Identification of Protein Domains That Control Proton and Calcium Sensitivity of ASIC1a

Abstract

The acid-sensing ion channels (ASICs) open in response to extracellular acidic pH, and individual subunits display differential sensitivity to protons and calcium. ASIC1a acts as a high affinity proton sensor, whereas ASIC2a requires substantially greater proton concentrations to activate. Using chimeras composed of ASIC1a and ASIC2a, we determined that two regions of the extracellular domain (residues 87-197 and 323-431) specify the high affinity proton response of ASIC1a. These two regions appear to undergo intersubunit interactions within the multimeric channel to specify proton sensitivity. Single amino acid mutations revealed that amino acids around Asp 357 play a prominent role in determining the pH dose response of ASIC1a. Within the same region, mutation F352L abolished PcTx1 modulation of ASIC1a. Surprisingly, we determined that another area of the extracellular domain was required for calcium-dependent regulation of ASIC1a activation, and this region functioned independently of high affinity proton sensing. These results indicate that specific regions play overlapping roles in pH-dependent gating and PcTx1-dependent modulation of ASIC1a activity, whereas a distinct region determines the calcium dependence of ASIC1a activation.The acid-sensing ion channels (ASICs) 3 are proton-gated ion channels expressed in neurons throughout the central and peripheral nervous system (1-3). ASICs are activated by extracellular acidosis, and protons act as ligands triggering channel opening (4). Disruption of the accn2 gene (which encodes ASIC1) dramatically reduces proton-gated currents in central neurons and alters a variety of behaviors, including fear, learning, and memory (5, 6). ASIC1 also contributes to neuronal damage and death during the prolonged acidosis accompanying cerebral ischemia (7). Specifically, mice with disruptions in the accn2 gene display 60% smaller lesion size compared with normal mice in models of stroke (8). Application of PcTx1, a venom peptide that prevents ASIC1a activation, is similarly neuroprotective, even when administered hours after injury (8, 9). Thus, ASIC1a represents a novel pharmacological target for the prevention of neuronal death following stroke.Mammals have four genes encoding ASICs (accn1 to -4) that encode at least six different ASIC subunits (1-3, 10). Like all members of the DEG/ENaC family, individual ASIC subunits have two transmembrane regions separated by a large cysteinerich extracellular region. Three ASIC subunits associate to form homomeric or heteromeric channels with distinct biophysical characteristics (11)(12)(13)(14). Specifically, ASIC1a homomeric channels activate at pH values much closer to neutral pH compared with ASIC2a homomeric channels. The high affinity proton sensitivity of ASIC1a plays a prominent role in acidosisinduced neuronal death, and modulators that alter the pH dose response of ASIC1a affect neuronal sensitivity to prolonged acidosis (8, 9, 15). For example, the neuroprotective venom peptide PcTx1 increases the proton sensitivity of the ...

A Combined Computational and Functional Approach Identifies New Residues Involved in pH-dependent Gating of ASIC1a

“…23,25,42), consistent with the hypothesis of the paper of the first ASIC structure (25) that the interaction between the thumb and the ␤-ball is critically involved in the activation process. Further confirming the importance of the thumb, residues at the lower end of the thumb helix ␣5 (Asp-357, Gln-358, and Glu-359) also affect ASIC activation when mutated (22,41). We show here that Glu-315 and Glu-355 in the thumb and Glu-235 and Glu-254 on different loops originating in the ␤-ball are involved in SSIN.…”

“…The D357N mutation did not shift pH 50 in rat ASIC1a but required higher amounts of cRNA for normal current expression (rASIC1a D355N (23)). However, mutation of Asp-357 to Ala in hASIC1a induced an acidic shift of Ϫ0.6 units in pH 50 (41), suggesting a potentially important role of Asp-357 in the pH dependence of activation. Mutation of Glu-427 to Gly was shown to result in an acidic shift of pH 50 of Ϫ0.15 units (rASIC1a E425G (20)), whereas the mutation of Asp-313 has not been described so far.…”

“…We show here that Glu-315 and Glu-355 in the thumb and Glu-235 and Glu-254 on different loops originating in the ␤-ball are involved in SSIN. Replacement of the residues downstream of ␣4, down to ␤10, and thus the short ␣4-␣5 loop and the ␣5 helix of the thumb as well as the loop connecting it to ␤10 on the palm, by the corresponding ASIC2a sequence (corresponding to residues 323-370 in hASIC1a), resulted in a channel with the same low pH 50 (ϳ4) as ASIC2a (41). The residue Trp-287 is part of the second loop connecting the thumb with the palm.…”

Acidotoxicity and acid-sensing ion channels contribute to motoneuron degeneration

“…32 ASIC2a can form heterodimeric channels with ASIC1a (ASIC2a/1a), which sense more acidic pH than ASIC1a homomeric channels and are PcTx1 insensitive. 27 While ASIC2b homomeric channels do not sense subtle changes in acidity, 33 recent data indicates that ASIC2b/1a heterodimeric channels may also contribute to acidosis-induced neuronal death. 32 Given our findings of increased ASIC2 levels in the ALS spinal cord, it is tempting to speculate that ASIC2/1a heterodimeric channels contribute to acidotoxicity in motoneurons, previously thought to be mediated by ASIC1a alone.…”

“…ASIC1a, the ASIC1 isoform expressed in the brain, is required for highaffinity sensing of acidosis, 6 and is known to have a causative role in neuronal damage induced by prolonged acidosis. 10,27 ASIC channels are activated in response to a marked decline in pH. 28 Moreover, gene deletion of asic1a has demonstrated neuroprotection in mouse models of stroke and multiple sclerosis.…”