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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 N-terminal P2X7R mutants. (Left) Representative traces of currents generated by a 40-s application of 100 μM BzATP by WT and mutant receptors expressed in HEK293 cells. Notice that BzATP induced monophasic currents in cells expressing T15E, T15K, and T15W mutants and biphasic current in all other mutants. Receptor deactivation was delayed in cells expressing T15E, T15K, T15W, and K17A mutants. Cells were bathed in KR buffer. (Middle and right) Comparison of the permeability of channels to NMDG+ (middle) and NMEA+ (right). Change in time courses are shown from left to right, and vertical dotted lines indicate the reversal potential for WT receptors at the beginning and at the end of a 40-s application of 100 μM BzATP. Cells were bathed in NMDG+- or NMEA+-containing KR buffer. Mean ± SEM values are shown in Table I.
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fig5: Characterization of N-terminal P2X7R mutants. (Left) Representative traces of currents generated by a 40-s application of 100 μM BzATP by WT and mutant receptors expressed in HEK293 cells. Notice that BzATP induced monophasic currents in cells expressing T15E, T15K, and T15W mutants and biphasic current in all other mutants. Receptor deactivation was delayed in cells expressing T15E, T15K, T15W, and K17A mutants. Cells were bathed in KR buffer. (Middle and right) Comparison of the permeability of channels to NMDG+ (middle) and NMEA+ (right). Change in time courses are shown from left to right, and vertical dotted lines indicate the reversal potential for WT receptors at the beginning and at the end of a 40-s application of 100 μM BzATP. Cells were bathed in NMDG+- or NMEA+-containing KR buffer. Mean ± SEM values are shown in Table I.

Mentions: All P2XRs have several putative protein kinase C phosphorylation sites, including the conserved 15TXK17 triplet of residues located in their N termini (P2X7R numbering). We next tested whether phosphorylation of Thr15 accounts for secondary current growth. Frequently, but not always, the introduction of Glu or Asp, or the introduction of positively charged residues, can mimic the effect of phosphorylation. In our experiments, cells bearing the T15E mutant responded to 100 μM BzATP, with a profile and amplitude of current comparable to that observed with the Δ18 mutant. Furthermore, the mutant was instantaneously permeable to NMDG+ and NMEA+, as indicated by reversal potential measured at the beginning of agonist application (Fig. 5 and Table I). In contrast to the Δ18 receptor, this mutant did not show additional changes in reversal potential during the 40-s agonist application. The permeability of cells expressing this receptor to YO-PRO-1 was not enhanced (not depicted), confirming that pannexin-1 channels do not account for increased permeability to NMDG+. Cells bearing the T15K mutant showed a similar pattern of current and permeability to NMDG+ and NMEA+ as the T15E mutant (Fig. 5 and Table I). Positive shifts in reversal potentials for the two mutants and the lack of biphasic responses suggest that the Thr15 residue also contributes to the secondary rise in current amplitude, presumably in a protein kinase C–dependent manner.


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 N-terminal P2X7R mutants. (Left) Representative traces of currents generated by a 40-s application of 100 μM BzATP by WT and mutant receptors expressed in HEK293 cells. Notice that BzATP induced monophasic currents in cells expressing T15E, T15K, and T15W mutants and biphasic current in all other mutants. Receptor deactivation was delayed in cells expressing T15E, T15K, T15W, and K17A mutants. Cells were bathed in KR buffer. (Middle and right) Comparison of the permeability of channels to NMDG+ (middle) and NMEA+ (right). Change in time courses are shown from left to right, and vertical dotted lines indicate the reversal potential for WT receptors at the beginning and at the end of a 40-s application of 100 μM BzATP. Cells were bathed in NMDG+- or NMEA+-containing KR buffer. Mean ± SEM values are shown in Table I.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2571973&req=5

fig5: Characterization of N-terminal P2X7R mutants. (Left) Representative traces of currents generated by a 40-s application of 100 μM BzATP by WT and mutant receptors expressed in HEK293 cells. Notice that BzATP induced monophasic currents in cells expressing T15E, T15K, and T15W mutants and biphasic current in all other mutants. Receptor deactivation was delayed in cells expressing T15E, T15K, T15W, and K17A mutants. Cells were bathed in KR buffer. (Middle and right) Comparison of the permeability of channels to NMDG+ (middle) and NMEA+ (right). Change in time courses are shown from left to right, and vertical dotted lines indicate the reversal potential for WT receptors at the beginning and at the end of a 40-s application of 100 μM BzATP. Cells were bathed in NMDG+- or NMEA+-containing KR buffer. Mean ± SEM values are shown in Table I.
Mentions: All P2XRs have several putative protein kinase C phosphorylation sites, including the conserved 15TXK17 triplet of residues located in their N termini (P2X7R numbering). We next tested whether phosphorylation of Thr15 accounts for secondary current growth. Frequently, but not always, the introduction of Glu or Asp, or the introduction of positively charged residues, can mimic the effect of phosphorylation. In our experiments, cells bearing the T15E mutant responded to 100 μM BzATP, with a profile and amplitude of current comparable to that observed with the Δ18 mutant. Furthermore, the mutant was instantaneously permeable to NMDG+ and NMEA+, as indicated by reversal potential measured at the beginning of agonist application (Fig. 5 and Table I). In contrast to the Δ18 receptor, this mutant did not show additional changes in reversal potential during the 40-s agonist application. The permeability of cells expressing this receptor to YO-PRO-1 was not enhanced (not depicted), confirming that pannexin-1 channels do not account for increased permeability to NMDG+. Cells bearing the T15K mutant showed a similar pattern of current and permeability to NMDG+ and NMEA+ as the T15E mutant (Fig. 5 and Table I). Positive shifts in reversal potentials for the two mutants and the lack of biphasic responses suggest that the Thr15 residue also contributes to the secondary rise in current amplitude, presumably in a protein kinase C–dependent manner.

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