Limits...
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.

Show MeSH

Related in: MedlinePlus

Effects of deletion of the P2X7R-specific Y358-E375 C-terminal sequence (Δ18) on the pattern of current and permeability to NMDG+. (A) Patterns of currents from cells bathed in normal (left) and NMDG+-containing KR buffer (right). (B) Changes in the reversal potential during the 40-s application of 100 μM BzATP from cells perfused with NMDG+-KR buffer.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2571973&req=5

fig4: Effects of deletion of the P2X7R-specific Y358-E375 C-terminal sequence (Δ18) on the pattern of current and permeability to NMDG+. (A) Patterns of currents from cells bathed in normal (left) and NMDG+-containing KR buffer (right). (B) Changes in the reversal potential during the 40-s application of 100 μM BzATP from cells perfused with NMDG+-KR buffer.

Mentions: Immediately after the second transmembrane domain, P2X7R contains a stretch of 18 amino acids, Y358–E375, not found in other P2XRs. Deletion of this sequence (Δ18 mutant) was reported to affect conductivity for NMDG+ (Jiang et al., 2005). In our hands, the mutant was unable to generate the biphasic current when cells were bathed in KR solution. The mutant initially responded to 100 μM BzATP with high amplitude of current that was reached rapidly, followed by a partial decline in the current amplitude during prolonged agonist application, and delayed deactivation after washout of agonist (Fig. 4 A, left trace). A similar pattern, but not amplitude of current, was observed in cells perfused with NMDG+-KR buffer (Fig. 4 A, right trace). The reversal potential for this mutant at the beginning of agonist application was significantly shifted when compared with WT receptors, although recordings were performed in cells bathed in identical NMDG+-KR buffer (Fig. 4 B and Table I). Such a difference was also observed in cells perfused with NMDG+ medium (−50.3 ± 0.9 mV for the Δ18 mutant vs. −66.9 ± 0.2 mV for the WT receptor; Table I), suggesting that the mutant was instantaneously permeable to organic cations. In further contrast to the WT receptor, this mutant initially showed a slow decrease in permeability to NMDG+, which temporally coincided with a decline in the current amplitude, followed by a gradual recovery in NMDG+ permeability during the 40-s agonist application (Fig. 4 B).


The P2X7 receptor channel pore dilates under physiological ion conditions.

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

Effects of deletion of the P2X7R-specific Y358-E375 C-terminal sequence (Δ18) on the pattern of current and permeability to NMDG+. (A) Patterns of currents from cells bathed in normal (left) and NMDG+-containing KR buffer (right). (B) Changes in the reversal potential during the 40-s application of 100 μM BzATP from cells perfused with NMDG+-KR buffer.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Effects of deletion of the P2X7R-specific Y358-E375 C-terminal sequence (Δ18) on the pattern of current and permeability to NMDG+. (A) Patterns of currents from cells bathed in normal (left) and NMDG+-containing KR buffer (right). (B) Changes in the reversal potential during the 40-s application of 100 μM BzATP from cells perfused with NMDG+-KR buffer.
Mentions: Immediately after the second transmembrane domain, P2X7R contains a stretch of 18 amino acids, Y358–E375, not found in other P2XRs. Deletion of this sequence (Δ18 mutant) was reported to affect conductivity for NMDG+ (Jiang et al., 2005). In our hands, the mutant was unable to generate the biphasic current when cells were bathed in KR solution. The mutant initially responded to 100 μM BzATP with high amplitude of current that was reached rapidly, followed by a partial decline in the current amplitude during prolonged agonist application, and delayed deactivation after washout of agonist (Fig. 4 A, left trace). A similar pattern, but not amplitude of current, was observed in cells perfused with NMDG+-KR buffer (Fig. 4 A, right trace). The reversal potential for this mutant at the beginning of agonist application was significantly shifted when compared with WT receptors, although recordings were performed in cells bathed in identical NMDG+-KR buffer (Fig. 4 B and Table I). Such a difference was also observed in cells perfused with NMDG+ medium (−50.3 ± 0.9 mV for the Δ18 mutant vs. −66.9 ± 0.2 mV for the WT receptor; Table I), suggesting that the mutant was instantaneously permeable to organic cations. In further contrast to the WT receptor, this mutant initially showed a slow decrease in permeability to NMDG+, which temporally coincided with a decline in the current amplitude, followed by a gradual recovery in NMDG+ permeability during the 40-s agonist application (Fig. 4 B).

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