Kinetic analysis of ASIC1a delineates conformational signaling from proton-sensing domains to the channel gate.

Acid-sensing ion channels (ASICs) are neuronal Na+ channels that are activated by a drop in pH. Their established physiological and pathological roles, involving fear behaviors, learning, pain sensation, and neurodegeneration after stroke, make them promising targets for future drugs. Currently, the ASIC activation mechanism is not understood. Here, we used voltage-clamp fluorometry (VCF) combined with fluorophore-quencher pairing to determine the kinetics and direction of movements. We show that conformational changes with the speed of channel activation occur close to the gate and in more distant extracellular sites, where they may be driven by local protonation events. Further, we provide evidence for fast conformational changes in a pathway linking protonation sites to the channel pore, in which an extracellular interdomain loop interacts via aromatic residue interactions with the upper end of a transmembrane helix and would thereby open the gate.

Structural and Functional Analysis of Gly212 Mutants Reveals the Importance of Intersubunit Interactions in ASIC1a Channel Function.

Acid-sensing ion channels (ASICs) act as pH sensors in neurons. ASICs contribute to pain sensation, learning, fear behavior and to neuronal death after ischemic stroke. Extracellular acidification induces a transient activation and subsequent desensitization of these Na+-selective channels. ASICs are trimeric channels made of identical or homologous subunits. We have previously shown that mutation of the highly conserved Gly212 residue of human ASIC1a to Asp affects the channel function. Gly212 is located in the proximity of a predicted Cl- binding site at a subunit interface. Here, we have measured the function of a series of Gly212 mutants. We show that substitution of Gly212 affects the ASIC1a pH dependence and current decay kinetics. Intriguingly, the mutations to the acidic residues Asp and Glu have opposing effects on the pH dependence and the current decay kinetics. Analysis of molecular dynamics simulation trajectories started with the coordinates of the closed conformation indicates that the immediate environment of residue 212 in G212E, which shifts the pH dependence to more alkaline values, adopts a conformation closer to the open state. The G212D and G212E mutants have a different pattern of intersubunit salt bridges, that, in the case of G212E, leads to an approaching of neighboring subunits. Based on the comparison of crystal structures, the conformational changes in this zone appear to be smaller during the open-desensitized transition. Nevertheless, MD simulations highlight differences between mutants, suggesting that the changed function upon substitution of residue 212 is due to differences in intra- and intersubunit interactions in its proximity.

A molecular view of the function and pharmacology of acid-sensing ion channels.

The pH in the different tissues and organs of our body is kept within tight limits. Local pH changes occur, however, temporarily under physiological conditions, as for example in synapses during neuronal activity. In pathological situations, such as in ischemia, inflammation, and tumor growth, long-lasting acidification develops. Acid-sensing ion channels (ASICs) are low pH-activated Na+-permeable ion channels that are widely expressed in the central and peripheral nervous systems. ASICs act as pH sensors, leading to neuronal excitation when the pH drops. Animal studies have shown that ASICs are involved in several physiological and pathological processes, such as pain sensation, learning, fear sensing, and neurodegeneration after ischemic stroke. ASIC inhibitors could be used as analgesic and anxiolytic drugs, and as drugs for the treatment of ischemic stroke. For these reasons, ASICs have recently attracted increasing attention. Currently, no drugs are clinically used as ASIC modulators. ASICs are however targets of several peptide toxins from animals. Much effort is invested in research studying the function of these channels. We review here the available pharmacological agents acting on ASICs, which include small molecules and animal toxins. We then discuss the current understanding of the molecular mechanisms by which pH controls ASIC activity. Knowledge of the function of ASICs at the molecular level should allow the development of new pharmacological strategies for targeting these promising ion channels.

Accelerated Current Decay Kinetics of a Rare Human Acid-Sensing ion Channel 1a Variant That Is Used in Many Studies as Wild Type.

Acid-sensing ion channels (ASICs) are neuronal Na+-permeable ion channels that are activated by extracellular acidification and are involved in fear sensing, learning, neurodegeneration after ischemia, and in pain sensation. We have recently found that the human ASIC1a (hASIC1a) wild type (WT) clone which has been used by many laboratories in recombinant expression studies contains a point mutation that occurs with a very low frequency in humans. Here, we compared the function and expression of ASIC1a WT and of this rare variant, in which the highly conserved residue Gly212 is substituted by Asp. Residue 212 is located at a subunit interface that undergoes changes during channel activity. We show that the modulation of channel function by commonly used ASIC inhibitors and modulators, and the pH dependence, are the same or only slightly different between hASIC1a-G212 and -D212. hASIC1a-G212 has however a higher current amplitude per surface-expressed channel and considerably slower current decay kinetics than hASIC1a-D212, and its current decay kinetics display a higher dependency on the type of anion present in the extracellular solution. We demonstrate for a number of channel mutants previously characterized in the hASIC1a-D212 background that they have very similar effects in the hASIC1a-G212 background. Taken together, we show that the variant hASIC1a-D212 that has been used as WT in many studies is, in fact, a mutant and that the properties of hASIC1a-D212 and hASIC1a-G212 are sufficiently close that the conclusions made in previous pharmacology and structure-function studies remain valid.

Conformational dynamics and role of the acidic pocket in ASIC pH-dependent gating.

Acid-sensing ion channels (ASICs) are proton-activated Na+ channels expressed in the nervous system, where they are involved in learning, fear behaviors, neurodegeneration, and pain sensation. In this work, we study the role in pH sensing of two regions of the ectodomain enriched in acidic residues: the acidic pocket, which faces the outside of the protein and is the binding site of several animal toxins, and the palm, a central channel domain. Using voltage clamp fluorometry, we find that the acidic pocket undergoes conformational changes during both activation and desensitization. Concurrently, we find that, although proton sensing in the acidic pocket is not required for channel function, it does contribute to both activation and desensitization. Furthermore, protonation-mimicking mutations of acidic residues in the palm induce a dramatic acceleration of desensitization followed by the appearance of a sustained current. In summary, this work describes the roles of potential pH sensors in two extracellular domains, and it proposes a model of acidification-induced conformational changes occurring in the acidic pocket of ASIC1a.

Extracellular Subunit Interactions Control Transitions between Functional States of Acid-sensing Ion Channel 1a.

Acid-sensing ion channels (ASICs) are neuronal, voltage-independent Na(+) channels that are transiently activated by extracellular acidification. They are involved in pain sensation, the expression of fear, and in neurodegeneration after ischemic stroke. Our study investigates the role of extracellular subunit interactions in ASIC1a function. We identified two regions involved in critical intersubunit interactions. First, formation of an engineered disulfide bond between the palm and thumb domains leads to partial channel closure. Second, linking Glu-235 of a finger loop to either one of two different residues of the knuckle of a neighboring subunit opens the channel at physiological pH or disrupts its activity. This suggests that one finger-knuckle disulfide bond (E235C/K393C) sets the channel in an open state, whereas the other (E235C/Y389C) switches the channel to a non-conducting state. Voltage-clamp fluorometry experiments indicate that both the finger loop and the knuckle move away from the ß-ball residue Trp-233 during acidification and subsequent desensitization. Together, these observations reveal that ASIC1a opening is accompanied by a distance increase between adjacent thumb and palm domains as well as a movement of Glu-235 relative to the knuckle helix. Our study identifies subunit interactions in the extracellular loop and shows that dynamic changes of these interactions are critical for normal ASIC function.

A novel Ca²âº-mediated cross-talk between endoplasmic reticulum and acidic organelles: implications for NAADP-dependent Ca²âº signalling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) serves as the ideal trigger of spatio-temporally complex intracellular Ca(2+) signals. However, the identity of the intracellular Ca(2+) store(s) recruited by NAADP, which may include either the endolysosomal (EL) or the endoplasmic reticulum (ER) Ca(2+) pools, is still elusive. Here, we show that the Ca(2+) response to NAADP was suppressed by interfering with either EL or ER Ca(2+) sequestration. The measurement of EL and ER Ca(2+) levels by using selectively targeted aequorin unveiled that the preventing ER Ca(2+) storage also affected ER Ca(2+) loading and vice versa. This indicates that a functional Ca(2+)-mediated cross-talk exists at the EL-ER interface and exerts profound implications for the study of NAADP-induced Ca(2+) signals. Extreme caution is warranted when dissecting NAADP targets by pharmacologically inhibiting EL and/or the ER Ca(2+) pools. Moreover, Ca(2+) transfer between these compartments might be essential to regulate vital Ca(2+)-dependent processes in both organelles.