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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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Effects of Thr15 substitution on the pattern of current and permeability to NMDG+ schematic models. (A) Two patterns of currents: biphasic (left) and high amplitude monophasic (right). The biphasic response reflects three steps in gating: closed (c), opened (o), and dilated (d), and is observed in cells expressing WT receptors and T15A, T15V, and T15S mutants. The high amplitude monophasic response reflects instantaneous transition from the closed to the dilated stage, and is observed in cells expressing T15W, T15E, and T15K mutants. (B) A shift in the reversal potential (RP) for WT and mutant receptors during sustained agonist application. Mean values for the reversal potential at the end of a 40-s recording for WT and six T15 mutant receptors were subtracted from the initial reversal potential value for the WT receptor (−36.5 mV; Table I, NMDG+-KR column). The side chains of amino acid residues used in substitution studies are shown at the bottom.
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fig7: Effects of Thr15 substitution on the pattern of current and permeability to NMDG+ schematic models. (A) Two patterns of currents: biphasic (left) and high amplitude monophasic (right). The biphasic response reflects three steps in gating: closed (c), opened (o), and dilated (d), and is observed in cells expressing WT receptors and T15A, T15V, and T15S mutants. The high amplitude monophasic response reflects instantaneous transition from the closed to the dilated stage, and is observed in cells expressing T15W, T15E, and T15K mutants. (B) A shift in the reversal potential (RP) for WT and mutant receptors during sustained agonist application. Mean values for the reversal potential at the end of a 40-s recording for WT and six T15 mutant receptors were subtracted from the initial reversal potential value for the WT receptor (−36.5 mV; Table I, NMDG+-KR column). The side chains of amino acid residues used in substitution studies are shown at the bottom.

Mentions: In summary, we suggest that channel pore opening of the WT receptor, as well as of the T15A, T15V, and T15S mutants, involves a two-step transition: (1) from closed to opened, and (2) from opened to dilated, leading to the generation of biphasic currents (Fig. 7 A). The steady-state size of the pore varies among these receptors, which is reflected by organic cation permeability (Fig. 7 B). In contrast, the enlargement of the channel pore for T15E, T15K, and T15W mutants involves a single transition step, from closed to dilated, resulting in the generation of high amplitude monophasic currents (Fig. 7 A, right). In the absence of crystal structure data, it is difficult to predict how Thr15 contributes to the control of gating. The T15XK17 triplet represents a putative protein kinase C phosphorylation site, but phosphorylation of the Thr15 residue (if it occurs) does not account for delayed current growth. There is a parallel between changes in permeability of receptors to NMDG+ and the steric size of the side chains of these residues (Fig. 7 B), suggesting that this phenomenon could underlie changes in the receptor behavior.


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 Thr15 substitution on the pattern of current and permeability to NMDG+ schematic models. (A) Two patterns of currents: biphasic (left) and high amplitude monophasic (right). The biphasic response reflects three steps in gating: closed (c), opened (o), and dilated (d), and is observed in cells expressing WT receptors and T15A, T15V, and T15S mutants. The high amplitude monophasic response reflects instantaneous transition from the closed to the dilated stage, and is observed in cells expressing T15W, T15E, and T15K mutants. (B) A shift in the reversal potential (RP) for WT and mutant receptors during sustained agonist application. Mean values for the reversal potential at the end of a 40-s recording for WT and six T15 mutant receptors were subtracted from the initial reversal potential value for the WT receptor (−36.5 mV; Table I, NMDG+-KR column). The side chains of amino acid residues used in substitution studies are shown at the bottom.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2571973&req=5

fig7: Effects of Thr15 substitution on the pattern of current and permeability to NMDG+ schematic models. (A) Two patterns of currents: biphasic (left) and high amplitude monophasic (right). The biphasic response reflects three steps in gating: closed (c), opened (o), and dilated (d), and is observed in cells expressing WT receptors and T15A, T15V, and T15S mutants. The high amplitude monophasic response reflects instantaneous transition from the closed to the dilated stage, and is observed in cells expressing T15W, T15E, and T15K mutants. (B) A shift in the reversal potential (RP) for WT and mutant receptors during sustained agonist application. Mean values for the reversal potential at the end of a 40-s recording for WT and six T15 mutant receptors were subtracted from the initial reversal potential value for the WT receptor (−36.5 mV; Table I, NMDG+-KR column). The side chains of amino acid residues used in substitution studies are shown at the bottom.
Mentions: In summary, we suggest that channel pore opening of the WT receptor, as well as of the T15A, T15V, and T15S mutants, involves a two-step transition: (1) from closed to opened, and (2) from opened to dilated, leading to the generation of biphasic currents (Fig. 7 A). The steady-state size of the pore varies among these receptors, which is reflected by organic cation permeability (Fig. 7 B). In contrast, the enlargement of the channel pore for T15E, T15K, and T15W mutants involves a single transition step, from closed to dilated, resulting in the generation of high amplitude monophasic currents (Fig. 7 A, right). In the absence of crystal structure data, it is difficult to predict how Thr15 contributes to the control of gating. The T15XK17 triplet represents a putative protein kinase C phosphorylation site, but phosphorylation of the Thr15 residue (if it occurs) does not account for delayed current growth. There is a parallel between changes in permeability of receptors to NMDG+ and the steric size of the side chains of these residues (Fig. 7 B), suggesting that this phenomenon could underlie changes in the receptor behavior.

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