Abstract
P2X receptors are cation-permeable ion channels gated by extracellular adenosine triphosphate (ATP). Available crystallographic data suggest that ATP-binding ectodomain is connected to the transmembrane pore domain by three structurally conserved linker regions, which additionally frame the lateral fenestrations through which permeating ions enter the channel pore. The role of these linker regions in relaying the conformational change evoked by ATP binding of the ectodomain to the pore-forming transmembrane domain has not been investigated systematically. Using P2X4R as our model, we employed alanine and serine replacement mutagenesis to determine how the side chain structure of these linker regions influences gating. The mutants Y54A/S, F198A/S, and W259A/S all trafficked normally to the plasma membrane of transfected HEK293 cells but were poorly responsive to ATP. Nevertheless, the function of the F198A/S mutants could be recovered by pretreatment with the known positive allosteric modulator of P2X4R, ivermectin (IVM), although the IVM sensitivity of this mutant was significantly impaired relative to wild type. The functional mutants Y195A/S, F200A/S, and F330A/S exhibited ATP sensitivities identical to wild type, consistent with these side chains playing no role in ATP binding. However, Y195A/S, F200A/S, and F330A/S all displayed markedly changed sensitivity to the specific effects of IVM on current deactivation, suggesting that these positions influence allosteric modulation of gating. Taken together, our data indicate that conserved amino acids within the regions linking the ectodomain with the pore-forming transmembrane domain meaningfully contribute to signal transduction and channel gating in P2X receptors.
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Abbreviations
- TM:
-
Transmembrane segment
- IVM:
-
Ivermectin
- EC50 :
-
Concentration for half-maximal response
- τ off :
-
Deactivation time constant
- I max :
-
The maximal amplitude of the current activated by ATP
- HEK293:
-
Human embryonic kidney 293
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Acknowledgments
We are grateful to Prof. Terrance M. Egan for providing the P2X4 plasmid.
This study was supported by the National Natural Science Foundation of China (81171037/H0903) and the National Key Basic Research Program of China (973 Program) (2012CB966400).
Conflict of interest
The authors declare that they have no conflict of interest.
Authorship contributions
Participated in Research design: Gao, Li.
Conducted experiments: Gao, Yu.
Contributed new reagents or analytic tools: Xu, Zhang, Ma.
Performed data analysis: Liu, Jie.
Wrote or contributed to the writing of the manuscript: Gao, Samways, Li.
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Gao, C., Yu, Q., Xu, H. et al. Roles of the lateral fenestration residues of the P2X4 receptor that contribute to the channel function and the deactivation effect of ivermectin. Purinergic Signalling 11, 229–238 (2015). https://doi.org/10.1007/s11302-015-9448-5
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DOI: https://doi.org/10.1007/s11302-015-9448-5