Extracellular trypsin increases ASIC1a selectivity for monovalent versus divalent cations

Acid (4), and a role for ASIC3 has been shown in pseudo-chronic hyperalgesia induced by repeated acid injections in muscle (5). In the central nervous system ASIC1a is the most abundant and most ubiquitously expressed of all ASICs. Evidence exists for a role of ASIC1a in modulating synaptic transmission, memory, and fear conditioning (6, 7). Recently it was shown that ASIC1a mediates cell injury induced by conditions associated with acidosis in the mammalian nervous system (8). On the cellular level, we and others have shown that ASIC activity modulates action potential signaling (9, 10). The ASICs themselves are targets of various regulatory mechanisms, one of them being serine proteases such as trypsin. Extracellular trypsin can cleave ASIC1a in the extracellular loop and thereby shift the pH dependence of channel activation and inactivation to more acidic pH (11). This regulation may adapt the ASIC1a pH dependence to situations in which the extracellular pH is constitutively lowered, as e.g. ischemia. In addition, the trypsin-modified ASIC1a channel shows a reduced permeability toward divalent cations (12).Identification of a protease cleavage site on ASIC1a would be a starting point for the search for endogenous proteases related to trypsin that may regulate ASIC function in vivo. With regard to ASIC function, the mechanism by which protons control ASIC gating is currently unknown. Knowledge of channel parts involved would make it possible to test hypotheses about gating mechanisms. The observation that trypsin cleaves ASIC1a in the extracellular loop and induces significant functional changes suggests that cleavage occurs at a functionally important site of the channel protein. The goal of this study was therefore first to verify that the cleavage event and the functional changes induced by trypsin are correlated and second to identify the cleavage site(s). The data show a correlation between cleavage and functional effect in ASIC1. The mutagenesis approach showed that trypsin cleaves ASIC1a in the N-terminal part of the extracellular loop, between a highly conserved domain and a channel domain that is critical for ASIC1a inhibition by the venom of the spider Psalmopoeus cambridgei. This channel portion is important for channel inactivation properties and undergoes conformational changes during channel inactivation. EXPERIMENTAL PROCEDURES Construction of Chimeras, Site-directed Mutagenesis, and Expression in Xenopus oocytes-TheASIC1a clone used in this study corresponds to expressed sequence tag clone Image 6849487 (National Institutes of Health) and was entirely sequenced. Its sequence is identical to the published mouse ASIC1a sequence BC067025. On the amino acid level its sequence is identical to the rat ASIC1a sequence and downstream of Val-186 to that of rat ASIC1b, except for a S476N substitution in the intracellular C terminus. Other cDNA constructs were kindly provided by M. Lazdunski (rat ASIC3) and S. Gründer (rat ASIC1b).Chimeras were made with ASIC1a and either ASIC1b or ASIC3. To make the cons...

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“…ischemia. In addition, the trypsin-modified ASIC1a channel shows a reduced permeability toward divalent cations (12).…”

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confidence: 99%

Trypsin Cleaves Acid-sensing Ion Channel 1a in a Domain That Is Critical for Channel Gating

Vukicevic

Weder

Boillat

et al. 2006

Journal of Biological Chemistry

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“…Average values of refined rates in these two settings, starting from identical initial rates and identical idealized data, are presented in Table 2. Despite differences between the values tachyphylaxis and precludes a contribution of Na + , since we have proved previously 26 that trypsin decreases ASIC1a permeability for Ca 2+ without altering permeability for monovalent cations (Na + and K + ). Other studies have also demonstrated the inhibitory effect of several PKC isoforms on open probability of ASIC1a channels inserted in artificial lipid bilayers, 55 as well as PKA phosphorylationmediated ASIC1a inhibition by interference with its C-terminus interaction with the PDZ domain of PICK1 (protein interacting with C-kinase 1).…”

Section: Resultsmentioning

confidence: 58%

“…Further, we assessed the action of amitriptyline following a mild trypsin pretreatment (50 μg/ml for 5 min), the effects of which were monitored by recording the cell membrane depolarization in current clamp mode, as described previously. 26 In these conditions, the activatory effect of amitriptyline was completely removed, while the inhibitory effect was attenuated, as proved by the increased halfinhibitory concentration (K inh = 781 μM) and the subunitary Hill coefficient (n Hill = 0.53) yielded by fitting the data with a classical Hill function (Fig. 2B).…”

Section: Resultsmentioning

confidence: 80%

“…Further studies 28 have revealed the trypsin cleavage site in ASIC1a, located at Arg145, in a proximal region of the extracellular loop controlling inactivation kinetics and co-determining the pH dependence of gating, and proved that psalmotoxin 1 (PcTX1) prevents enzyme access to this site. Neaga et al 26 have shown that trypsin pretreatment reduced ASIC1a Ca 2+ permeability and unitary conductance from 5.7 to 2.7 pS, without altering Na + and K + permeabilities. However, the physiological and clinical relevance of these findings requires further assessment, given that several endogenous serine proteases, like tissue plasminogen activator (t-PA), urokinase-type plasminogen activator, thrombin, chymotrypsin B, neuropsin and neurotrypsin, are present in the central nervous system and their activity may be modulated by brain injury.…”

Section: Discussionmentioning

confidence: 99%

“…17 ASIC-mediated cell Ca 2+ entry was correlated with a sustained proton-activated current component 8,9,18,19 and assumed to account for neurodegeneration consequent to ASIC activation in ischemic conditions. [20][21][22][23][24][25] Moreover, we have shown that ASIC1a Ca 2+ permeability is decreased by extracellular proteases, 26 presumably due to direct action at a cleavage site located in the extracellular loop of the channel. 27,28 Accurate single-channel recordings have reveled a wealth of details concerning the gating of ASICs: the existence of distinct gating modes and subconductance states, 29 stepwise activation by protonation of residues involved in Ca 2+ binding, 30 a long-lived desensitized state triggered by proton inflow.…”

Section: Introductionmentioning

confidence: 99%

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ASIC1a activation by amitriptyline and FMRF-amide is removed by serine proteases

Marin¹,

Prica²,

Amuzescu³

et al. 2008

Channels

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Analgesia induced by certain tricyclic antidepressants has been largely used for decades, yet the mechanisms involved are incompletely understood. Starting from previously reported dual effects of amitriptyline on wild-type ENaC (see ref. 39), we extended our study to ASIC1a by performing a series of whole cell and singlechannel recordings of proton-activated currents in HEK293 cells. Acid pulses were applied at 2 or 5 min intervals, and amitriptyline (1-500 μM) was applied at a holding pH of 7.4 or 8.4 between pulses. Dose-response plots were fitted with dual Hill type functions, yielding a half-activatory constant of 0.3 μM and a half-inhibitory constant of 382 μM at pH 7.4. At pH 8.4 both constants were shifted to higher values (0.5 and 444 μM, respectively). In whole-cell experiments, FMRF-amide increased the peak amplitude of ASIC1a transients at 0.1 μM and decreased it at 1 and 100 μM. Single-channel recordings were idealized and fitted using an 8-state linear connectivity model comprising four consecutive activation steps. Both amitriptyline (1 μM) and FMRF-amide (0.1 μM) increased the unitary current amplitude, and modified the opening and closing rates of the first gating mode. They also increased the transition rate from the second to the first gating mode, and the rate of final closure. The activatory effect of both compounds vanished after a mild trypsin pretreatment, suggesting the existence of activatory sites for FMRF-amide and amitriptyline in the outer vestibule of ASIC1a, which can be removed by exo-or endogenous serine-proteases.

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Modulation of Slow Desensitization (Tachyphylaxis) of Acid-Sensing Ion Channel (ASIC)1a

et al. 2022

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Extracellular trypsin increases ASIC1a selectivity for monovalent versus divalent cations

Cited by 19 publications

References 35 publications

Trypsin Cleaves Acid-sensing Ion Channel 1a in a Domain That Is Critical for Channel Gating

Trypsin Cleaves Acid-sensing Ion Channel 1a in a Domain That Is Critical for Channel Gating

ASIC1a activation by amitriptyline and FMRF-amide is removed by serine proteases

Modulation of Slow Desensitization (Tachyphylaxis) of Acid-Sensing Ion Channel (ASIC)1a

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