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Acid sensing ion channels (ASICs) are members of the epithelial sodium channel and degenerins (ENaC/DEG) family of proteins1. The first members of the family were discovered in the early 90s, shortly thereafter various other members of the family were identified using sequence homology, this lead to the discovery of the first ASIC by Waldmann et al. in 19972. In the few years following this, several other ASICs were discovered. All members of the ENaC/DEG family are ion channels that are usually selective for sodium ions and have a common tertiary subunit structure of a short cytoplasmic N-terminal tail followed by a transmembrane helix, a large extracellular section - accounting for over 60% of the protein, a second transmembrane helix, which forms the pore, and a short cytoplasmic C-terminal tail1,3.
ASICs are cation (usually sodium) channels which open in response to a decrease in extracellular pH. If the low pH persists the ASIC will enter a desensitised closed state4. The pH required for the channel to open has been shown to be as high as 6.7 (ASIC3) or as low as 4.4 (ASIC2a)5. The time that the channel takes to enter the desensitised state has been observed as being anywhere between less than 100ms (in fish) to up to 10 seconds (in mammals), both of these properties vary according to the subunits that make up the channel, which can be homomeric or hetromeric6.
There are four genes that code for ASICs, so far three of these have had splice variants discovered, leading to a wide variety of known ASIC subunits across various species, many of these subunits have had a few names over the years. The subunits identified in humans are ASIC1a (AKA ASIC1, ASIC-α, BNaC2), ASIC2a (AKA MDEG1, BNC1, BNaC1), ASIC3a, ASIC3b, ASIC3c (AKA DRASIC), and ASIC4 (AKA SPASIC). ASIC1b (AKA ASIC-β), ASIC1b2 (AKA ASIC-β2), and ASIC2b (AKA MDEG2) have also frequently been used in research over the past 10 or so years into the structure, function, regulation of, and drug effect on ASICs7. ASIC2b and ASIC4 homomeric proteins are not pH sensitive, but they are able to modulate the pH sensitivity in hetromeric proteins8.
ASICs can be found in various different parts of both the central and peripheral nervous system, the expression of different subunits results in different cellular responses to a drop in pH. They have been shown to be involved in many conditions and cellular functions, including but not limited to: tissue inflammation, ischemic strokes, neurotrauma, epilepsy, glioma, nociception, synaptic plasticity, and taste4,5. Knowledge of ASICs and ways to modulate their function or expression could therefore, prove invaluable.
Since the discovery of ASICs, many studies have been conducted in an attempt to further knowledge, it has become clear from these studies that despite being activated by the simplest ligand possible (hydrogen ions), ASICs, and members of the ENaC/DEG family in general, are not simple, so elucidating their mechanism has proven difficult. Arguably the greatest leap in knowledge of the ENaC/DEG family occurred when Jasti et al. published a crystal structure of chicken ASIC1a in 20079. This was the first and so far only crystal structure of the family and has shed a considerable amount of light on various hypotheses, and allowed many more to be made, not only on ASICs, but due to the similarities between all proteins in the family information on one area can be useful to research on another3. The one thing Jasti's structure has failed to do so far though, is to provide an obvious mechanism. The protein was crystallised at a physiologically low pH, so will have been in a desensitised state, making predictions on the conformation of an open state and how the protein is able to change between the three states (sensitive closed, open, desensitised closed) difficult.
The crystal structure of cASIC1a subunit A. The transmembrane domain is coloured red, the palm green, the knuckle orange, the finger yellow, the thumb purple, and the ball blue. The structure was downloaded from RCSB PDB and the image rendered with Swiss PDB Viewer and MS paint.Early research into the structure of the protein suggested that it could contain anywhere up to nine subunits, with four being a common structure5. Because of the cASIC1a structure it is now seen as most likely that all ASICs form trimers, rather than tetramers or higher (up to nonamers) as previously believed. Jasti et al. likened the shape of an individual subunit to that of a forearm and clenched right fist: the transmembrane domain forming the arm section, the extracellular domain forming the fist, with two short loops between them, forming the wrist. Motifs within the extracellular domain were named the palm domain (a large β-sheet), the knuckle domain (2 small α-helices), the finger domain (3 slightly larger α-helices) the thumb domain (2 larger α-helices), and the central ball domain (a small β-sheet)9.
The thumb domain contains 15 highly conserved cystine residues, 14 of which form disulphide bridges, a line can be roughly drawn from the finger through each of these disulphide bridges to a highly conserved tryptophan residue in the wrist. The thumb also contains a chloride ion, these two features give the thumb a fairly rigid structure, it was suggested therefore, that the thumb may be involved in the transduction of a conformational change from one part of the protein to another and the stabilisation of conformational states9.
Jasti et al also noted the existence of pairs of acidic residues (asp238 and asp350, glu239 and asp346, glu219 and asp407) with the side chains in very close proximity to each other, so close that to avoid charge hindrances, at least one of the pairs must be protonated. They suggested that these pairs could act as the H+ binding site, when the pH drops one or both of the residues become protonated, allowing them to rotate towards each other, inducing a conformational change in the rest of the protein9.
Another feature noted by Jasti et al. is that the six transmembrane helices (two from each subunit) form an hour glass like shape which may be involved in the selectivity filter9. It had previously been shown by Paukert et al. that Ca2+ reduces the affinity of the protein for H+, and that two residues on the second transmembrane helix (glu425, asp432) formed a calcium binding site12. They suggested that Ca2+ blocked the pore, indeed mapping these residues on to the cASIC1a structure shows that they are just above the centre of the hourglass, so a Ca2+ ion bound to this position would prevent any other ion from passing through. A decrease in pH protonates these two residues, removing the Ca2+ block, allowing the pore to open, they also showed that this site was likely to not be the only Ca2+ binding site on the protein, and later identified four residues that could form a second site or could interact in other ways6.
This second study by Paukert et al into the effects of Calcium ions on ASICs revealed four more residues which may form a second binding site (glu63, his72/his73, asp78 on the palm)4,6, in some ASICs both histidine residues are present, on others only one exists. E63 is a fair distance from the other 2/3 residues, so will not be involved in the same interactions. Cushman et al. Noted that D78 and is highly conserved and only present on acid sensitive ASICs (not 2b or 4)8. Paukert et al. suggested that E63 is the second Ca2+ binding site and that after a conformational change caused by the displacement of Ca2+ from both sites, D78 rotates and stabilises the desensitised conformation by forming a salt bridge to H72/H73. The same study also looked into the role of the acid pairs identified by Jasti et al. they appeared to have a fairly low affinity for Ca2+, so could bind it in the closed state to slightly increase stability and the mutation of at least one residue in at least two pairs reduced the stability of the desensitised state, Paukert et al. suggested that Jasti's pairs did not cause any conformational change but did stabilise certain conformations6.
Various other studies have noted many other residues which also seem to be involved in H+ sensitivity and/or state stabilisation8,13,14, the exact role of each residue that appears to have an effect should slowly come to light over the next 10 years. Using the cASIC1a structure Paukert et al. came up with a possible mechanism. The protein starts in the closed state, the pH drops, His 73 is protonated, this causes a conformational change which, along with the pH drop, causes the displacement of Ca2+, opening the channel. The conformational change continues to the desensitised state, which is stabilised by a salt bridge between H73 and D78 and Jasti's acidic pairs6. Because only a desensitised structure exists it is difficult to predict the exact nature of the conformational changes but it is likely that they are transmitted by the thumb. This is only a first attempt at describing a mechanism and although it is likely that it is essentially correct, minor changes and corrections will probably be suggested over the next decade.
The majority of research into ASIC permeability has shown that although they have a significant level of permeability to potassium, calcium, beryllium, magnesium and hydrogen ions, the majority are mainly selective for sodium, although ASIC3 and ASIC2b show roughly equal levels of permeability to potassium and sodium15, and ASIC1a shows highest permeability to protons2. The selectivity filter in ENaCs is formed by a short G/SXS motif found near the centre of the hourglass10. The Serine OH groups are believed to point to the centre of the pore, co-ordinating sodium ions as they pass through, other ions such as potassium are too large so cannot pass through11. Similar motifs have been found in other proteins within the ENaC/DEG family. This motif is not present in ASICs, and the nature of the selectivity filter is currently unknown.
The inhibition of ASICs has been shown to be a promising area for medical research; it has been shown to alleviate many of the symptoms of conditions mentioned earlier4,5. Three inhibitors have been looked into in recent years: Amiloride, PcTX1 (Tarantula psalmtoxin), and A-3175675,16. They have been shown to reduce the amount of damage after an ischemic stroke5,17 and prevent epileptic fits5. It has also been demonstrated that they have anxiolytic17 and anti-depressive16 activity, which ASIC1a activity has been directly linked to15. All ENaCs can be inhibited by amiloride1,19, but it is non-specific so would not be suitable as a drug5,19. Kuduk et al recently investigated the inhibitory nature of many amiloride analogues, some of which could eventually lead to the production of amiloride based drugs19, while PcTX1 and A-317567 are both in early preclinical trials16.
Information on ASICs, their structure, function, mechanisms, and clinical significance has shown many promising advances over the past decade or so since their discovery, various theories have been postulated over the years and many of these have started to come together since Jasti et al. published a crystal structure of cASIC1a, the next few years should show many more advances in knowledge surrounding ASICs, one piece of information that could finally crack the mechanism would be a crystal structure of an ASIC in a closed conformation, further still an ASIC in an open conformation would be very beneficial but this structure may prove impossible to obtain due to desensitisation occurring after a few seconds. Maybe desensitisation could be prevented through mutagenesis, but would this still allow the channel to open in such a way that is still relevant to the wild type ASICs?
Note: The various papers referenced in this essay used different ASICs for their research, residues mentioned in each paper may vary in primary structure slightly but their equivalents in other ASICs can easily be deduced through comparison with the structure published by Jasti et al.