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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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Two phases of activation in rat P2X7R-expressing cells bathed in KR-like buffer. (A) Repetitive short-term stimulation with 100 μM BzATP. Traces shown on the left and right panels are from two different cells. Inset shows the expanded time scale of the initial current response. Horizontal dotted lines indicate the peak amplitude of currents reached during initial agonist application. (B, left) Short-term and subsequent long-term stimulation with 100 μM BzATP. (Right) Calcium response to prolonged application of BzATP in cells loaded with Fura-FF. (C) Dose-dependent effects of BzATP and ATP on the rate of sustained current growth. Traces shown are from different cells during initial agonist application. In B and C, dotted lines separate rapid phase (I) from slow phase (II) in receptor response. Electrophysiological experiments were performed in HEK293 cells, and [Ca2+]i measurements were performed in GT1 cells expressing recombinant P2X7Rs. Gray areas and bottom numbers indicate duration of BzATP application. Unless otherwise specified, in this and all figures, recordings were performed in cells held at −60 mV. Traces shown are representative of at least five recordings.
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fig1: Two phases of activation in rat P2X7R-expressing cells bathed in KR-like buffer. (A) Repetitive short-term stimulation with 100 μM BzATP. Traces shown on the left and right panels are from two different cells. Inset shows the expanded time scale of the initial current response. Horizontal dotted lines indicate the peak amplitude of currents reached during initial agonist application. (B, left) Short-term and subsequent long-term stimulation with 100 μM BzATP. (Right) Calcium response to prolonged application of BzATP in cells loaded with Fura-FF. (C) Dose-dependent effects of BzATP and ATP on the rate of sustained current growth. Traces shown are from different cells during initial agonist application. In B and C, dotted lines separate rapid phase (I) from slow phase (II) in receptor response. Electrophysiological experiments were performed in HEK293 cells, and [Ca2+]i measurements were performed in GT1 cells expressing recombinant P2X7Rs. Gray areas and bottom numbers indicate duration of BzATP application. Unless otherwise specified, in this and all figures, recordings were performed in cells held at −60 mV. Traces shown are representative of at least five recordings.

Mentions: Cells clamped at −60 mV and stimulated with 100 μM BzATP repeatedly for 0.5–2 s responded with comparable current peaks (Fig. 1 A, left). However, this was not the case when cells were repeatedly stimulated with 100 μM BzATP for 4 s, separated by 2-min intervals. Under such experimental conditions, there was a progressive increase in the peak amplitude of current (Fig. 1 A, right). Growth of current was also evident during longer agonist applications. Fig. 1 B illustrates the pattern of current in response to 4- and 40-s application of 100 μM BzATP. During both applications, there was a rapid rise in current of comparable amplitudes, with the 10–90% rise time completed within 198 ± 9 ms; however, longer exposure to agonist was followed by a secondary growth of current. We termed such a pattern of response as biphasic, and the dotted line in Fig. 1 separates the rapid phase of current growth (I) from the slow phase of current growth (II). To avoid the possible side effects of repetitive exposure to agonists for a prolonged period on pattern of current responses, further recordings were performed only during the initial agonist application (naive receptors).


The P2X7 receptor channel pore dilates under physiological ion conditions.

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

Two phases of activation in rat P2X7R-expressing cells bathed in KR-like buffer. (A) Repetitive short-term stimulation with 100 μM BzATP. Traces shown on the left and right panels are from two different cells. Inset shows the expanded time scale of the initial current response. Horizontal dotted lines indicate the peak amplitude of currents reached during initial agonist application. (B, left) Short-term and subsequent long-term stimulation with 100 μM BzATP. (Right) Calcium response to prolonged application of BzATP in cells loaded with Fura-FF. (C) Dose-dependent effects of BzATP and ATP on the rate of sustained current growth. Traces shown are from different cells during initial agonist application. In B and C, dotted lines separate rapid phase (I) from slow phase (II) in receptor response. Electrophysiological experiments were performed in HEK293 cells, and [Ca2+]i measurements were performed in GT1 cells expressing recombinant P2X7Rs. Gray areas and bottom numbers indicate duration of BzATP application. Unless otherwise specified, in this and all figures, recordings were performed in cells held at −60 mV. Traces shown are representative of at least five recordings.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2571973&req=5

fig1: Two phases of activation in rat P2X7R-expressing cells bathed in KR-like buffer. (A) Repetitive short-term stimulation with 100 μM BzATP. Traces shown on the left and right panels are from two different cells. Inset shows the expanded time scale of the initial current response. Horizontal dotted lines indicate the peak amplitude of currents reached during initial agonist application. (B, left) Short-term and subsequent long-term stimulation with 100 μM BzATP. (Right) Calcium response to prolonged application of BzATP in cells loaded with Fura-FF. (C) Dose-dependent effects of BzATP and ATP on the rate of sustained current growth. Traces shown are from different cells during initial agonist application. In B and C, dotted lines separate rapid phase (I) from slow phase (II) in receptor response. Electrophysiological experiments were performed in HEK293 cells, and [Ca2+]i measurements were performed in GT1 cells expressing recombinant P2X7Rs. Gray areas and bottom numbers indicate duration of BzATP application. Unless otherwise specified, in this and all figures, recordings were performed in cells held at −60 mV. Traces shown are representative of at least five recordings.
Mentions: Cells clamped at −60 mV and stimulated with 100 μM BzATP repeatedly for 0.5–2 s responded with comparable current peaks (Fig. 1 A, left). However, this was not the case when cells were repeatedly stimulated with 100 μM BzATP for 4 s, separated by 2-min intervals. Under such experimental conditions, there was a progressive increase in the peak amplitude of current (Fig. 1 A, right). Growth of current was also evident during longer agonist applications. Fig. 1 B illustrates the pattern of current in response to 4- and 40-s application of 100 μM BzATP. During both applications, there was a rapid rise in current of comparable amplitudes, with the 10–90% rise time completed within 198 ± 9 ms; however, longer exposure to agonist was followed by a secondary growth of current. We termed such a pattern of response as biphasic, and the dotted line in Fig. 1 separates the rapid phase of current growth (I) from the slow phase of current growth (II). To avoid the possible side effects of repetitive exposure to agonists for a prolonged period on pattern of current responses, further recordings were performed only during the initial agonist application (naive receptors).

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