Abstract
Crystallization of the zebrafish P2X4 receptor in both open and closed states revealed conformational differences in the ectodomain structures, including the dorsal fin and left flipper domains. Here, we focused on the role of these domains in receptor activation, responsiveness to orthosteric ATP analogue agonists, and desensitization. Alanine scanning mutagenesis of the R203-L214 (dorsal fin) and the D280-N293 (left flipper) sequences of the rat P2X4 receptor showed that ATP potency/efficacy was reduced in 15 out of 26 alanine mutants. The R203A, N204A, and N293A mutants were essentially non-functional, but receptor function was restored by ivermectin, an allosteric modulator. The I205A, T210A, L214A, P290A, G291A, and Y292A mutants exhibited significant changes in the responsiveness to orthosteric analog agonists 2-(methylthio)adenosine 5′-triphosphate, adenosine 5′-(γ-thio)triphosphate, 2′(3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate, and α,β-methyleneadenosine 5′-triphosphate. In contrast, the responsiveness of L206A, N208A, D280A, T281A, R282A, and H286A mutants to analog agonists was comparable to that of the wild type receptor. Among these mutants, D280A, T281A, R282A, H286A, G291A, and Y292A also exhibited increased time-constant of the desensitizing current response. These experiments, together with homology modeling, indicate that residues located in the upper part of the dorsal fin and left flipper domains, relative to distance from the channel pore, contribute to the organization of the ATP binding pocket and to the initiation of signal transmission towards residues in the lower part of both domains. The R203 and N204 residues, deeply buried in the protein, may integrate the output signal from these two domains towards the gate. In addition, the left flipper residues predominantly account for the control of transition of channels from an open to a desensitized state.
Highlights
The purinergic P2X receptors (P2XRs) are ATP-gated ion channels that are permeable to Na+, K+, Ca2+, and small organic cations
We examined the hypothesis that the dorsal fin (DF) and left flipper (LF) domains may influence the organization of the ATP binding pocket, transmission of ATP-induced signal from ATP binding pocket to the gate, and receptor desensitization
Mutants I205A, L214A, D280A, R282A, and P290A showed a significant reduction (p,0.01) in Imax, and with exception of P290A, their EC50 values were approximately 10fold rightward shifted when compared to the WT receptor
Summary
The purinergic P2X receptors (P2XRs) are ATP-gated ion channels that are permeable to Na+, K+, Ca2+, and small organic cations. Seven subunits of P2XRs have been identified in mammals [1], and functional receptors are composed of three homologous or heterologous subunits [2]. Each subunit consists of a large, glycosylated, and cystine-rich extracellular domain that contributes to the formation of the intersubunit ATP binding sites, two transmembrane domains that form the pore of the channel, and intracellular N- and C- termini that contribute to gating specificity [3]. In contrast to the large number of studies using the native ligand, ATP, there are very few studies providing structural information derived from the use of orthosteric ATP analog agonists. Understanding receptor interactions with these analog agonists may provide significant insights aiding the design of drugs that compete with the native ligand
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