Zebrafish Acid-sensing Ion Channel (ASIC) 4, Characterization of Homo- and Heteromeric Channels, and Identification of Regions Important for Activation by H+

Abstract

There are four genes for acid-sensing ion channels (ASICs) in the genome of mammalian species. Whereas ASIC1 to ASIC3 form functional H ؉ -gated Na ؉ channels, ASIC4 is not gated by H ؉ , and its function is unknown. Zebrafish has two ASIC4 paralogs: zASIC4.1 and zASIC4.2. Whereas zASIC4.1 is gated by extracellular H ؉ , zASIC4.2 is not. This differential response to H ؉ makes zASIC4 paralogs a good model to study the properties of this ion channel. In this study, we found that surface expression of homomeric zASIC4.2 is higher than that of zASIC4. . Upon a drop in extracellular pH, ASICs rapidly open, depolarizing the cell, and during sustained acidification they desensitize. The genome of mammals, such as mice and humans, contains four asic genes that code for at least six distinct ASIC subunits (1). ASIC subunits contain two transmembrane domains, a large extracellular loop and relatively short intracellular N and C termini (2). Functional ASICs are homo-or hetero-oligomeric assemblies of individual subunits (3-9).The ASIC1a subunit is broadly expressed throughout the central and peripheral nervous system and contributes to synaptic transmission (10, 11). ASIC1b, a splice variant of ASIC1a, is specifically expressed in the peripheral nervous system and might contribute to the perception of pain (12, 13). ASIC2a is broadly expressed in the brain and contributes to synaptic transmission by the formation of heteromeric channels with ASIC1a (3,6,8). ASIC2b, a splice variant of ASIC2a, does not generate functional channels on its own but forms heteromeric channels with other ASIC subunits, notably ASIC3 (14). ASIC3, like ASIC1b, is specifically expressed in the peripheral nervous system (15), and there is evidence for a role in the perception and processing of painful stimuli (16 -19). ASIC4 has the most restricted expression of all ASIC subunits. In humans, ASIC4 mRNA is strongly expressed only in the pituitary gland; expression in other parts of the brain is faint (20). Like ASIC2b, ASIC4 does not form a functional homomeric channel (20, 21), and unlike ASIC2b, it does apparently also not form functional heteromeric channels with other ASIC subunits (20). These properties distinguish ASIC4 from other ASIC subunits, and, whereas possible functions are emerging for all other ASIC subunits, the function of ASIC4 is still unknown.Recently, we have reported the cloning of a family of ASICs from the zebrafish (22). Like their mammalian homologs, zASICs are broadly expressed in the zebrafish central nervous system (22). We identified six ASIC subunits, zASIC1.1, 1.2, 1.3, 2, 4.1, and 4.2, that are encoded by six different genes (22). zASIC1.2 and 1.3 are paralogs of zASIC1.1, and zASIC4.2 is a paralog of zASIC4.1, respectively. The existence of paralogs in zebrafish can be at least partly explained by the fish-specific genome duplication in ray-finned fish that happened ϳ350 million years ago (23). The amino acid sequences of zASIC4.1 and 4.2 are 68% identical; within the ectodomains identity is even 83%. Unlike rat AS...

The role of acid-sensing ion channels (ASICs) in epithelial Na+ uptake in adult zebrafish (Danio rerio).

“…The mechanism by which VHA activity promotes Na + uptake is not yet known; however, two alternatives may exist: firstly, VHA charges the apical membrane potential thereby increasing the electrochemical gradient for Na + to enter in the cell through ASIC, and secondly, VHA provides a gating signal to ASIC by acidification of the boundary layer, since ASICs are gated by extracellular H + ions that activate several amino acid residues located in different extracellular domains (Bonifacio et al, 2014;Paukert et al, 2008). One complicating factor is that ASIC4.2 has been demonstrated to be insensitive to extracellular H + (Chen et al, 2007). However, there is evidence that ASICs assemble into homoor heteromeric trimers (Jasti et al, 2007), therefore it is possible that ASIC4.2 forms a channel with other ASIC subunits that show extracellular H + gating.…”

Acid-sensing ion channels are involved in epithelial Na+ uptake in the rainbow trout Oncorhynchus mykiss

“…It is believed that, similar to ENaCs, functional ASICs are trimers (20), although their configuration has not been resolved. It has been demonstrated that, with a few exceptions, ASICs function as homomers (forming channels of identical subunits) or heteromers (forming channels with other ASIC subunits) (4,23). ASICs have been characterized by using oocyte expression in zebrafish (4,35) and dogfish shark (ASIC1b only) (42).…”

“…It has been demonstrated that, with a few exceptions, ASICs function as homomers (forming channels of identical subunits) or heteromers (forming channels with other ASIC subunits) (4,23). ASICs have been characterized by using oocyte expression in zebrafish (4,35) and dogfish shark (ASIC1b only) (42). It has been found that coexpression of zASIC4.1 with zASIC1.3 in Xenopus oocytes increased the current amplitude and abundance (ϳ15-fold) of the channel at the cell surface, indicating that these two ASIC subunits form a functional channel (4).…”

“…Na ϩ uptake occurs via specialized mitochondrion-rich cells (MRCs), located on the fish gill epithelium (26,29), and was initially proposed to be linked to NH 4 ϩ /H ϩ excretion (26). Subsequent studies have described two models of Na ϩ transport (for review see Refs.…”

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

“…Experiments using chimeras composed of proton-insensitive and proton-sensitive ASICs suggest that specific residues within the wrist domain of ASICs are required for proton-dependent activation (35)(36)(37). Channels that are unresponsive to protons are produced when these residues are mutated, and because some of these residues can be titrated by protons, it has been suggested that they represent the proton-binding site(s).…”