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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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Agonist-induced biphasic current response is independent of pannexin-1 channels. (A) Agonist-induced diffusion of extracellular YO-PRO-1 into GT1 cells. Fluorescence intensities expressed in arbitrary units (λex = 488 nm) are shown in response to application of BzATP only (left trace) and ATP followed by BzATP (right trace). (B) Normalized fluorescence intensities (gray, λex = 380 nm; black, λex = 340 nm) in GT1 cells loaded with Fura-FF (top) or Fura-2 (bottom) and stimulated with 100 μM BzATP. (C and D) BzATP-induced biphasic response in GT1 (C) and C6 single cells (D). (E) The lack of effects of CBX on pattern of current response in HEK293 cells; control (left) and 30 μM CBX-treated cells (right). In experiments shown in A, C, D, and E, cells were bathed in KR buffer. In experiments shown in B, cells were bathed in Ca2+-deficient KR buffer.
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fig3: Agonist-induced biphasic current response is independent of pannexin-1 channels. (A) Agonist-induced diffusion of extracellular YO-PRO-1 into GT1 cells. Fluorescence intensities expressed in arbitrary units (λex = 488 nm) are shown in response to application of BzATP only (left trace) and ATP followed by BzATP (right trace). (B) Normalized fluorescence intensities (gray, λex = 380 nm; black, λex = 340 nm) in GT1 cells loaded with Fura-FF (top) or Fura-2 (bottom) and stimulated with 100 μM BzATP. (C and D) BzATP-induced biphasic response in GT1 (C) and C6 single cells (D). (E) The lack of effects of CBX on pattern of current response in HEK293 cells; control (left) and 30 μM CBX-treated cells (right). In experiments shown in A, C, D, and E, cells were bathed in KR buffer. In experiments shown in B, cells were bathed in Ca2+-deficient KR buffer.

Mentions: The averaged ratio between the rapid and slow currents, the latter measured at the end of 40-s applications of 100 μM BzATP, was 1:2.4 (1,011 ± 78 pA vs. 2,390 ± 133 pA; n = 52). The biphasic response was also observed in amphotericin-perforated cells, suggesting that the second phase in current growth does not reflect dialysis of cells during the whole cell current recording (not depicted). To rule out other possible artifacts, we used intact cells and single cell calcium measurements. Previous experiments with Fura-2 revealed inability to report high [Ca2+]i in cells with activated P2X7Rs (Koshimizu et al., 2000), prompting us to use the less sensitive Fura-FF dye. Because HEK293 cells endogenously express Ca2+-mobilizing P2Y receptors (He et al., 2003), GT1 cells were used as an expression system. In these cells, 100 μM BzATP also induced biphasic [Ca2+]i response (Fig. 1 B, right), with the averaged ratio between the rapid and slow phase of 1:2.8 comparable to that observed in current measurements (F340/F380: 0.83 ± 0.04 vs. 2.32 ± 0.14; n = 23; basal [Ca2+]i subtracted). However, the leak of Fura-FF (see Fig. 3) could influence estimates of the F340/F380 ratio during sustained agonist application.


The P2X7 receptor channel pore dilates under physiological ion conditions.

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

Agonist-induced biphasic current response is independent of pannexin-1 channels. (A) Agonist-induced diffusion of extracellular YO-PRO-1 into GT1 cells. Fluorescence intensities expressed in arbitrary units (λex = 488 nm) are shown in response to application of BzATP only (left trace) and ATP followed by BzATP (right trace). (B) Normalized fluorescence intensities (gray, λex = 380 nm; black, λex = 340 nm) in GT1 cells loaded with Fura-FF (top) or Fura-2 (bottom) and stimulated with 100 μM BzATP. (C and D) BzATP-induced biphasic response in GT1 (C) and C6 single cells (D). (E) The lack of effects of CBX on pattern of current response in HEK293 cells; control (left) and 30 μM CBX-treated cells (right). In experiments shown in A, C, D, and E, cells were bathed in KR buffer. In experiments shown in B, cells were bathed in Ca2+-deficient KR buffer.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Agonist-induced biphasic current response is independent of pannexin-1 channels. (A) Agonist-induced diffusion of extracellular YO-PRO-1 into GT1 cells. Fluorescence intensities expressed in arbitrary units (λex = 488 nm) are shown in response to application of BzATP only (left trace) and ATP followed by BzATP (right trace). (B) Normalized fluorescence intensities (gray, λex = 380 nm; black, λex = 340 nm) in GT1 cells loaded with Fura-FF (top) or Fura-2 (bottom) and stimulated with 100 μM BzATP. (C and D) BzATP-induced biphasic response in GT1 (C) and C6 single cells (D). (E) The lack of effects of CBX on pattern of current response in HEK293 cells; control (left) and 30 μM CBX-treated cells (right). In experiments shown in A, C, D, and E, cells were bathed in KR buffer. In experiments shown in B, cells were bathed in Ca2+-deficient KR buffer.
Mentions: The averaged ratio between the rapid and slow currents, the latter measured at the end of 40-s applications of 100 μM BzATP, was 1:2.4 (1,011 ± 78 pA vs. 2,390 ± 133 pA; n = 52). The biphasic response was also observed in amphotericin-perforated cells, suggesting that the second phase in current growth does not reflect dialysis of cells during the whole cell current recording (not depicted). To rule out other possible artifacts, we used intact cells and single cell calcium measurements. Previous experiments with Fura-2 revealed inability to report high [Ca2+]i in cells with activated P2X7Rs (Koshimizu et al., 2000), prompting us to use the less sensitive Fura-FF dye. Because HEK293 cells endogenously express Ca2+-mobilizing P2Y receptors (He et al., 2003), GT1 cells were used as an expression system. In these cells, 100 μM BzATP also induced biphasic [Ca2+]i response (Fig. 1 B, right), with the averaged ratio between the rapid and slow phase of 1:2.8 comparable to that observed in current measurements (F340/F380: 0.83 ± 0.04 vs. 2.32 ± 0.14; n = 23; basal [Ca2+]i subtracted). However, the leak of Fura-FF (see Fig. 3) could influence estimates of the F340/F380 ratio during sustained agonist application.

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