ASICS AND NEUROPEPTIDES

Abstract

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.

1.1 Introduction

The acid sensing ion channels (ASICs) are proton-gated cation channels and members of the degenerin/epithelial sodium channel (DEG/ENaC) super family (1). There are four ASIC genes (ACCN1-4) which encode six known subunit isoforms including ASIC1a and ASIC1b, ASIC2a and ASIC2b, ASIC3, and ASIC4 (2-11). 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 (12-15). ASICs are enriched in the dorsal root ganglia (DRG), olfactory bulbs, hippocampus, amygdala, cerebellum, and cerebral cortex (16). 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 (17). ASICs are activated by reductions in extracellular pH and depolarize the membrane. Recent work shows that ASICs are activated during synaptic transmission (18,19). 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 (19-21). Furthermore, acidic pH fluctuations are a major form of neuromodulation in the retina (22). Thus, protons and ASICs represent a neurotransmitter system that functions in concert with more traditional neurotransmitters, such as glutamate, to mediate neuronal signaling.

Mice with disruptions in individual ASIC genes (ACCN1-3) are healthy, reproduce, and display no obvious signs of dysfunction (17,23,24). Moreover, simultaneous disruption of ASIC1, ASIC2, and ASIC3 results in viable animals (25). 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 (26-32).Interestingly, disruption of ASIC2 has similar effects suggesting that both ASIC1a and ASIC2 are essential for proper function in the brain (33). 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 (23,24).

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 (34,35). 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 (36-39), autoimmune inflammation (40-42), and traumatic injury (43). ASIC1 and ASIC3 also contribute to hyperalgesia (44-48). 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 (48,49).

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 (50,51). When ASICs are exposed to mildly acidic extracellular pH, which itself is not sufficient for robust activation, they become desensitized at steady state (52). 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 (53,54). 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(12). 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 (12,55-58). 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 (12,59). The wrist domain connects the extracellular to the transmembrane domains and is involved in transducing extracellular signals into changes in gating characteristics (60-63). 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 (64). 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 (12,59,60,63,65).

1.2 ASICs and Neuropeptides

The DEG/ENaC superfamily includes FaNaC, a molluscan ion channel that is gated by the neuropeptide FMRFamide (Phe-Met-Arg-Phe-amide) and modulated by acidic pH (9). FaNaC-like receptors have been identified in mollusks and hydra (66,67) 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 (68,69). 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 (68,70-73). The pharmacology of the FMRFamide-related peptides in mammals is thought to arise from the activation of five distinct G protein-coupled receptors (74). However, the FMRFamide-related peptides also have effects that are independent of G protein-coupled receptor activation (75-80). ASICs, like FaNaC, are affected by the FMRFamide-related peptides, but while FaNaC is activated by neuropeptides and modulated by acidic pH, ASICs are activated by acidic pH and modulated by neuropeptides (81,82).

Several aspects of ASIC current are affected by FMRFamide and the FMRFamide-related peptides (Figure 1). Most notably, FMRFamide slows inactivation and induces a pH-dependent sustained current in ASIC1 and ASIC3 (81). 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 (81,83). FMRFamide affects both homomeric ASIC1 and ASIC3 as well as heteromericASIC1b/ASIC3, ASIC1a/2a, ASIC1a/2b, and ASIC3/2a (81,84-87). With ASIC3/2a, FMRFamide slows inactivation and enhances the amplitude of the transient current (84). Homomeric ASIC2a or heteromeric ASIC2a/2b are unaffected by FMRFamide.

Figure 1.

Amino acid sequenced of the FMRFamide-related peptided and dynorphins, (A) The sequence of FMRFamide and human FMRFamide-related neuropeptided. The “RFamide” moiety is shown in red. (B) Amino acid sequences of the dunorphins. The smallest dynorphin peptide that modulates ASIC is highlighted in red.

In DRG neurons, an extensive complement of ASIC1, ASIC2, and ASIC3 are expressed (14). The increase in the sustained current by FMRFamide is dependent on ASIC3 in DRG neurons, as disruption of ASIC3 eliminates the effect of FMRFamide (81,88,89). Furthermore, while loss of ASIC2 is without effect, loss of ASIC1 from mouse DRG neurons potentiates the effects of FMRFamide (87). 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 (83). FMRFamide also decreases the proton sensitivity of steady state desensitization of ASIC1b/ASIC3 (85) and ASIC1a (86). Specifically, FMRFamide reduces the proton-sensitivity of steady state desensitization such that more acidic pH is required to induce desensitization (Figure 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 (87). 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 FMRFamide-related 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.

Figure 2. FRRFamide, FMRFamide, and Dynorphin A Modulation of Human ASIC1a.

Representative traces of human ASICa expressed in Xenopus oocytes showing the response to FRRFamide (A), FMRFamide (B), or Dynorphin A(1-17) (C), Note that all three peptided inhibit steady-sate desenitization of ASIC and allow activation after incubation with Ph 6.8. Only FMRF Famide and FRRFamide slow inactivation and induce a sustained current (red arrows). Dynorphin A, FRRF amide, and FMRFamide were applied fro two minutes at Ph 7.9 (50100μ) and during the Ph 6.8 condition solution indicated in grey boxes above the trace. The pH 5.0 test solution was applied for 30s as indicated in white boxes the trace.

FMRFamide action generally requires the peptide to be applied before the extracellular solution is acidified. For example, if 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 (Figure 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 (81,85). 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 proton-gated current in rat DRG neurons (90). The effects of the FMRFamide-related peptides are also pH dependent, being most pronounced at pH 5, and eliminated by increasing extracellular calcium (85,90). 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 FMRFamide-related 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 (β₁₁-β₁₂linker) hydrophobically interacts with I307 in α₇ of the thumb domain, V367 in β₁₀ of the palm domain, and L280 in β₉ 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 FMRFamide-related peptides affect the lower palm domain by interfering with the conformational changes necessary for inactivation (91). 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 (Figure 1). Specifically, the dynorphins are expressed in the amygdala and contribute to fear learning (92). Both big dynorphin and dynorphin A decrease the proton-sensitivity of steady state desensitization of ASIC1a and ASIC1b independent of opioid receptor activation (Figure 2C). Big dynorphin also decreases the proton sensitivity of steady state desensitization of proton-gated currents in mouse cortical neurons. Big dynorphin is nearly 1,000 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 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 (93). 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 (49,94) and ASIC1a/2b (95). 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 (44).

The interactions between neuropeptides and ASICs could have a variety of physiological and pathological consequences. The FMRF-amide 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 FMRFamide-related 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 (1) 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 and (2) 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.

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 (93). Interestingly, mutations in the dynorphin A peptide cause neurodegeneration in spinocerebellar ataxia 23 (SCA23) through non-opioid receptor dependent mechanisms (96). Dynorphins can modulate NMDA receptors as well as ASICs suggesting dynorphins may interact with additional channels (97). Yet, our results suggested that ASICs could play a role (98), and recent work indicates that ASIC1a contributes to neuronal damage in spinocerebellar ataxia and suggest inhibition of ASIC1a activity could provide a novel strategy to attenuate neurological injury in this genetic disease (99). 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.

• The acid sensing ion channels (ASICs) are important for normal behaviors.

• ASICs also induce neuronal degeneration in pathological conditions.

• ASICs are modulated by neuropeptides.

• Dynorphins and RFamide-related peptides can enhance ASIC activity.

• These effects are independent of GPCRs.

• Neuropeptides may affect ASICs role in pain, behavior, and neuronal injury.