Electrophysiological characterization of the acid sensing ion channel shark ASIC1b and identification of amino acids controlling the gating of ASIC1

Acid sensing ion channels (ASICs) are sodium-selective and proton-sensitive members of the DEG/ENaC gene family and are expressed in the chordate lineage while being absent in evolutionary older animals. Although members of the DEG/ENaC family share similarities with respect to topology, selectivity for sodium and sensitivity to the blocking agent amiloride, the family comprises ion channels of various functions and diverse gating mechanisms. So far, four ASIC genes have been identified in mammals (asic1-asic4) that code for at least six different ASIC subunits. Amino acid sequences of the members of the ASIC subfamily are at least 45% identical and they are composed, like all members of the DEG/ENaC family, of two transmembrane domains, a large extracellular loop domain and rather short intracellular termini. So far, ASICs have been cloned from urochordates, jawless vertebrates, cartilaginous shark and bony fish, from chicken and different mammals. Proton-sensitivity, however, was postulated to have evolved with the rise of bony fish and ASICs from lower chordate species were characterized as proton-insensitive. Since the crystal structure of chicken ASIC1 was resolved in 2007 it is known that functional ASIC channels are trimeric structures that assemble in a homo- or heteromeric fashion in vivo and in vitro. Depending on the subunit composition they exhibit different functional features regarding proton sensitivity or gating kinetics. Some ASICs, like the abundant ASIC1a, are broadly expressed in the peripheral as well as in the central nervous system whereas the expression pattern of other ASICs, for example ASIC1b and ASIC3, is restricted to the peripheral nervous system. Predominantly located at the postsynaptic membrane, ASICs are supposedly implicated in modulation of synaptic transmission and pain perception, but they also contribute to pathophysiological processes like ischemia, epilepsy as well as to axonal degeneration during neuroinflammation. Moreover, knockout of the asic1 gene leads to deficits in spatial memory and learned fear, suggesting a contribution to higher brain functions. Because of such important implications and the limited pharmacological toolkit for ASIC modulation, it is desirable to find new specific drugs for ASIC modulation and understanding of the gating process of ASICs would be of great benefit for designing new highly specific drugs. The first part of this work shows that proton-sensitivity evolved latest in cartilaginous fish and that ASIC1b from shark (Squalus acanthias) indeed responds to extracellular acidification. Furthermore, a detailed characterization of homomeric shark ASIC1b channels revealed that the current of these channels exhibit unique features and that it can be divided into two components. The fast transient current component shows a time constant of desensitization (tau) of less than 50 ms and is followed by a highly proton sensitive sustained current component that does not completely desensitize as long as protons are present in the extracellular solution. In addition, the second part of this thesis elucidates the amino acids that are crucial for the unique sustained current component of shark ASIC1b. An amino acid triplet (M109DS) in the proximal region of the extracellular domain that is located in the linker region between two beta-sheets partially controls the time constant of desensitization and is necessary for the generation of the sustained current of shark ASIC1b. Additionally, it is shown that the same triplet is also sufficient to introduce a sustained current in rat ASIC1a, a channel that usually completely desensitizes during prolonged acidification. Moreover, when the most critical residue of this triplet at position 110 is mutated to cysteine, different MTS-modification rates at this position in the closed and the desensitized state, respectively, provide evidence that this residue is moving during the gating transition. Finally, engineering of a cysteine at position 110 (position 82 in rat) and at an adjacent position in the beta11-beta12-linker leads to spontaneous formation of a disulfide bond that traps shark ASIC1a and rat ASIC1a in the desensitized conformation. Collectively the results presented in this work suggest that the beta1-beta2 and beta11-beta12 linkers are dynamic during gating and tightly oppose each other during desensitization gating. Obstruction of this tight opposition leads to reopening of the channel. It results that the beta1-beta2 and beta11-beta12 linkers modulate gating movements of ASIC1 and may thus be drug targets for modulation of ASIC activity

The Interaction between Two Extracellular Linker Regions Controls Sustained Opening of Acid-sensing Ion Channel 1*

Activation of acid-sensing ion channels (ASICs) contributes to neuronal death during stroke, to axonal degeneration during neuroinflammation, and to pain during inflammation. Although understanding ASIC gating may help to modulate ASIC activity during these pathologic situations, at present it is poorly understood. The ligand, H+, probably binds to several sites, among them amino acids within the large extracellular domain. The extracellular domain is linked to the two transmembrane domains by the wrist region that is connected to two anti-parallel β-strands, β1 and β12. Thus, the wrist region together with those β-strands may have a crucial role in transmitting ligand binding to pore opening and closing. Here we show that amino acids in the β1-β2 linker determine constitutive opening of ASIC1b from shark. The most crucial residue within the β1-β2 linker (Asp110), when mutated from aspartate to cysteine, can be altered by cysteine-modifying reagents much more readily when channels are closed than when they are desensitized. Finally, engineering of a cysteine at position 110 and at an adjacent position in the β11-β12 linker leads to spontaneous formation of a disulfide bond that traps the channel in the desensitized conformation. Collectively, our results suggest that the β1-β2 and β11-β12 linkers are dynamic during gating and tightly appose to each other during desensitization gating. Hindrance of this tight apposition leads to reopening of the channel. It follows that the β1-β2 and β11-β12 linkers modulate gating movements of ASIC1 and may thus be drug targets to modulate ASIC activity

An acid-sensing ion channel from shark (Squalus acanthias) mediates transient and sustained responses to protons

Acid-sensing ion channels (ASICs) are proton-gated Na+ channels. They are implicated in synaptic transmission, detection of painful acidosis, and possibly sour taste. The typical ASIC current is a transient, completely desensitizing current that can be blocked by the diuretic amiloride. ASICs are present in chordates but are absent in other animals. They have been cloned from urochordates, jawless vertebrates, cartilaginous shark and bony fish, from chicken and different mammals. Strikingly, all ASICs that have so far been characterized from urochordates, jawless vertebrates and shark are not gated by protons, suggesting that proton gating evolved relatively late in bony fish and that primitive ASICs had a different and unknown gating mechanism. Recently, amino acids that are crucial for the proton gating of rat ASIC1a have been identified. These residues are completely conserved in shark ASIC1b (sASIC1b), prompting us to re-evaluate the proton sensitivity of sASIC1b. Here we show that, contrary to previous findings, sASIC1b is indeed gated by protons with half-maximal activation at pH 6.0. sASIC1b desensitizes quickly but incompletely, efficiently encoding transient as well as sustained proton signals. Our results show that the conservation of the amino acids crucial for proton gating can predict proton sensitivity of an ASIC and increase our understanding of the evolution of ASICs