Strong modulation by RFamide neuropeptides of the ASIC1b/3 heteromer in competition with extracellular calcium

Abstract

Acid-sensing ion channels are excitatory receptors for extracellular H⁺. Since the extracellular H⁺ concentration can significantly increase during an inflammation, one of the proposed functions for ASICs is peripheral perception of pain. The ASIC1b and ASIC3 subunits are specifically expressed in sensory ganglia neurons and are candidate sensors of peripheral acidosis. However, the function of these ASIC subunits is limited by their steady-state desensitization during a small but persistent increase of the H⁺ concentration and by their desensitization after stronger H⁺ stimuli. Here we show that ASIC1b and ASIC3 form a heteromeric channel that, at steady-state, desensitizes at more acidic values than either homomeric ASIC1b or homomeric ASIC3 alone. Moreover, we show that RFamide neuropeptides, putative modulators of ASIC activity during inflammation, drastically slow down the desensitization of the ASIC1b/3 heteromer with an apparent dissociation constant of ∼24 μM. The apparent affinity for RFamide-induced effects was about 3-fold higher at low extracellular calcium concentrations. Our results suggest that the ASIC1b/3 heteromer is a possible target for RFamide neuropeptides in the peripheral nervous system.

Introduction

Acid-sensing ion channels (ASICs) are ion channels that are gated by extracellular H⁺ (Waldmann and Lazdunski, 1998, Krishtal, 2003). Although the extracellular pH is usually maintained within a narrow range of around pH 7.4, it can significantly decrease after the release of the acidic content of synaptic vesicles, during metabolic acidosis that often accompanies tissue inflammation, and after ingestion of acidic food. Indeed, experimental evidence has confirmed a contribution of ASICs to synaptic transmission (Wemmie et al., 2002), peripheral perception of pain (Sutherland et al., 2001, Voilley et al., 2001, Chen et al., 2002, Mamet et al., 2002), and perception of taste (Ugawa et al., 2003).

The genome of mammals contains four genes coding for ASICs: asic1–asic4 (Waldmann and Lazdunski, 1998, Gründer et al., 2000). Alternative first exons of the asic1 gene lead to the two variants ASIC1a and ASIC1b (Chen et al., 1998, Bässler et al., 2001). ASIC1a is broadly expressed in the central and peripheral nervous system (Waldmann et al., 1997b), whereas ASIC1b, like ASIC3 (Waldmann et al., 1997a), is specifically expressed in the peripheral nervous system (Chen et al., 1998). Therefore, ASIC1b and ASIC3 could have a role in pain perception. Indeed, ASIC3 has been proposed as an important H⁺ sensor in cardiac afferents (Sutherland et al., 2001) and has been implicated in the development of mechanical hyperalgesia (Sluka et al., 2003) and the modulation of high-intensity pain sensations (Chen et al., 2002). Elimination of all functional ASIC currents in mice results in paradoxical hypersensitivity to a number of pain modalities (Mogil et al., 2005). About 40–50% of the neuropeptidergic and non-peptidergic C fibers, most of which are nociceptors, express ASIC1b and a similar proportion expresses ASIC3 (Voilley et al., 2001). Recently it has been reported that approximately 10% of the ASIC1b-positive DRG neurons express ASIC3 (Ugawa et al., 2005), demonstrating co-expression of ASIC1b and ASIC3 in a small subset of sensory neurons.

Functional ASICs are homo- or hetero-oligomeric assemblies of individual subunits (Sutherland et al., 2001, Baron et al., 2002, Benson et al., 2002, Xie et al., 2002, Askwith et al., 2004, Hesselager et al., 2004, Wu et al., 2004). Most likely, four subunits assemble to form the functional channel (Coscoy et al., 1998, Firsov et al., 1998).

ASIC1b and ASIC3 homomers activate rapidly upon H⁺ application (τ_act about 10 ms at pH 6 for ASIC1b) (Bässler et al., 2001) and desensitize completely in the continuous presence of H⁺ (at pH 6, τ_desens about 1 s for ASIC1b and 0.3 s for ASIC3) (Sutherland et al., 2001). Moreover, during a small but persistent decrease of the resting pH, ASIC1b and ASIC3 become desensitized at steady-state (Benson et al., 1999, Babini et al., 2002). Desensitization and steady-state desensitization probably reflect the same molecular process, just at different time scales. They set strong limits to the capacity of these ASIC subunits to serve as peripheral H⁺ receptors during an inflammation, when the pH persistently decreases.

H⁺-gated ASICs are evolutionarily related to another channel that is gated by an extracellular ligand, the FMRFamide receptor FaNaCh (Lingueglia et al., 1995). FaNaCh, which has been cloned from mollusks, is directly gated by the neuropeptide FMRFamide (FMRFa), a common neurotransmitter in mollusks. FaNaCh is the only known peptide-gated ion channel. Mammalian species do not use FMRFa but related peptides, like neuropeptide FF (NPFF, Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide). NPFF belongs to the group of RFamide peptides, which are characterized by a C-terminal Arg–Phe sequence and amidation of the C-terminus. NPFF immunoreactivity is particularly high in the peripheral layers of the dorsal horn of rat spinal cord (Roumy and Zajac, 1998). However, lower level expression was also found in small- and medium-diameter DRG neurons, suggesting that NPFF could be released from sensory neurons (Allard et al., 1999). Moreover, in rats, RFamide peptides are algogenic (Yudin et al., 2004).

FMRFa directly activates the mollusk channel FaNaCh. In contrast, RFamide neuropeptides do not activate ASICs on their own, but rather modulate H⁺-activated ASIC currents (Askwith et al., 2000, Catarsi et al., 2001, Deval et al., 2003, Xie et al., 2003, Ostrovskaya et al., 2004). The main effect of the peptides on ASICs, which has been reported, is to slow down the desensitization of H⁺-induced currents. The slower desensitization of ASICs induced by RFamide peptides increases the depolarizing current and co-application of RFamide peptides with H⁺ indeed increases sensory neuron excitability (Deval et al., 2003). Analysis of knock-out mice suggested that ASIC1 and ASIC3 are the crucial ASIC subunits to mediate the effect of RFamides on H⁺-gated currents in the peripheral nervous system (Xie et al., 2003). Although this suggests that the ASIC1b/3 heteromer is a potential target of neuropeptide modulation, the effect of RFamide peptides on this heteromer has not been investigated so far. Moreover, a mechanism for the action of RFamide peptides has not yet been proposed.

The present study had two main purposes. First, we investigated the characteristics of the ASIC1b/3 heteromer and its modulation by RFa peptides. Second, we studied in detail the mechanism of ASIC modulation by RFa peptides and its dependence on extracellular calcium.