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Identification of functionally important residues of the rat P2X4 receptor by alanine scanning mutagenesis of the dorsal fin and left flipper domains.

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'-(?-thio)triphosphate, 2'(3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, and ?,?-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.

SUBMITTER: Tvrdonova V

PROVIDER: S-EPMC4232510 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Identification of functionally important residues of the rat P2X4 receptor by alanine scanning mutagenesis of the dorsal fin and left flipper domains.

Tvrdonova Vendula V Rokic Milos B MB Stojilkovic Stanko S SS Zemkova Hana H

PloS one 20141114 11

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 w ...[more]

PMID: 25398027

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Project description:Neuronal membrane glycoprotein M6a (Gpm6a) is a protein with four transmembrane regions and the N- and the C-ends facing the cytosol. It functions in processes of neuronal development, outgrowth of neurites, and formation of filopodia, spines, and synapsis. Molecular mechanisms by which Gpm6a acts in these processes are not fully comprehended. Structural similarities of Gpm6a with tetraspanins led us to hypothesize that, similarly to tetraspanins, the cytoplasmic tails function as connections with cytoskeletal and/or signaling proteins. Here, we demonstrate that the C- but not the N-terminal cytosolic end of Gpm6a is required for the formation of filopodia by Gpm6a in cultured neurons from rat hippocampus and in neuroblastoma cells N2a. Further immunofluorescence microcopy and flow cytometry analysis show that deletion of neither the N- nor the C-terminal intracellular domains interferes with the recognition of Gpm6a by the function-blocking antibody directed against the extracellular part of Gpm6a. Expression levels of both truncation mutants were not affected but we observed decrease in the amount of both truncated proteins on cell surface suggesting that the incapacity of the Gpm6a lacking C-terminus to induce filopodium formation is not due to the lower amount of Gpm6a on cell surface. Following colocalization assays shows that deletion of the C- but not the N-terminus diminishes the association of Gpm6a with clathrin implying involvement of clathrin-mediated trafficking events. Next, using comprehensive alanine scanning mutagenesis of the C-terminus we identify K250, K255, and E258 as the key residues for the formation of filopodia by Gpm6a. Substitution of these charged residues with alanine also diminishes the amount of Gpm6a on cell surface and in case of K255 and E258 leads to the lower amount of total expressed protein. Subsequent bioinformatic analysis of Gpm6a amino acid sequence reveals that highly conserved and functional residues cluster preferentially within the C- and not within the N-terminus and that K250, K255, and E258 are predicted as part of sorting signals of transmembrane proteins. Altogether, our results provide evidence that filopodium outgrowth induced by Gpm6a requires functionally critical residues within the C-terminal cytoplasmic tail.

Dataset Information

Identification of functionally important residues of the rat P2X4 receptor by alanine scanning mutagenesis of the dorsal fin and left flipper domains.

Publications

Identification of functionally important residues of the rat P2X4 receptor by alanine scanning mutagenesis of the dorsal fin and left flipper domains.

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