The Thumb Domain Mediates Acid-sensing Ion Channel Desensitization

Abstract

Acid-sensing ion channels (ASICs) are cation-selective proton-gated channels expressed in neurons that participate in diverse physiological processes, including nociception, synaptic plasticity, learning, and memory. ASIC subunits contain intracellular N and C termini, two transmembrane domains that constitute the pore, and a large extracellular loop with defined domains termed the finger, ␤-ball, thumb, palm, and knuckle. Here we examined the contribution of the finger, ␤-ball, and thumb domains to activation and desensitization through the analysis of chimeras and the assessment of the effect of covalent modification of introduced Cys at the domain-domain interfaces. Our studies with ASIC1a-ASIC2a chimeras showed that swapping the thumb domain between subunits results in faster channel desensitization. Likewise, the covalent modification of Cys residues at selected positions in the ␤-ball-thumb interface accelerates the desensitization of the mutant channels. Studies of accessibility with thiol-reactive reagents revealed that the ␤-ball and thumb domains reside apart in the resting state but that they become closer to each other in response to extracellular acidification. We propose that the thumb domain moves upon continuous exposure to an acidic extracellular milieu, assisting with the closing of the pore during channel desensitization.Acid-sensing ion channels (ASICs) 2 are voltage-insensitive cation-permeable channels expressed in neurons of the peripheral and central nervous systems that respond to sudden changes in extracellular pH. Four genes that encode for six ASIC subunits and splice variants have been identified in mammals (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4) (1-10). In the central nervous system, disruption of ASIC1a expression causes a number of learning and memory-related phenotypes (11)(12)(13)(14). Conversely, activation of ASIC1a has been linked to neurotoxicity in a mouse model of brain ischemia (15) and in neurodegenerative diseases such as multiple sclerosis (16), Huntington disease (17), Parkinson disease (18), and spinal cord injury (19). There is a substantial body of research that indicates that ASIC subunits participate in nociception in peripheral neurons (20 -22) as well as in pain processing in the central nervous system (23-25). Therefore, ASICs represent novel therapeutic targets to treat pain as well as neurological diseases.ASIC subunits assemble to form homo-and heterotrimers that display distinct proton sensitivities for activation and desensitization kinetics (26 -29). Although the molecular determinants for channel activation have been extensively investigated, less is known about the mechanisms that promote channel desensitization in the continual presence of protons. ASIC subunits contain two membrane-spanning helices that form the pore of the channel and connect to cytoplasmic N-and C-terminal regions and to a large extracellular loop with defined domains termed the wrist, thumb, finger, ␤-ball, knuckle, and palm (see Fig. 1) (30). In the solved structure o...

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

“…Some mutants displayed up to twofold accelerated kinetics of current decay (Fig. S1A), in accordance with previous studies that described altered current kinetics of AcP mutants (15,16). Some mutants showed a sustained current, which was, however, small in all mutants except AcP16b (Fig.…”

“…These observations are consistent with an important regulatory, but not essential, role of the AcP in ASIC activation. The AcP also controls the kinetics and pH dependence of desensitization (15,16,27), a role that is underlined here by the slow ΔF signals, which may reflect conformational changes leading to desensitization. In ENaC, the AcP may also have a regulatory role, because it was shown to contain a binding site for Na + involved in channel inhibition (31).…”

Cryo-EM structure of the ASIC1a–mambalgin-1 complex reveals that the peptide toxin mambalgin-1 inhibits acid-sensing ion channels through an unusual allosteric effect

“…Voltage clamp fluorometry (VCF) analysis provided evidence of conformational changes in the acidic pocket correlated to activation and desensitization, and proposed a likely sequence of conformational changes in the acidic pocket upon extracellular acidification 42 . Furthermore, studies with ASIC1a–ASIC2a chimeras showed that swapping the thumb domain between subunits results in faster channel desensitization 43 . Likewise, the covalent modification of Cys residues at selected positions in the β-ball–thumb interface accelerated the desensitization of the mutant channels 43 .…”

Thumb domains of the three epithelial Na+ channel subunits have distinct functions

“…For example, intersubunit cross-linking between thumb and palm domains alters ENaC activity in a length-dependent manner (52). Furthermore, there is evidence that ASIC1 thumb domain interactions with the ␤-ball and palm domains influence channel gating (53,54). The recently published closed state structure of ASIC1 highlights a prominent role of the thumb domains in channel gating (55).…”