Revealing the arrangement of the intracellular termini of ASIC1a using transition metal ion FRET.

Abstract

Acid-sensing ion channels (ASICs) are proton gated, sodium selective, trimeric ion channels primarily located in the central and peripheral nervous systems. Recently, it has been shown that the C-terminus of ASIC1a binds to the protein receptor interacting serine/threonine kinase 1 (RIPK1) under prolonged acidic conditions and this association triggers necroptotic cell death. It has been hypothesized that at neutral pH, the N- and C-termini interact which prevents the association of RIPK1 with the C-terminus. To test this, we performed microscale thermophoresis using a combination of peptides and purified proteins. We found that the ASIC1a C-terminus binds with high affinity to the RIPK1 death domain but does not bind to the ASIC1a N-terminus. We further investigated the proposed interaction of the ASIC1a N- and C-termini in intact channels in cell membranes using transition metal ion FRET (tmFRET) where we measure quenching of an incorporated small fluorophore unnatural amino acid 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (L-Anap) by metal ions bound to a cysteine reactive chelator. First, we demonstrate that we are able to successfully incorporate L-ANAP at a number of positions in both the N- and C-termini of ASIC1a. Second, we measure tmFRET between multiple positions in the termini which show that the N- and C-termini are not in particularly close proximity and during acidosis they undergo only a small rearrangement that, in fact, moves them closer together. Given this result, we go on to map the topology and dynamics of these regions relative to metal ions incorporated into the plasma membrane via a lipid with an NTA head group. Our results suggest that there must be an alternative mechanism by which ASIC1a binds to RIPK1 only after prolonged acidosis.