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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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Permeability of P2X7R to NMDG+ depends on duration of agonist application. (A) Pattern of 100 μM BzATP-induced current and reversal potential in cells bathed in 10% Na+ and 90% NMDG+-containing KR buffer during the initial agonist application. (A, left) Current recording during a 40-s agonist application at −60 mV holding potential. (Middle) Time course of agonist-induced currents in cells under the ramp protocol. (Right) Positive shift in reversal potential observed during the initial 40-s application of BzATP; 0.485-s voltage ramps were delivered twice per second. (B) Patterns of BzATP-induced currents (left and middle) and shift in the reversal potential (right) in cells bathed in KR buffer, in which Na+ was completely substituted with NMDG+. (C) Time course of BzATP-induced P2X7R current in cells bathed in NMDG+ buffer (left and middle) and the accompanied changes in the reversal potential (right). In this and all figures, only 15 out of 100 traces for the current voltage relationship with the equal time intervals are shown. Notice the lack of receptor deactivation in C compared with records shown in A (left) and B (left). (D) Increase in NMDG+ permeability occurs in cells bathed in physiological solution for agonist application of >∼10 s. Application of agonist was initially performed in cells bathed in KR buffer for 4 (left) and 40 s (middle) and was continued in NMDG+ medium (gray areas). Notice a difference in responses when replacement of KR buffer with NMDG+ buffer was performed during the phase I (left) and II (middle) of current growth. Right panel illustrates the mean ± SEM values of the peak currents reached after replacement of KR buffer with NMDG+ buffer in two time points. All experiments were performed in HEK293 cells.
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fig2: Permeability of P2X7R to NMDG+ depends on duration of agonist application. (A) Pattern of 100 μM BzATP-induced current and reversal potential in cells bathed in 10% Na+ and 90% NMDG+-containing KR buffer during the initial agonist application. (A, left) Current recording during a 40-s agonist application at −60 mV holding potential. (Middle) Time course of agonist-induced currents in cells under the ramp protocol. (Right) Positive shift in reversal potential observed during the initial 40-s application of BzATP; 0.485-s voltage ramps were delivered twice per second. (B) Patterns of BzATP-induced currents (left and middle) and shift in the reversal potential (right) in cells bathed in KR buffer, in which Na+ was completely substituted with NMDG+. (C) Time course of BzATP-induced P2X7R current in cells bathed in NMDG+ buffer (left and middle) and the accompanied changes in the reversal potential (right). In this and all figures, only 15 out of 100 traces for the current voltage relationship with the equal time intervals are shown. Notice the lack of receptor deactivation in C compared with records shown in A (left) and B (left). (D) Increase in NMDG+ permeability occurs in cells bathed in physiological solution for agonist application of >∼10 s. Application of agonist was initially performed in cells bathed in KR buffer for 4 (left) and 40 s (middle) and was continued in NMDG+ medium (gray areas). Notice a difference in responses when replacement of KR buffer with NMDG+ buffer was performed during the phase I (left) and II (middle) of current growth. Right panel illustrates the mean ± SEM values of the peak currents reached after replacement of KR buffer with NMDG+ buffer in two time points. All experiments were performed in HEK293 cells.

Mentions: When 90% of Na+ was replaced with NMDG+ (Na+/NMDG+-KR buffer), 100 μM BzATP still induced biphasic current in naive cells held at −60 mV, with kinetics that resembled growth of current observed in cells bathed in Na+-containing KR medium (Fig. 2 A, left vs. Fig. 1). Under repetitive 485-ms voltage ramp pulses from −80 to +80 mV, delivered twice per second, there was also a progressive increase in the peak amplitude of inward current at −80 mV (Fig. 2 A, middle). Current voltage curves constructed from ramp voltage commands revealed a shift in reversal potential during the 40-s agonist application from −28.2 ± 0.5 mV to a steady-state of −17.3 ± 1.3 mV (Fig. 2 A, right panel, from left to right). Such a shift was observed in all cells studied (n = 9). We next repeated these experiments in cells bathed in KR medium with NMDG+ replacing all the extracellular Na+ (NMDG+-KR buffer). Under these ionic conditions, the rate of the secondary growth in current was comparable to that observed in cells perfused with Na+/NMDG+-KR solution, and the 100 μM BzATP–evoked current reversal potential changed from −36.5 ± 0.3 mV to −24.6 ± 0.8 mV (Fig. 2 B). However, in cells clamped at −60 mV and exposed to BzATP for 40 s, the peak amplitude of current differed in two ionic conditions: (1) Na+/NMDG+-KR = 1.3 ± 0.2 nA (n = 6), 54% of that observed in cells bathed in KR buffer; (2) NMDG+-KR = 0.59 ± 0.08 nA (n = 4), 24% of that observed in cells bathed in KR buffer. These results suggest that NMDG+ only partially substitutes for Na+ as the conducted ion.


The P2X7 receptor channel pore dilates under physiological ion conditions.

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

Permeability of P2X7R to NMDG+ depends on duration of agonist application. (A) Pattern of 100 μM BzATP-induced current and reversal potential in cells bathed in 10% Na+ and 90% NMDG+-containing KR buffer during the initial agonist application. (A, left) Current recording during a 40-s agonist application at −60 mV holding potential. (Middle) Time course of agonist-induced currents in cells under the ramp protocol. (Right) Positive shift in reversal potential observed during the initial 40-s application of BzATP; 0.485-s voltage ramps were delivered twice per second. (B) Patterns of BzATP-induced currents (left and middle) and shift in the reversal potential (right) in cells bathed in KR buffer, in which Na+ was completely substituted with NMDG+. (C) Time course of BzATP-induced P2X7R current in cells bathed in NMDG+ buffer (left and middle) and the accompanied changes in the reversal potential (right). In this and all figures, only 15 out of 100 traces for the current voltage relationship with the equal time intervals are shown. Notice the lack of receptor deactivation in C compared with records shown in A (left) and B (left). (D) Increase in NMDG+ permeability occurs in cells bathed in physiological solution for agonist application of >∼10 s. Application of agonist was initially performed in cells bathed in KR buffer for 4 (left) and 40 s (middle) and was continued in NMDG+ medium (gray areas). Notice a difference in responses when replacement of KR buffer with NMDG+ buffer was performed during the phase I (left) and II (middle) of current growth. Right panel illustrates the mean ± SEM values of the peak currents reached after replacement of KR buffer with NMDG+ buffer in two time points. All experiments were performed in HEK293 cells.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Permeability of P2X7R to NMDG+ depends on duration of agonist application. (A) Pattern of 100 μM BzATP-induced current and reversal potential in cells bathed in 10% Na+ and 90% NMDG+-containing KR buffer during the initial agonist application. (A, left) Current recording during a 40-s agonist application at −60 mV holding potential. (Middle) Time course of agonist-induced currents in cells under the ramp protocol. (Right) Positive shift in reversal potential observed during the initial 40-s application of BzATP; 0.485-s voltage ramps were delivered twice per second. (B) Patterns of BzATP-induced currents (left and middle) and shift in the reversal potential (right) in cells bathed in KR buffer, in which Na+ was completely substituted with NMDG+. (C) Time course of BzATP-induced P2X7R current in cells bathed in NMDG+ buffer (left and middle) and the accompanied changes in the reversal potential (right). In this and all figures, only 15 out of 100 traces for the current voltage relationship with the equal time intervals are shown. Notice the lack of receptor deactivation in C compared with records shown in A (left) and B (left). (D) Increase in NMDG+ permeability occurs in cells bathed in physiological solution for agonist application of >∼10 s. Application of agonist was initially performed in cells bathed in KR buffer for 4 (left) and 40 s (middle) and was continued in NMDG+ medium (gray areas). Notice a difference in responses when replacement of KR buffer with NMDG+ buffer was performed during the phase I (left) and II (middle) of current growth. Right panel illustrates the mean ± SEM values of the peak currents reached after replacement of KR buffer with NMDG+ buffer in two time points. All experiments were performed in HEK293 cells.
Mentions: When 90% of Na+ was replaced with NMDG+ (Na+/NMDG+-KR buffer), 100 μM BzATP still induced biphasic current in naive cells held at −60 mV, with kinetics that resembled growth of current observed in cells bathed in Na+-containing KR medium (Fig. 2 A, left vs. Fig. 1). Under repetitive 485-ms voltage ramp pulses from −80 to +80 mV, delivered twice per second, there was also a progressive increase in the peak amplitude of inward current at −80 mV (Fig. 2 A, middle). Current voltage curves constructed from ramp voltage commands revealed a shift in reversal potential during the 40-s agonist application from −28.2 ± 0.5 mV to a steady-state of −17.3 ± 1.3 mV (Fig. 2 A, right panel, from left to right). Such a shift was observed in all cells studied (n = 9). We next repeated these experiments in cells bathed in KR medium with NMDG+ replacing all the extracellular Na+ (NMDG+-KR buffer). Under these ionic conditions, the rate of the secondary growth in current was comparable to that observed in cells perfused with Na+/NMDG+-KR solution, and the 100 μM BzATP–evoked current reversal potential changed from −36.5 ± 0.3 mV to −24.6 ± 0.8 mV (Fig. 2 B). However, in cells clamped at −60 mV and exposed to BzATP for 40 s, the peak amplitude of current differed in two ionic conditions: (1) Na+/NMDG+-KR = 1.3 ± 0.2 nA (n = 6), 54% of that observed in cells bathed in KR buffer; (2) NMDG+-KR = 0.59 ± 0.08 nA (n = 4), 24% of that observed in cells bathed in KR buffer. These results suggest that NMDG+ only partially substitutes for Na+ as the conducted ion.

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