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The homomeric acid-sensing ion channel 1a (ASIC1a) is a H+-activated ion channel with important physiological functions and pathophysiological impact in the central nervous system. Here we show that homomeric ASIC1a is distinguished from other ASICs by a reduced response to successive acid stimulations. Such a reduced response is called tachyphylaxis. We show that tachyphylaxis depends on H+ permeating through ASIC1a, that tachyphylaxis is attenuated by extracellular Ca2+, and that tachyphylaxis is probably linked to Ca2+ permeability of ASIC1a. Moreover, we provide evidence that tachyphylaxis is probably due to a long-lived inactive state of ASIC1a. A deeper understanding of ASIC1a tachyphylaxis may lead to pharmacological control of ASIC1a activity that could be of potential benefit for the treatment of stroke.
ASIC1a is an ASIC subunit that seems to have important functions for the physiology and pathophysiology of the central nervous system. It contributes to the most abundant ASICs in the brain: homomeric ASIC1a and heteromeric ASIC1a/2a (Baron et al. 2002; Askwith et al. 2004; Vukicevic & Kellenberger, 2004) and knockout of the asic1 gene leads to deficits in spatial memory and learned fear (Wemmie et al. 2002; Wemmie et al. 2003), suggesting a contribution to higher brain functions. Moreover, homomeric ASIC1a seems to be the only ASIC which is permeable for Ca2+ (Waldmann et al. 1997b; Bässler et al. 2001; Yermolaieva et al. 2004). Recently, it has been shown that activation of ASIC1a channels during brain ischaemia leads to Ca2+ influx in neurons and contributes to neuronal death associated with ischaemia (Xiong et al. 2004). This result suggests that the control of ASIC1a activity may be of clinical relevance in the treatment of stroke.
In an early characterization of ASIC currents in mesencephalic neurons and oligodendrocytes, it was noted that the currents showed a run-down phenomenon (Sontheimer et al. 1989). More recently it was noted that ASIC1a channels endogenously expressed in a skeletal muscle cell line (Gitterman et al. 2005) or HEK293 cells (Neaga et al. 2005) or heterologously expressed in Xenopus oocytes (Paukert et al. 2004b) showed tachyphylaxis, a decreased response to successive applications of the ligand. In the present study, we show that tachyphylaxis is specific for homomeric ASIC1a and propose a model in which H+, permeating through the channels, contribute to the induction of a conformational change of the pore that leads to a long-lived inactive state. A detailed understanding of this mechanism could be useful for a pharmacological control of ASIC1a activity.
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Homomeric ASIC1a is the only ASIC that shows tachyphylaxis (Fig. 1). Tachyphylaxis of the ASIC1a current had already previously been noted (Paukert et al. 2004a; Gitterman et al. 2005; Neaga et al. 2005). However, our study for the first time investigates this phenomenon systematically. Beyond a detailed description, our results also provide some clues to the mechanism of tachyphylaxis. First, they show that tachyphylaxis depends on the concentration of extracellular H+. Second, the amiloride and voltage dependence of tachyphylaxis suggest that H+, at least partially, exerts its effect by permeating through the channel. Probably permeating H+ locally decreases the cytoplasmic pH, which somehow induces tachyphylaxis. In agreement with this model, intracellular acidification by a CO2–HCO−3 buffer facilitated tachyphylaxis despite the fact that it reduced the driving force for H+ permeation through ASIC1a.
But why should permeating H+ induce tachyphylaxis when also the related ASIC1b is permeable to H+, yet does not show tachyphylaxis? One clue to this apparent paradox comes from the chimeras, which identify an intracellular region that is probably close to the inner ion permeation pathway (Coscoy et al. 1999; Bässler et al. 2001) and somehow controls tachyphylaxis in ASIC1 (Fig. 10). This region could structure the inner pore in a way that is unique to ASIC1a. Since the same intracellular region is responsible for the Ca2+ permeability of ASIC1a (Bässler et al. 2001), tachyphylaxis and Ca2+ permeability, two features that distinguish homomeric ASIC1a from other ASICs, would be linked by a unique pore structure of ASIC1a. Such a relation between tachyphylaxis and Ca2+ permeability is supported by a recent study reporting that treatment of ASIC1a with trypsin reduces tachyphylaxis and Ca2+ permeability (Neaga et al. 2005). Exactly how this unique structure favours tachyphylaxis is presently unknown. We speculate that H+ may interact with the inner pore of ASIC1a to induce tachyphylaxis. It is even possible that Ca2+ exerts its negative effect on tachyphylaxis (Fig. 7) by interacting with this same site, competing with the interaction of H+. At present we have, however, no direct evidence for an intracellular or ion pore effect of Ca2+ on tachyphylaxis. Therefore, although our results suggest that Ca2+ did not affect tachyphylaxis by changing steady-state desensitization, H+ activation or blocking the pore, we cannot rule out that Ca2+ affects tachyphylaxis via an extracellular mechanism.
We propose that binding of H+ induces with a low efficacy a conformational change, leading to a long-lived desensitized state. We think this state is long-lived because we did not obtain consistent evidence for recovery from tachyphylaxis (not shown). The hypothesis of a long-lived blocked state of the channel is consistent with the unchanged single channel amplitude during repetitive stimulation in excised patches (Fig. 3). Interestingly, tachyphylaxis could be induced by either extra- or intracellular acidification only when the pore was open. This suggests that the putative site where binding of H+ induces tachyphylaxis is accessible only in the open conformation, but not in the closed and desensitized conformations of the channel.
The following simple scheme illustrates the relation of the long-lived desensitized state D2 to the other basic states of the channel:
According to this scheme, upon binding of H+, channels would open. From the open state, O, the majority of the channels would undergo acute desensitization entering state D1. A smaller fraction, however, would enter the long-lived desensitized state D2. Since the fraction of channels that enters D2 is pH dependent, channels would bind additional H+ during the transition from O to D2 (not shown in the scheme). This explains why high H+ concentrations would increase the fraction of channels entering the long-lived state D2 whereas an increased rate for acute desensitization (transition from O to D1), as observed in ASIC1a mutant SQL(83-85)PLM (Fig. 5B), would increase the fraction of channels entering state D1, attenuating tachyphylaxis. Although this scheme does not take into account the existence of multiple closed, open and desensitized states, it illustrates the basic features of the long-lived state D2.
We confirmed an earlier finding (Waldmann et al. 1997b) that ASIC1a is permeable for H+. Moreover, ASIC1b is also H+ permeable (not shown). Since the related ENaC is also H+ permeable (Gilbertson et al. 1993), H+ permeability may be a general feature of ASICs. Usually, the H+ concentration is small compared with the Na+ concentration, and therefore the contribution of the H+ current to the total ASIC current will be small. However, under some circumstances, H+ could substantially contribute to the depolarizing current passing through ASICs. For example, ASIC2a/2b heteromers are expressed in rat taste cells (Ugawa et al. 2003) and H+ permeating through this channel could lead to excitation of the taste cell even when there is only a small Na+ concentration on the tongue.
Another finding of our study is the dependence of ASIC1a tachyphylaxis on the intracellular pH (pHi). Several studies reported an intracellular acidification of mammalian neurons associated with Ca2+ permeation through NMDA receptors (Irwin et al. 1994; Wang et al. 1994; Canzoniero et al. 1996) or through TRPV1 (Hellwig et al. 2004). Our study suggests that a reduced pHi in neuronal cells would negatively feed back on ASIC1a activity, contributing to the control of ASIC1a activity. Very recently, inhibition by acidic pHi has indeed been shown for ASICs from mouse cortical neurons (Wang et al. 2006). Such an inhibition could be especially relevant in nociceptors where ASIC1a is co-expressed with TRPV1 (Olson et al. 1998; Alvarez De La Rosa et al. 2002; Ugawa et al. 2005; Poirot et al. 2006). TRPV1 shares the property of H+ permeability with ASIC1a, and TRPV1 activation leads to a robust decrease of pHi (Hellwig et al. 2004). Thus, TRPV1 activity could control ASIC1a activity in nociceptors.
Our study shows that the impact of tachyphylaxis on ASIC1a activity depends on many parameters (pHo, pHi, [Ca2+]o, duration of the open state, number of stimulations). Therefore, the impact of tachyphylaxis under in vivo conditions is hard to predict. At normal body temperature, where acute desensitization is faster than at the temperature at which our experiments have been performed (approximately 22°C) (Askwith et al. 2001), tachyphylaxis is probably attenuated. However, understanding tachyphylaxis of ASIC1a may be a way to control ASIC1a activity, which is of potential therapeutic value for the treatment of stroke (Xiong et al. 2004).