Protons and Psalmotoxin-1 reveal nonproton ligand stimulatory sites in chicken acid-sensing ion channel: Implication for simultaneous modulation in ASICs

Abstract

Acid-sensing ion channels (ASICs) are proton-sensitive, sodium-selective channels expressed in the nervous system that sense changes in extracellular pH. These ion channels are sensitive to an increasing number of nonproton ligands that include natural venom peptides and guanidine compounds. In the case of chicken ASIC1, the spider toxin Psalmotoxin-1 (PcTx1) activates the channel, resulting in an inward current. Furthermore, a growing class of ligands containing a guanidine group has been identified that stimulate peripheral ASICs (ASIC3), but exert subtle influence on other ASIC subtypes. The effects of the guanidine compounds on cASIC1 have not been the focus of previous study. Here, we investigated the interaction of the guanidine compound 2-guanidine-4-methylquinazoline (GMQ) on cASIC1 proton activation and PcTx1 stimulation. Exposure of expressed cASIC1 to PcTx1 resulted in biphasic currents consisting of a transient peak followed by an irreversible cASIC1 PcTx1 persistent current. This cASIC1 PcTx1 persistent current may be the result of locking the cASIC1 protein into a desensitized transition state. The guanidine compound GMQ increased the apparent affinity of protons on cASIC1 and decreased the half-maximal constant of the cASIC1 steady-state desensitization profile. Furthermore, GMQ stimulated the cASIC1 PcTx1 persistent current in a concentration-dependent manner, which resulted in a non-desensitizing inward current. Our data suggests that GMQ may have multiple sites within cASIC1 and may act as a "molecular wedge" that forces the PcTx1-desensitized ASIC into an open state. Our findings indicate that guanidine compounds, such as GMQ, may alter acid-sensing ion channel activity in combination with other stimuli, and that additional ASIC subtypes (along with ASIC3) may serve to sense and mediate signals from multiple stimuli.

Keywords: 2-guanidine-4-methylquinazoline; Psalmotoxin-1; acid-sensing ion channel; gating; ion channel; neuroprotection; nonproton ligand; pain; venom peptides.

Figure 1. cASIC1 is maximally sensitive to 30 nM PcTx1. (A) Representative whole- cell patch clamp recordings of separate cells subjected to PcTx1 (3, 10, or 30 nM) normalized to a pH 6.0 (pre-PcTx1) control pulse, for comparison, are shown (holding potential: -70 mV). Test solutions were applied for 5 s and returned to the normal bath solution (pH 7.35). Whole-cell recordings are scaled for comparison. Horizontal scale bar is in seconds (s). (B) Summary of normalized peak current for 3, 10 and 30 nM (0.02 ± 0.01; 0.35 ± 0.05; 0.40 ± 0.04 of the pH 6.0 control, respectively) PcTx1 in pH 7.35 following pH 6.0 (pre-PcTx1) control is shown. Data are presented as mean ± SEM of at least 3 individual cells.

Figure 2. cASIC1 PcTx1 persistent current is sensitive to pH. (A) Whole-cell patch clamp recordings of pH 6.0 (pre-PcTx1), PcTx1 mediated, and pH 6.0 (post-PcTx1) in cASIC1 are shown (holding potential maintained at -70 mV). Test solutions were applied for 5 s and returned to the normal bath solution (pH 7.35). Horizontal and vertical scale bars are seconds (s) and picoAmperes (pA), respectively. (B) Summary of normalized peak current of the PcTx1 transient current (0.40 ± 0.04, n = 7) and the pH 6.0 following the establishment of the cASIC1 PcTx1 persistent current (0.23 ± 0.03, n = 7) are shown. (C) Summary of the cASIC1 PcTx1 persistent current (0.22 ± 0.03, n = 7) are shown. Data are presented as mean ± SEM of at least seven individual cells and significance was determined using paired the Student t test (*, P < 0.05 compared with control). (PcTx1-PC, PcTx1 persistent current).

Figure 3. The cASIC1 PcTx1 persistent current is similar to the ASIC3 low calcium environment current (A) Whole-cell recording of rASIC3 following a shift from calcium containing external (1 mM) to a nominal calcium external solution (~0 mM CaCl₂) at pH 7.35 are shown. Scale bars: time, in seconds (horizontal) and nanoAmperes, nA (vertical). (B) Representative whole-cell recording of cASIC1 showing a 5 s application of PcTx1 (30 nM) at pH 7.35 is presented. The duration of the recording is 5 min. Scale bars: time, in seconds (horizontal) and picoAmperes, pA (vertical).

Figure 4. Structures of guanidinium compounds. The nonproton ligand chemical structure of (A) 2-guanidine-4-methylquinazoline (GMQ) and the common ASIC antagonist (B), amiloride are shown. The guanidinium group within both compounds is highlighted with a dashed box.

Figure 5. GMQ fails to directly activate cASIC1. (A) Patch-clamp recordings of GMQ (0.3 mM) at pH 7.35 following a pH 6.0 control test pulse are shown. (B) Patch-clamp currents of GMQ (0.3 mM) at pH 8.0 following a pH 6.6 control test pulse are shown. No measurable current was observed in either condition Data are representative of n ≥ 3 cells.

Figure 6. GMQ shifts the proton sensitivity and steady-state desensitization of cASIC1. (A) Representative outside-out patch clamp recordings for WT cASIC1 activation for selected test pulses are shown. Conditioning pH for each pulse is pH 8.0. (B) Summary of pH dependence of cASIC1 activation (solid line) and steady-state desensitization, or SSD, (dashed line) profiles under control conditions for WT cASIC1 are shown. The half-maximal pH response (pH₅₀) and Hill slope (nH) values for the cASIC1 activation were 6.65 ± 0.01 and 4.55 ± 0.54, respectively. The mean pH₅₀ and Hill slope (nH) values for cASIC1 steady-state desensitization were 7.53 ± 0.00 and 5.82 ± 0.08, respectively. Data are presented with normalized current (I/I_max) as a function of pH. (C) Representative outside-out patch clamp recordings for cASIC1 activation in the presence of GMQ (0.3 mM) for selected test pulses are shown. (D) Summary of pH dependence of cASIC1 activation (solid line) and steady-state desensitization, or SSD, (dashed line) in the presence of GMQ (300 μM) are shown. The half-maximal pH response (pH₅₀) and Hill slope (nH) values in the presence of GMQ were 6.98 ± 0.03 and 2.21 ± 0.38, respectively, while the mean SSD pH₅₀ and SSD Hill slope (nH) values for the GMQ SSD curve are 7.48 ± 0.00 and 5.75 ± 0.19, respectively. Data are presented as the mean ± SEM of at least 6 patched cells with normalized current (I/I_{pH 6.0}) as a function of pH.

Figure 7. GMQ stimulates the cASIC1 PcTx1 protein complex. (A) Whole cell recording of pH 6.0, PcTx1 (30 nM), and GMQ concentration (0.1, 0.3, 1 mM). Horizontal and vertical scale bars are in seconds (s, horizontal axis) and picoAmperes (pA, vertical axis), respectively. The zero current (blackened dashed lines) and cASIC1 PcTx1 persistent current (red dashed line) are indicated. (B) Summary of concentration-dependent GMQ stimulation of PcTx1-cASIC1 current at 0.01 mM (0.02 ± 0.01), 0.3 mM (0.19 ± 0.05), and 1 mM (0.50 ± 0.11). The GMQ peak current amplitude was normalized to the pH 6.0 control current. Data are presented as mean ± SEM of at least 4 individual cells and significance was determined using unpaired Student t test (*, P < 0.05 compared with control).

Figure 8. Amiloride influences the cASIC1 PcTx1 persistent state in 2 ways. (A) Whole-cell recordings of the cASIC1 PcTx1 persistent current at low pH with amiloride. Amiloride (0.01, 0.1, and 0.5 mM) inhibited the cASIC1 PcTx1 persistent current. (B) Summary of amiloride rebound current and low pH peak current. Observed amiloride rebound current was normalized to pH 6.0 peak current. Compared with the control response, normalized amiloride peak current at 0.01, 0.1, and 0.5 mM were 0.10 ± 0.04, 0.51 ± 0.20, and 1.14 ± 0.4, respectively. Observed amiloride rebound current was normalized to the PcTx1 persistent current. Compared with the PcTx1 persistent current, the normalized amiloride peak current at 0.01, 0.1, and 0.5 mM were 0.88 ± 0.45, 4.19 ± 1.96, and 9.44 ± 4.06, respectively. Data are presented as mean ± SEM of at least 3 individual cells.

Figure 9. Proposed model of PcTx1 mediated gating of cASIC1. Hypothetical toxin-mediated cASIC1 gating pathway is shown. PcTx1 activates ASIC1 inducing an expanded transmembrane (TM) domain region (left). After the initial PcTx1 application, the toxin-cASIC1 complex moves to a non-conducting conformation (middle). Furthermore, the central vestibule may collapse to mediate the non-conduction of current. The transmembrane domain occludes the pore (possibly similar to the desensitized channel conformation). Nonproton ligands, such as GMQ, may act to pry apart the central vestibule, like a “molecular wedge,” to open the channel complex (right). The ASIC gating schemes are depicted as the following: immovable protein scaffold (blue), central vestibule region (green), and TM domains (orange). The solved PcTx1-ASIC1 protein crystal structure is highlighted (dashed box). Model was generated with similar terminology and design for comparison to the previous models of channel gating (for review, see ref. 32).