The receptor site of the spider toxin PcTx1 on the proton-gated cation channel ASIC1a

Abstract

Acid-sensing ion channels (ASICs) are excitatory neuronal cation channels, involved in physiopathological processes related to extracellular pH fluctuation such as nociception, ischaemia, perception of sour taste and synaptic transmission. The spider peptide toxin psalmotoxin 1 (PcTx1) has previously been shown to inhibit specifically the proton-gated cation channel ASIC1a. To identify the binding site of PcTx1, we produced an iodinated form of the toxin ((125)I-PcTx1Y(N)) and developed a set of binding and electrophysiological experiments on several chimeras of ASIC1a and the PcTx1-insensitive channels ASIC1b and ASIC2a. We show that (125)I-PcTx1Y(N) binds specifically to ASIC1a at a single site, with an IC(50) of 128 pM, distinct from the amiloride blocking site. Results obtained from chimeras indicate that PcTx1 does not bind to ASIC1a transmembrane domains (M1 and M2), involved in formation of the ion pore, but binds principally on both cysteine-rich domains I and II (CRDI and CRDII) of the extracellular loop. The post-M1 and pre-M2 regions, although not involved in the binding site, are crucial for the ability of PcTx1 to inhibit ASIC1a current. The linker domain between CRDI and CRDII is important for their correct spatial positioning to form the PcTx1 binding site. These results will be useful for the future identification or design of new molecules acting on ASICs.

Figure 1. PcTx1-induced inhibition of ASIC1a current recorded in COS-7 cells

A, PcTx1-induced inhibition of ASIC1a current depends on the total duration of the PcTx1 application, independent of the number of current activations during this application. The percentage of normalized inhibition of ASIC1a current induced by 1 nm PcTx1 is plotted as a function of the duration of PcTx1 application and is fitted by a single exponential: y= 100−122 e^−x/60.9, R²= 0.9. ○, the first currents recorded after PcTx1 applications for different durations; ●, currents recorded after repeated activation by successive pH drops. Results were obtained from 11 different cells. Currents were activated by pH drops from pH 7.4 at a holding potential of −50 mV. Inset, original currents traces showing the inhibition of ASIC1a current by 1 nm PcTx1 (Tx 1) after 60 s application (first recorded current) and 180 s. B, original current traces recorded from the same cell showing the inhibition induced by different durations of 10 nm PcTx1 (Tx 10) application (top and middle panels). The first recorded currents are indicated (1st). The washout of PcTx1 10 nm inhibition is illustrated in the bottom traces. Currents were activated by pH drops from pH 7.4 to pH 6 every 2 min at a holding potential of −50 mV. Variation of control current amplitude is due to a spontaneous run-down of ASIC1a current throughout the experiment. C, PcTx1-induced inhibition of ASIC1a current is independent of the pH reached during the pH drop. ASIC1a currents were activated by pH drops from a resting pH of 7.4 down to the pH value indicated below (test pH). Holding potential, −50 mV. The amplitude of 1 nm PcTx1-inhibited ASIC1a current was measured at steady state, expressed as a percentage of control current and plotted as mean ± s.e.m. (n = 5–8). D, PcTx1-induced inhibition of ASIC1a current is independent of the holding potential. ASIC1a currents were activated by pH drops from a resting pH 7.4 down to pH 5 at the holding potentials indicated below. The amplitude of ASIC1a currents in the presence of 1 nm PcTx1 (filled bars) or 10 nm PcTx1 (shaded bars) was measured at steady state, expressed as a percentage of control current and plotted as mean ± s.e.m. (n = 3–14). E, PcTx1-induced inhibition of ASIC1a current is dependent on the resting pH value. ASIC1a currents were activated by pH drops from the resting pH values indicated below each bar down to pH 5. Holding potential, −50 mV. The amplitude of ASIC1a currents in the presence of 1 nm PcTx1 (filled bars) or 10 nm PcTx1 (shaded bars) or 100 nm PcTx1 (open bar) was measured at steady state, expressed as a percentage of control current and plotted as mean ± s.e.m. (n = 4–14). **P < 0.01 and ***P < 0.005 compared with respective control values.

Figure 2. Characterization of iodinated PcTx1

A, concentration–response curve showing the inhibition of ASIC1a currents in Xenopus oocytes by wild-type PcTx1 (IC₅₀= 1.17 ± 0.11 nm) and the tyrosine variant PcTx1Y_N(IC₅₀= 1.98 ± 0.24 nm). Inset, current traces showing ASIC1a current in a Xenopus oocyte in the absence (control) and presence of 10 nm PcTx1Y_N. B, graph showing the pH-dependent binding of ¹²⁵I-PcTx1Y_N to rat brain membranes (▵) and ASIC1a/CHO cell lysates (●) (n = 2–3). ¹²⁵I-PcTx1Y_N concentrations of around 100 pm (50–200 pm depending on specific activity) were used. C, saturation curve of ¹²⁵I-PcTx1Y_N binding to ASIC1a/CHO lysate (K_d= 213.7 ± 35.2 pm, Maximum Binding, B_max= 36.9 ± 3.2 fmol mg⁻¹, n = 3); inset, linear Scatchard plot indicating a single family of binding sites. D, saturation curve of ¹²⁵I-PcTx1Y_N binding to rat brain membranes (K_d= 371.6 ± 48.5 pm, B_max= 49.5 ± 10.2 fmol mg⁻¹, n = 4); inset, linear Scatchard plot indicating a single family of binding sites. E, selectivity of ¹²⁵I-PcTx1Y_N (1 nm) binding to lysates of CHO cells expressing different ASIC subtypes (n = 2). In this experiment, the cell lysates came from a different transfection to that in panel C, thus explaining the change in B_max value. F, other known inhibitors of ASIC1a (amiloride (Amil) and flurbiprofen (Flurb)) and of ASIC3 (diclofenac (Dicl)), up to a concentration of 1 mm, failed to compete with ¹²⁵I-PcTx1Y_N (100 pm) binding to rat brain membranes (n = 2). ¹²⁵I-PcTx1Y_N binding was displaced by 50 nm PcTx1 (Tx). G, FMRFamide, a positive modulator of ASIC1a and ASIC3, up to a concentration of 500 μm, failed to compete with ¹²⁵I-PcTx1Y_N (100 pm) binding to rat brain membranes (n = 2). ¹²⁵I-PcTx1Y_N binding was displaced by 50 nm PcTx1 (Tx).

Figure 3. ASIC structure–function relationships and chimeras design

A, alignment of the amino acid sequences of rat ASIC1a with ASIC1b and ASIC2a. The two transmembrane segments, M1 and M2, are indicated by an open rectangle. Identical residues are enclosed in filled boxes, moderately conserved related residues are in dark-grey boxes, and similar related residues are in light-grey boxes. The ASIC1a/ASIC1b splice junction divides the extracellular loop into two parts. The first part is much more variable than the highly conserved second part. Each part is then subdivided into three domains for chimeric constructions. His72 (●), which is important for pH sensing (Baron et al. 2001), and a domain related to the kinetics of desensitization (♡♡♡) (Coric et al. 2003) are indicated in domain 1. Domain 2 corresponds to a highly variable region in size and sequence, and carries a small sequence (♦♦) crucial for pH activation and steady-state inactivation (Babini et al. 2002). In domain 2 of ASIC1a, lysine-133 corresponds to the high-affinity site for Zn²⁺ inhibition (Chu et al. 2004). Domain 3 frames approximately Cysteine-Rich Domain I (CRDI) , which contains a histidine residue (▪) related to the Zn²⁺ coactivator effect on ASIC2a (Baron et al. 2001). The FMRFamide-dependent activation domain (Cottrell et al. 2001) and the regulatory trypsin site (Poirot et al. 2004) belong to this first part of the loop (domains 1, 2 and 3) but have not been identified to date. Domain 4 is very conserved among all the ASIC family members, and Cys193 (indicated by ©193) is potentially involved in a disulphide bridge with Cys93 (©93) (domain 1) as described for ENaC. Domain 5 frames CRDII and also has a histidine residue (▪) related to the Zn²⁺ coactivator effect on ASIC2a (Baron et al. 2001). Its size is variable in ASIC3 (16 amino acids more) and in ASIC4 (4 amino acids more) (not shown). Cysteine residues involved in a disulphide bridge in ENaC correspond to Cys323 (©323) and Cys335 (©335) in the ASIC1a sequence. Domain 6 covers the most highly conserved region and contains the degenerin site (♠) just before the second transmembrane domain (Hong & Driscoll, 1994; Champigny et al. 1998), and two amino acids (♣) crucial for Ca²⁺ block of ASIC1a (Paukert et al. 2004). Domains involved in selectivity and gating on the N-terminus region (Bassler et al. 2001), and the regulatory phosphorylation site (℗) (Baron et al. 2002a) are noted. The amiloride site (↑) and selectivity filter on the first and second transmembrane domain are also indicated (Ji et al. 2001; Poet et al. 2001; Kellenberger & Schild, 2002). B and C, nomenclature and schematic representation of ASIC1b/1a and ASIC2a/1a chimeric constructions, respectively.

Figure 4. ASIC1b/1a chimeras

A, binding of PcTx1Y_N. Competition between ¹²⁵I-PcTx1Y_N and unlabelled PcTx1 for binding to ASIC1a or ASIC1b/1a chimeras in CHO cell lysates (n = 2–5). Sigmoïdal curves were fitted with a Hill number of 1. A concentration of ¹²⁵I-PcTx1Y_N around 100 pm (50–200 pm depending on specific activity) was used. B, effect of 10 nm PcTx1 on ASIC1b/1a chimera current. Currents were activated by pH drops from 7.4 to 5 and 10 nm PcTx1 was applied between pH drops. Holding potential, −50 mV. The amplitude of the PcTx1-inhibited current was measured at steady state, expressed as a percentage of control current and plotted as the mean ±s.e.m (n = 3–10); *P < 0.05 compared with ASIC1b (no inhibition). The 100% no-inhibition level is indicated by a dashed line. C, concentration–inhibition curves of PcTx1-inhibited ASIC1b/1a chimera currents. The amplitude of the PcTx1-inhibited current was measured at steady state, expressed as a percentage of control current, and the mean ± s.e.m. (n = 3–13) was plotted as a function of PcTx1 concentration (log [PcTx1]). The maximal concentration of PcTx1 tested was 100 nm. Sigmoïdal curves were fitted with a Hill number of 1. D, original current traces showing the inhibition of ASIC1b/1a:3, ASIC1b/1a:13 and ASIC1b/1a:123 currents, activated by pH drops from 7.4 to 5, by 10 nm PcTx1 (Tx 10). PcTx1 was applied between pH drops. Holding potential, −50 mV. Note the significant slowing of ASIC1b/1a:123 inactivation compared with ASIC1a (Table 1).

Figure 5. ASIC2a/1a chimeras

A, binding of PcTx1Y_N. Competition between ¹²⁵I-PcTx1Y_N and unlabelled PcTx1 for binding to ASIC1a or ASIC2a/1a chimeras in CHO cell lysates (n = 3–5). Sigmoidal curves were fitted with a Hill number of 1. A concentration of ¹²⁵I-PcTx1Y_N around 100 pm (50–200 pm depending on specific activity) was used. B, effect of 10 nm PcTx1 on ASIC2a/1a chimera current. Currents were activated by pH drops from 7.4 to 5 and 10 nm PcTx1 was applied between pH drops. Holding potential, −50 mV. The amplitude of the PcTx1-inhibited current was measured at steady state, expressed as a percentage of control current and plotted as the mean ± s.e.m. (n = 4–11); *P < 0.05 compared with ASIC2a (no inhibition). The 100% no-inhibition level is indicated by a dashed line. C, concentration–inhibition curves of PcTx1-inhibited ASIC2a/1a chimera currents. The amplitude of the PcTx1-inhibited current was measured at steady state, expressed as a percentage of control current and the mean ± s.e.m. (n = 3–13) was plotted as a function of PcTx1 concentration (log [PcTx1]). The maximal concentration of PcTx1 tested was 100 nm. Sigmoïdal curves were fitted with a Hill number of 1. D, original current traces showing the effect of 10 nm (Tx 10) or 100 nm (Tx 100) PcTx1 on domain 5-containing ASIC2a/1a currents activated by pH drops from 7.4 to 5. PcTx1 was applied between pH drops. Holding potential, −50 mV. ASIC2a/1a:123456 and ASIC2a/1a:12345 currents were inhibited by PcTx1 whereas ASIC2a/1a:1235 and ASIC2a/1a:12356 currents were increased. Note the significant acceleration of ASIC2a/1a:12345 and ASIC2a/1a:1235 inactivation compared with ASIC1a (Table 1) and the PcTx1-induced slowing of inactivation associated with ASIC2a/1a:1235 and ASIC2a/1a:12356 current increases.

Figure 6. The PcTx1 binding site on the ASIC1a channel and important structural determinants of the ENaC/DEG/ASIC family

Only two subunits of the tetrameric channel are shown to simplify representation in two dimensions. The six different ASIC1a extracellular domains defined in our structure–function approach are numbered from 1 to 6. Domains directly involved in the PcTx1 binding site on ASIC1a, domains 3 (CDRI) and 5 (CRDII), are indicated in pink. The ASIC1a domain 2 (in green) is involved in PcTx1 binding and also in the ability of PcTx1 to modify channel gating. ASIC1a domains 1, 4 and 6 (in blue) are not directly involved in the binding site but are necessary for PcTx1 to modify channel gating thus leading to inhibition. ASIC1a domain 4 is probably essential in regulating the positioning of CRDI and CRDII, the global shape being imposed by the disulphide bridge between domains 1 and 4. The pre-M1 domain has been shown to control ion permeability of ASIC1a (Bassler et al. 2001), with the conserved motif His-Gly involved in the gating mechanism of the ENaC (Grunder et al. 1999). A PKC phosphorylation site located before the M1 domain allows the modulation of ASIC2a activity (Baron et al. 2002a). The first transmembrane domain M1 of FaNaC was shown to be part of a large aqueous cavity, with the charge selectivity filter in the outer vestibule and the ion gate located close to the interior (Poet et al. 2001). In the M2 segment, some amino acids were involved in the amiloride binding site and the selectivity filter of the ENaC (Schild et al. 1997; Kellenberger et al. 1999). The degenerin mutation (DEG), which causes a persistent channel activation, affects a residue proposed to lie upstream of the M2 segment in or near the pore of ASICs (Waldmann et al. 1996; Champigny et al. 1998). Close to this residue, two amino acids are crucial for Ca²⁺ block of ASIC1a (Paukert et al. 2004). The intracellular post-M2 domain of the ENaC contributes to ion permeation, suggesting that multiple sites contribute to ion selectivity (Ji et al. 2001).