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Voltage- and [ATP]-dependent gating of the P2X(2) ATP receptor channel.

Fujiwara Y, Keceli B, Nakajo K, Kubo Y - J. Gen. Physiol. (2009)

Bottom Line: We investigated its structural basis by substituting a glycine residue (G344) in the second transmembrane (TM) helix, which may provide a kink that could mediate "gating." We found that, instead of a gradual increase, the inward current through the G344A mutant increased instantaneously upon hyperpolarization, whereas a G344P mutant retained an activation phase that was slower than the wild type (WT).These results demonstrate that the flexibility of G344 contributes to the voltage-dependent gating.We then executed simulation analyses using the calculated rate constants and successfully reproduced the results observed experimentally, voltage-dependent activation that is accelerated by increases in [ATP].

View Article: PubMed Central - PubMed

Affiliation: Division of Biophysics and Neurobiology, Department of Molecular Physiology, National Institute for Physiological Sciences, Aichi, Japan. fujiwara@phys2.med.osaka-u.ac.jp

ABSTRACT
P2X receptors are ligand-gated cation channels activated by extracellular adenosine triphosphate (ATP). Nonetheless, P2X(2) channel currents observed during the steady-state after ATP application are known to exhibit voltage dependence; there is a gradual increase in the inward current upon hyperpolarization. We used a Xenopus oocyte expression system and two-electrode voltage clamp to analyze this "activation" phase quantitatively. We characterized the conductance-voltage relationship in the presence of various [ATP], and observed that it shifted toward more depolarized potentials with increases in [ATP]. By analyzing the rate constants for the channel's transition between a closed and an open state, we showed that the gating of P2X(2) is determined in a complex way that involves both membrane voltage and ATP binding. The activation phase was similarly recorded in HEK293 cells expressing P2X(2) even by inside-out patch clamp after intensive perfusion, excluding a possibility that the gating is due to block/unblock by endogenous blocker(s) of oocytes. We investigated its structural basis by substituting a glycine residue (G344) in the second transmembrane (TM) helix, which may provide a kink that could mediate "gating." We found that, instead of a gradual increase, the inward current through the G344A mutant increased instantaneously upon hyperpolarization, whereas a G344P mutant retained an activation phase that was slower than the wild type (WT). Using glycine-scanning mutagenesis in the background of G344A, we could recover the activation phase by introducing a glycine residue into the middle of second TM. These results demonstrate that the flexibility of G344 contributes to the voltage-dependent gating. Finally, we assumed a three-state model consisting of a fast ATP-binding step and a following gating step and estimated the rate constants for the latter in P2X(2)-WT. We then executed simulation analyses using the calculated rate constants and successfully reproduced the results observed experimentally, voltage-dependent activation that is accelerated by increases in [ATP].

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Simulation analyses of the activation phase evoked by a voltage step in the P2X2 WT channel. (A) Reproduction of the activation phase by simulation. The activation phases evoked by step pulses from −60 to −160 mV in the presence of various [ATP] were simulated. Rate constants used are shown in Table II (A and B). The applied [ATP] relative to Kd is indicated. (B) Comparison of the simulations of the activation phases using various ATP-binding and unbinding rate constants in the presence of an [ATP] that equals the Kd. The kunbind values used are shown. Rate constants used are shown in Table II (A and C). Red dashed lines indicate lines fitted by a single exponential function for each current trace. (C) Summary of the simulation of the activation kinetics at various voltages and [ATP]. The activation phases evoked by a voltage step from −60 mV to each voltage were simulated using the rate constants in Table II (A and B). The activation phases of the simulated currents could be fitted satisfactorily with a single exponential function, and the time constants of the fittings at various [ATP] relative to Kd were plotted versus membrane potential. (D) Reproduction of [ATP]-dependent changes in the activation kinetics by a simulation assuming that kbind is voltage dependent and that kon and koff are voltage independent. The activation phases evoked by the step pulse from −60 to −160 mV in the presence of high and low [ATP] were simulated. The rate constants used are shown in Table II D. In the case of low [ATP] here, Kd is equal to 100 × [ATP] at −60 mV and 10 × [ATP] at −160 mV due to the voltage-dependent change of kbind. In the high [ATP] case, Kd is equal to 10 × [ATP] at −60 mV and 1 × [ATP] at −160 mV.
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fig11: Simulation analyses of the activation phase evoked by a voltage step in the P2X2 WT channel. (A) Reproduction of the activation phase by simulation. The activation phases evoked by step pulses from −60 to −160 mV in the presence of various [ATP] were simulated. Rate constants used are shown in Table II (A and B). The applied [ATP] relative to Kd is indicated. (B) Comparison of the simulations of the activation phases using various ATP-binding and unbinding rate constants in the presence of an [ATP] that equals the Kd. The kunbind values used are shown. Rate constants used are shown in Table II (A and C). Red dashed lines indicate lines fitted by a single exponential function for each current trace. (C) Summary of the simulation of the activation kinetics at various voltages and [ATP]. The activation phases evoked by a voltage step from −60 mV to each voltage were simulated using the rate constants in Table II (A and B). The activation phases of the simulated currents could be fitted satisfactorily with a single exponential function, and the time constants of the fittings at various [ATP] relative to Kd were plotted versus membrane potential. (D) Reproduction of [ATP]-dependent changes in the activation kinetics by a simulation assuming that kbind is voltage dependent and that kon and koff are voltage independent. The activation phases evoked by the step pulse from −60 to −160 mV in the presence of high and low [ATP] were simulated. The rate constants used are shown in Table II D. In the case of low [ATP] here, Kd is equal to 100 × [ATP] at −60 mV and 10 × [ATP] at −160 mV due to the voltage-dependent change of kbind. In the high [ATP] case, Kd is equal to 10 × [ATP] at −60 mV and 1 × [ATP] at −160 mV.

Mentions: The activation phase evoked by the voltage step was simulated using Igor Pro (WaveMetrics, Inc.) software (Fig. 11). Simulation was performed based on the two-step model in Fig. 10 A. Transitions from each state were represented by differential equations and plotted every 1 ms using numerical integration (Fig. 11). The gating rate constants kon and koff were taken from the data in Fig. 10 B, and the rate constants for the ATP-binding step were determined with reference to the single-channel analysis data, kbind = 2.6 × 107 [M−1s−1] and kunbind = 1.1 × 103 [s−1] (Ding and Sachs, 1999). We used a kunbind value of 1,100, and the value of [ATP] × kbind was set depending on the [ATP] relative to Kd. The rate constants used for simulation are shown in Table II.


Voltage- and [ATP]-dependent gating of the P2X(2) ATP receptor channel.

Fujiwara Y, Keceli B, Nakajo K, Kubo Y - J. Gen. Physiol. (2009)

Simulation analyses of the activation phase evoked by a voltage step in the P2X2 WT channel. (A) Reproduction of the activation phase by simulation. The activation phases evoked by step pulses from −60 to −160 mV in the presence of various [ATP] were simulated. Rate constants used are shown in Table II (A and B). The applied [ATP] relative to Kd is indicated. (B) Comparison of the simulations of the activation phases using various ATP-binding and unbinding rate constants in the presence of an [ATP] that equals the Kd. The kunbind values used are shown. Rate constants used are shown in Table II (A and C). Red dashed lines indicate lines fitted by a single exponential function for each current trace. (C) Summary of the simulation of the activation kinetics at various voltages and [ATP]. The activation phases evoked by a voltage step from −60 mV to each voltage were simulated using the rate constants in Table II (A and B). The activation phases of the simulated currents could be fitted satisfactorily with a single exponential function, and the time constants of the fittings at various [ATP] relative to Kd were plotted versus membrane potential. (D) Reproduction of [ATP]-dependent changes in the activation kinetics by a simulation assuming that kbind is voltage dependent and that kon and koff are voltage independent. The activation phases evoked by the step pulse from −60 to −160 mV in the presence of high and low [ATP] were simulated. The rate constants used are shown in Table II D. In the case of low [ATP] here, Kd is equal to 100 × [ATP] at −60 mV and 10 × [ATP] at −160 mV due to the voltage-dependent change of kbind. In the high [ATP] case, Kd is equal to 10 × [ATP] at −60 mV and 1 × [ATP] at −160 mV.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2606937&req=5

fig11: Simulation analyses of the activation phase evoked by a voltage step in the P2X2 WT channel. (A) Reproduction of the activation phase by simulation. The activation phases evoked by step pulses from −60 to −160 mV in the presence of various [ATP] were simulated. Rate constants used are shown in Table II (A and B). The applied [ATP] relative to Kd is indicated. (B) Comparison of the simulations of the activation phases using various ATP-binding and unbinding rate constants in the presence of an [ATP] that equals the Kd. The kunbind values used are shown. Rate constants used are shown in Table II (A and C). Red dashed lines indicate lines fitted by a single exponential function for each current trace. (C) Summary of the simulation of the activation kinetics at various voltages and [ATP]. The activation phases evoked by a voltage step from −60 mV to each voltage were simulated using the rate constants in Table II (A and B). The activation phases of the simulated currents could be fitted satisfactorily with a single exponential function, and the time constants of the fittings at various [ATP] relative to Kd were plotted versus membrane potential. (D) Reproduction of [ATP]-dependent changes in the activation kinetics by a simulation assuming that kbind is voltage dependent and that kon and koff are voltage independent. The activation phases evoked by the step pulse from −60 to −160 mV in the presence of high and low [ATP] were simulated. The rate constants used are shown in Table II D. In the case of low [ATP] here, Kd is equal to 100 × [ATP] at −60 mV and 10 × [ATP] at −160 mV due to the voltage-dependent change of kbind. In the high [ATP] case, Kd is equal to 10 × [ATP] at −60 mV and 1 × [ATP] at −160 mV.
Mentions: The activation phase evoked by the voltage step was simulated using Igor Pro (WaveMetrics, Inc.) software (Fig. 11). Simulation was performed based on the two-step model in Fig. 10 A. Transitions from each state were represented by differential equations and plotted every 1 ms using numerical integration (Fig. 11). The gating rate constants kon and koff were taken from the data in Fig. 10 B, and the rate constants for the ATP-binding step were determined with reference to the single-channel analysis data, kbind = 2.6 × 107 [M−1s−1] and kunbind = 1.1 × 103 [s−1] (Ding and Sachs, 1999). We used a kunbind value of 1,100, and the value of [ATP] × kbind was set depending on the [ATP] relative to Kd. The rate constants used for simulation are shown in Table II.

Bottom Line: We investigated its structural basis by substituting a glycine residue (G344) in the second transmembrane (TM) helix, which may provide a kink that could mediate "gating." We found that, instead of a gradual increase, the inward current through the G344A mutant increased instantaneously upon hyperpolarization, whereas a G344P mutant retained an activation phase that was slower than the wild type (WT).These results demonstrate that the flexibility of G344 contributes to the voltage-dependent gating.We then executed simulation analyses using the calculated rate constants and successfully reproduced the results observed experimentally, voltage-dependent activation that is accelerated by increases in [ATP].

View Article: PubMed Central - PubMed

Affiliation: Division of Biophysics and Neurobiology, Department of Molecular Physiology, National Institute for Physiological Sciences, Aichi, Japan. fujiwara@phys2.med.osaka-u.ac.jp

ABSTRACT
P2X receptors are ligand-gated cation channels activated by extracellular adenosine triphosphate (ATP). Nonetheless, P2X(2) channel currents observed during the steady-state after ATP application are known to exhibit voltage dependence; there is a gradual increase in the inward current upon hyperpolarization. We used a Xenopus oocyte expression system and two-electrode voltage clamp to analyze this "activation" phase quantitatively. We characterized the conductance-voltage relationship in the presence of various [ATP], and observed that it shifted toward more depolarized potentials with increases in [ATP]. By analyzing the rate constants for the channel's transition between a closed and an open state, we showed that the gating of P2X(2) is determined in a complex way that involves both membrane voltage and ATP binding. The activation phase was similarly recorded in HEK293 cells expressing P2X(2) even by inside-out patch clamp after intensive perfusion, excluding a possibility that the gating is due to block/unblock by endogenous blocker(s) of oocytes. We investigated its structural basis by substituting a glycine residue (G344) in the second transmembrane (TM) helix, which may provide a kink that could mediate "gating." We found that, instead of a gradual increase, the inward current through the G344A mutant increased instantaneously upon hyperpolarization, whereas a G344P mutant retained an activation phase that was slower than the wild type (WT). Using glycine-scanning mutagenesis in the background of G344A, we could recover the activation phase by introducing a glycine residue into the middle of second TM. These results demonstrate that the flexibility of G344 contributes to the voltage-dependent gating. Finally, we assumed a three-state model consisting of a fast ATP-binding step and a following gating step and estimated the rate constants for the latter in P2X(2)-WT. We then executed simulation analyses using the calculated rate constants and successfully reproduced the results observed experimentally, voltage-dependent activation that is accelerated by increases in [ATP].

Show MeSH
Related in: MedlinePlus