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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 V, Rokic MB, Stojilkovic SS, Zemkova H - PLoS ONE (2014)

Bottom Line: 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 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 addition, the left flipper residues predominantly account for the control of transition of channels from an open to a desensitized state.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Neuroendocrinology, Institute of Physiology Academy of Sciences of the Czech Republic, Prague, Czech Republic; Department of Physiology of Animals, Faculty of Science, Charles University, Prague, Czech Republic.

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.

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DF and LF mutants exhibit a rightward shift in EC50.(A, B) Example records of ATP-induced currents from cells expressing the WT receptor and I205A, T210A, and L214A DF mutants (A) and D280A, R282A, H286A, and G291A LF mutants (B). Currents were stimulated by a short (2–5 s) application of different concentrations of ATP (1–1000 µM), indicated by horizontal bars above the traces. Experiments were performed on naïve receptors, and traces from different cells are shown. (C, D) Concentration response curves for WT, I205A, T210A, and L214A DF mutants (C) and D280A, R282A, H286A, and G291A LF mutants (D). Data points are presented as the mean ± SEM from 7–35 measurements per mutant, per concentration and 78 measurements for WT.
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pone-0112902-g003: DF and LF mutants exhibit a rightward shift in EC50.(A, B) Example records of ATP-induced currents from cells expressing the WT receptor and I205A, T210A, and L214A DF mutants (A) and D280A, R282A, H286A, and G291A LF mutants (B). Currents were stimulated by a short (2–5 s) application of different concentrations of ATP (1–1000 µM), indicated by horizontal bars above the traces. Experiments were performed on naïve receptors, and traces from different cells are shown. (C, D) Concentration response curves for WT, I205A, T210A, and L214A DF mutants (C) and D280A, R282A, H286A, and G291A LF mutants (D). Data points are presented as the mean ± SEM from 7–35 measurements per mutant, per concentration and 78 measurements for WT.

Mentions: To address the structure-function relationship between the LF and DF regions of rP2X4R, we performed single-point mutagenesis on sequences encompassing the LF and DF regions R203-L214 (DF) and D280-N293 (LF) (Fig. 1A). The crystal structure of the zfP2X4R showed elevated B-factor values in these regions, indicating conformational flexibility (Fig. 1B). For the initial electrophysiological characterization, we examined the EC50 and Imax values to determine ATP potency and efficacy, respectively. The results from experiments on both mutant and WT receptors are summarized in Figs. 2A and 3 and Table S1.


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

Tvrdonova V, Rokic MB, Stojilkovic SS, Zemkova H - PLoS ONE (2014)

DF and LF mutants exhibit a rightward shift in EC50.(A, B) Example records of ATP-induced currents from cells expressing the WT receptor and I205A, T210A, and L214A DF mutants (A) and D280A, R282A, H286A, and G291A LF mutants (B). Currents were stimulated by a short (2–5 s) application of different concentrations of ATP (1–1000 µM), indicated by horizontal bars above the traces. Experiments were performed on naïve receptors, and traces from different cells are shown. (C, D) Concentration response curves for WT, I205A, T210A, and L214A DF mutants (C) and D280A, R282A, H286A, and G291A LF mutants (D). Data points are presented as the mean ± SEM from 7–35 measurements per mutant, per concentration and 78 measurements for WT.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4232510&req=5

pone-0112902-g003: DF and LF mutants exhibit a rightward shift in EC50.(A, B) Example records of ATP-induced currents from cells expressing the WT receptor and I205A, T210A, and L214A DF mutants (A) and D280A, R282A, H286A, and G291A LF mutants (B). Currents were stimulated by a short (2–5 s) application of different concentrations of ATP (1–1000 µM), indicated by horizontal bars above the traces. Experiments were performed on naïve receptors, and traces from different cells are shown. (C, D) Concentration response curves for WT, I205A, T210A, and L214A DF mutants (C) and D280A, R282A, H286A, and G291A LF mutants (D). Data points are presented as the mean ± SEM from 7–35 measurements per mutant, per concentration and 78 measurements for WT.
Mentions: To address the structure-function relationship between the LF and DF regions of rP2X4R, we performed single-point mutagenesis on sequences encompassing the LF and DF regions R203-L214 (DF) and D280-N293 (LF) (Fig. 1A). The crystal structure of the zfP2X4R showed elevated B-factor values in these regions, indicating conformational flexibility (Fig. 1B). For the initial electrophysiological characterization, we examined the EC50 and Imax values to determine ATP potency and efficacy, respectively. The results from experiments on both mutant and WT receptors are summarized in Figs. 2A and 3 and Table S1.

Bottom Line: 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 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 addition, the left flipper residues predominantly account for the control of transition of channels from an open to a desensitized state.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Neuroendocrinology, Institute of Physiology Academy of Sciences of the Czech Republic, Prague, Czech Republic; Department of Physiology of Animals, Faculty of Science, Charles University, Prague, Czech Republic.

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.

Show MeSH
Related in: MedlinePlus