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The P2X7 receptor channel pore dilates under physiological ion conditions.

Yan Z, Li S, Liang Z, Tomić M, Stojilkovic SS - J. Gen. Physiol. (2008)

Bottom Line: The biphasic current was preserved in N-terminal T15A, T15S, and T15V mutants that have low or no permeability to organic cations, reflecting enhanced permeability to inorganic cations.In contrast, the T15E, T15K, and T15W mutants, and the Delta18 mutant with deleted P2X(7) receptor-specific 18-amino acid C-terminal segment, were instantaneously permeable to organic cations and generated high amplitude monophasic currents.These results indicate that the P2X(7) receptor channel dilates under physiological ion conditions, leading to generation of biphasic current, and that this process is controlled by residues near the intracellular side of the channel pore.

View Article: PubMed Central - PubMed

Affiliation: Section on Cellular Signaling, Program in Developmental Neuroscience, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Activation of the purinergic P2X(7) receptor leads to the rapid opening of an integral ion channel that is permeable to small cations. This is followed by a gradual increase in permeability to fluorescent dyes by integrating the actions of the pannexin-1 channel. Here, we show that during the prolonged agonist application a rapid current that peaked within 200 ms was accompanied with a slower current that required tens of seconds to reach its peak. The secondary rise in current was observed under different ionic conditions and temporally coincided with the development of conductivity to larger organic cations. The biphasic response was also observed in cells with blocked pannexin channels and in cells not expressing these channels endogenously. The biphasic current was preserved in N-terminal T15A, T15S, and T15V mutants that have low or no permeability to organic cations, reflecting enhanced permeability to inorganic cations. In contrast, the T15E, T15K, and T15W mutants, and the Delta18 mutant with deleted P2X(7) receptor-specific 18-amino acid C-terminal segment, were instantaneously permeable to organic cations and generated high amplitude monophasic currents. These results indicate that the P2X(7) receptor channel dilates under physiological ion conditions, leading to generation of biphasic current, and that this process is controlled by residues near the intracellular side of the channel pore.

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Characterization of the T15A-P2X7R mutant expressed in HEK293 cells. (A) Patterns of current in response to repetitive application of 100 μM BzATP, followed by a washout period of 5 min. (B and C) 100 μM BzATP-induced biphasic response from cells bathed in NMDG+-KR buffer (B) and NMDG+ buffer (C). (D and E) Lack of channel permeability to NMDG+ (D) and NMEA+ (E) when cells were bathed in KR buffer in which all Na+ was substituted with organic cations. (F) A shift in the reversal potential observed during a 40-s application of 100 μM BzATP from cells bathed in NMDG+ medium.
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fig6: Characterization of the T15A-P2X7R mutant expressed in HEK293 cells. (A) Patterns of current in response to repetitive application of 100 μM BzATP, followed by a washout period of 5 min. (B and C) 100 μM BzATP-induced biphasic response from cells bathed in NMDG+-KR buffer (B) and NMDG+ buffer (C). (D and E) Lack of channel permeability to NMDG+ (D) and NMEA+ (E) when cells were bathed in KR buffer in which all Na+ was substituted with organic cations. (F) A shift in the reversal potential observed during a 40-s application of 100 μM BzATP from cells bathed in NMDG+ medium.

Mentions: We next substituted Thr15 with valine and serine, as these amino acids are closest in structure to threonine. We also substituted this residue with smaller alanine. The T15V and T15S mutants responded to prolonged agonist application with biphasic currents. Permeability to NMDG+ and NMEA+ was reduced but not abolished in these mutants (Fig. 5 and Table I). The T15A mutant also responded to longer BzATP application by generating a biphasic current in cells bathed in KR buffer (Fig. 6 A), as well as in cells bathed in NMDG+-KR buffer (Fig. 6 B). As in control cells (Fig. 2 C, left), a rapid and transient outward current was observed in cells bathed in NMDG+ medium, followed by a shift from outward to inward current (Fig. 6 C). The initial reversal potential for this mutant bathed in NMDG+-KR and NMEA+-KR buffers was comparable to that observed in cells expressing WT receptors. In contrast to controls, prolonged application of agonist was not associated with a rightward shift in reversal potential (Fig. 6, D and E), indicating that the T15A mutant was not permeable to larger organic cations. In cells bathed in NMDG+ buffer, there was a positive shift in reversal potential by +15.1 mV (Fig. 6 F) compared with the +22.5 mV shift observed in cells expressing the WT receptor.


The P2X7 receptor channel pore dilates under physiological ion conditions.

Yan Z, Li S, Liang Z, Tomić M, Stojilkovic SS - J. Gen. Physiol. (2008)

Characterization of the T15A-P2X7R mutant expressed in HEK293 cells. (A) Patterns of current in response to repetitive application of 100 μM BzATP, followed by a washout period of 5 min. (B and C) 100 μM BzATP-induced biphasic response from cells bathed in NMDG+-KR buffer (B) and NMDG+ buffer (C). (D and E) Lack of channel permeability to NMDG+ (D) and NMEA+ (E) when cells were bathed in KR buffer in which all Na+ was substituted with organic cations. (F) A shift in the reversal potential observed during a 40-s application of 100 μM BzATP from cells bathed in NMDG+ medium.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2571973&req=5

fig6: Characterization of the T15A-P2X7R mutant expressed in HEK293 cells. (A) Patterns of current in response to repetitive application of 100 μM BzATP, followed by a washout period of 5 min. (B and C) 100 μM BzATP-induced biphasic response from cells bathed in NMDG+-KR buffer (B) and NMDG+ buffer (C). (D and E) Lack of channel permeability to NMDG+ (D) and NMEA+ (E) when cells were bathed in KR buffer in which all Na+ was substituted with organic cations. (F) A shift in the reversal potential observed during a 40-s application of 100 μM BzATP from cells bathed in NMDG+ medium.
Mentions: We next substituted Thr15 with valine and serine, as these amino acids are closest in structure to threonine. We also substituted this residue with smaller alanine. The T15V and T15S mutants responded to prolonged agonist application with biphasic currents. Permeability to NMDG+ and NMEA+ was reduced but not abolished in these mutants (Fig. 5 and Table I). The T15A mutant also responded to longer BzATP application by generating a biphasic current in cells bathed in KR buffer (Fig. 6 A), as well as in cells bathed in NMDG+-KR buffer (Fig. 6 B). As in control cells (Fig. 2 C, left), a rapid and transient outward current was observed in cells bathed in NMDG+ medium, followed by a shift from outward to inward current (Fig. 6 C). The initial reversal potential for this mutant bathed in NMDG+-KR and NMEA+-KR buffers was comparable to that observed in cells expressing WT receptors. In contrast to controls, prolonged application of agonist was not associated with a rightward shift in reversal potential (Fig. 6, D and E), indicating that the T15A mutant was not permeable to larger organic cations. In cells bathed in NMDG+ buffer, there was a positive shift in reversal potential by +15.1 mV (Fig. 6 F) compared with the +22.5 mV shift observed in cells expressing the WT receptor.

Bottom Line: The biphasic current was preserved in N-terminal T15A, T15S, and T15V mutants that have low or no permeability to organic cations, reflecting enhanced permeability to inorganic cations.In contrast, the T15E, T15K, and T15W mutants, and the Delta18 mutant with deleted P2X(7) receptor-specific 18-amino acid C-terminal segment, were instantaneously permeable to organic cations and generated high amplitude monophasic currents.These results indicate that the P2X(7) receptor channel dilates under physiological ion conditions, leading to generation of biphasic current, and that this process is controlled by residues near the intracellular side of the channel pore.

View Article: PubMed Central - PubMed

Affiliation: Section on Cellular Signaling, Program in Developmental Neuroscience, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Activation of the purinergic P2X(7) receptor leads to the rapid opening of an integral ion channel that is permeable to small cations. This is followed by a gradual increase in permeability to fluorescent dyes by integrating the actions of the pannexin-1 channel. Here, we show that during the prolonged agonist application a rapid current that peaked within 200 ms was accompanied with a slower current that required tens of seconds to reach its peak. The secondary rise in current was observed under different ionic conditions and temporally coincided with the development of conductivity to larger organic cations. The biphasic response was also observed in cells with blocked pannexin channels and in cells not expressing these channels endogenously. The biphasic current was preserved in N-terminal T15A, T15S, and T15V mutants that have low or no permeability to organic cations, reflecting enhanced permeability to inorganic cations. In contrast, the T15E, T15K, and T15W mutants, and the Delta18 mutant with deleted P2X(7) receptor-specific 18-amino acid C-terminal segment, were instantaneously permeable to organic cations and generated high amplitude monophasic currents. These results indicate that the P2X(7) receptor channel dilates under physiological ion conditions, leading to generation of biphasic current, and that this process is controlled by residues near the intracellular side of the channel pore.

Show MeSH
Related in: MedlinePlus