Measuring Interactions Between the Intracellular Domains of the Acid-Sensing Ion Channel

Abstract

Acid-sensing ion channels (ASICs) are sodium selective, proton activated channels primarily located in the central and peripheral nervous system. To date, there have been numerous structures solved of ASIC1 in the putative open, closed and desensitized states. These structures reveal the trimeric nature and overall fold of the channel but are missing information about the intracellular N- and C- termini which are known to be important for proper channel gating, trafficking and selectivity. Deletion of the channel's N-terminus causes ASICs to become non-functional. Despite a lack of current, channels appear to traffic to the membrane normally. Deletion of the C-terminus causes the channels to enter a prolonged desensitized state sometimes referred to as tachyphylaxis. Here we examine the role of the intracellular domains in ASIC function using a number of approaches. We hypothesize that the intracellular domains make interactions that are important for channel gating. We can show that isolated peptides of the N- and C-terminus interact with a surprisingly high affinity using microscale thermophoresis. We then try to examine this interaction with two techniques in intact channels. First, we use transition metal ion FRET (tmFRET) to measure the proximity between these two domains. To get specific fluorescent labelling of the N-terminus, we use the non-canonical amino acid L-Anap which we have successfully incorporated at many positions along the N-terminus. We then incorporate transition metals using a cysteine reactive high-affinity metal chelator called TETAC at individual sites along the C-terminus of the channel. FRET between these two domains suggests regions where the two domains may be in close proximity and that the position changes with pH. Second, we are using crosslinking to confirm our results and to examine how restricting the movement of these domains might alter channel function.