas Sherwood for technical assistance and Kirk Mykytyn for helpful discussions.

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