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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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Ivermectin rescues the Imax of low-functioning mutants.(A) Acute effect of 3 µM ivermectin (IVM) applied for 10 s (gray areas) during ongoing stimulation with 100 µM ATP for 30 s (horizontal bars) in cells expressing the WT, the DF mutants (R203A, N204A, I205A, and L214A), or the LF mutants (D280A, R282A, P290A and N293A). Recordings are examples of traces similar to 3–5 traces per mutant and 30 per WT receptor. (B) Summary data showing the potentiating effect of IVM preapplication (for 4–6 min) on Imax in WT and alanine mutant receptors. The Imax values were derived from measurements taken in the absence (open bars) or in the presence (filled bars) of IVM. Values are presented as the mean ± SEM from 5–8 measurements per mutant and 15 measurements per WT. IVM treatment rescued the Imax of all low-functioning receptors, except in the case of N293A, which is an ATP binding mutant. The statistical significance was determined by an ANOVA comparing the WT Imax and the Imax of mutant receptors in the presence of IVM. **, p<0.01.
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pone-0112902-g004: Ivermectin rescues the Imax of low-functioning mutants.(A) Acute effect of 3 µM ivermectin (IVM) applied for 10 s (gray areas) during ongoing stimulation with 100 µM ATP for 30 s (horizontal bars) in cells expressing the WT, the DF mutants (R203A, N204A, I205A, and L214A), or the LF mutants (D280A, R282A, P290A and N293A). Recordings are examples of traces similar to 3–5 traces per mutant and 30 per WT receptor. (B) Summary data showing the potentiating effect of IVM preapplication (for 4–6 min) on Imax in WT and alanine mutant receptors. The Imax values were derived from measurements taken in the absence (open bars) or in the presence (filled bars) of IVM. Values are presented as the mean ± SEM from 5–8 measurements per mutant and 15 measurements per WT. IVM treatment rescued the Imax of all low-functioning receptors, except in the case of N293A, which is an ATP binding mutant. The statistical significance was determined by an ANOVA comparing the WT Imax and the Imax of mutant receptors in the presence of IVM. **, p<0.01.

Mentions: The effect of IVM on Imax was tested initially during ongoing responses to 100 µM ATP to determine whether the low current amplitudes observed in R203A, N204A, I205A, L214A, D280A, R282A, P290A, and N293A mutants could be rescued. The application of 3 µM IVM increased immediately the amplitude of ATP-induced responses in all low-functioning mutants (Fig. 4A). Next, we performed quantitative analysis of Imax in WT and all alanine mutants before and after 4–6 min pretreatment with IVM (Table S1). The WT receptor was potentiated 1.5-fold by IVM, while the low-functioning mutants were potentiated 3.7- to 16-fold. In the presence of IVM, the Imax values of all low-functioning mutants were comparable to those of WT receptors, except for N293A (Fig. 4B). These experiments indicate that R203, N204, I205, L214, D280, R282, R290, and N293 residues play a critical role in agonist binding and/or channel gating.


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)

Ivermectin rescues the Imax of low-functioning mutants.(A) Acute effect of 3 µM ivermectin (IVM) applied for 10 s (gray areas) during ongoing stimulation with 100 µM ATP for 30 s (horizontal bars) in cells expressing the WT, the DF mutants (R203A, N204A, I205A, and L214A), or the LF mutants (D280A, R282A, P290A and N293A). Recordings are examples of traces similar to 3–5 traces per mutant and 30 per WT receptor. (B) Summary data showing the potentiating effect of IVM preapplication (for 4–6 min) on Imax in WT and alanine mutant receptors. The Imax values were derived from measurements taken in the absence (open bars) or in the presence (filled bars) of IVM. Values are presented as the mean ± SEM from 5–8 measurements per mutant and 15 measurements per WT. IVM treatment rescued the Imax of all low-functioning receptors, except in the case of N293A, which is an ATP binding mutant. The statistical significance was determined by an ANOVA comparing the WT Imax and the Imax of mutant receptors in the presence of IVM. **, p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4232510&req=5

pone-0112902-g004: Ivermectin rescues the Imax of low-functioning mutants.(A) Acute effect of 3 µM ivermectin (IVM) applied for 10 s (gray areas) during ongoing stimulation with 100 µM ATP for 30 s (horizontal bars) in cells expressing the WT, the DF mutants (R203A, N204A, I205A, and L214A), or the LF mutants (D280A, R282A, P290A and N293A). Recordings are examples of traces similar to 3–5 traces per mutant and 30 per WT receptor. (B) Summary data showing the potentiating effect of IVM preapplication (for 4–6 min) on Imax in WT and alanine mutant receptors. The Imax values were derived from measurements taken in the absence (open bars) or in the presence (filled bars) of IVM. Values are presented as the mean ± SEM from 5–8 measurements per mutant and 15 measurements per WT. IVM treatment rescued the Imax of all low-functioning receptors, except in the case of N293A, which is an ATP binding mutant. The statistical significance was determined by an ANOVA comparing the WT Imax and the Imax of mutant receptors in the presence of IVM. **, p<0.01.
Mentions: The effect of IVM on Imax was tested initially during ongoing responses to 100 µM ATP to determine whether the low current amplitudes observed in R203A, N204A, I205A, L214A, D280A, R282A, P290A, and N293A mutants could be rescued. The application of 3 µM IVM increased immediately the amplitude of ATP-induced responses in all low-functioning mutants (Fig. 4A). Next, we performed quantitative analysis of Imax in WT and all alanine mutants before and after 4–6 min pretreatment with IVM (Table S1). The WT receptor was potentiated 1.5-fold by IVM, while the low-functioning mutants were potentiated 3.7- to 16-fold. In the presence of IVM, the Imax values of all low-functioning mutants were comparable to those of WT receptors, except for N293A (Fig. 4B). These experiments indicate that R203, N204, I205, L214, D280, R282, R290, and N293 residues play a critical role in agonist binding and/or channel gating.

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