Fujiwara Y et al. (2009), Voltage- and [ATP]-dependent gating of the P2X(...

XB-ART-38982

J Gen Physiol 2009 Jan 01;1331:93-109. doi: 10.1085/jgp.200810002.

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

Fujiwara Y , Keceli B , Nakajo K , Kubo Y .

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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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Species referenced: Xenopus laevis
Genes referenced: cd59 p2rx2

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	Figure 1. Voltage-dependent activation of P2X2 during the steady state after application of ATP. Macroscopic currents recorded under two-electrode voltage clamp using Xenopus oocytes in Na-based external solution during the steady state after application of ATP. (A) Current traces from an identical oocyte evoked by a hyperpolarizing step pulse (from −40 to −100 mV) in the absence (dashed line) and presence (solid line) of ATP. The actual membrane potentials recorded are indicated in the bottom panel. The instantaneous component (a), activation component (b), and deactivation component (c) are illustrated. (B) Comparison of macroscopic currents evoked by hyperpolarizing step pulses from two different prepulse potentials, +60 and −60 mV. Pulse protocols are indicated below. These current traces were recorded from an identical oocyte and shown after subtracting data obtained in the absence of ATP. (C) Current traces from an identical oocyte evoked by hyperpolarizing step pulses to −120 from −30-mV prepulses of different durations. The pulse protocols are indicated below. Current traces were overlaid by arranging them relative to the beginning of the prepulse. (D) Comparison of macroscopic currents evoked by hyperpolarizing step pulses during the steady state after the application of various [ATP]. The pulse protocols are indicated on the right. These current traces were recorded from an identical oocyte and shown after subtracting data obtained in the absence of ATP. (E) Iactivation/Isteady ratio from an identical oocyte after different prepulse potentials and [ATP]. Data points obtained at the same [ATP] are connected by lines.
	Figure 2. Macroscopic current recordings through P2X2 evoked by step pulses during the steady state after the application of various [ATP]. Macroscopic currents through WT P2X2 evoked by step pulses in the presence of various [ATP]. The holding potential was −40 mV. Step pulses from +60 to −160 mV were applied in 20-mV decrements. Tail currents were recorded at −60mV, and their enlarged images are shown in the insets. The pulse protocol is indicated at the bottom. These current traces were recorded from an identical oocyte and shown after subtracting data obtained in the absence of ATP.
	Figure 3. Analyses of the voltage-dependent gating of P2X2 in the presence of various [ATP]. (A) Dependence of the activation kinetics on voltage and [ATP]. The activation phases of the currents shown in Fig. 2 were fitted with a single exponential function, and the time constants of the fittings at each membrane potential are plotted. (B) [ATP] response relationships are derived from the recordings in Fig. 2. Current amplitudes were measured at the test pulse. Data were fitted with Hill's equation as described in Materials and methods. (C) [ATP] response relationships in B were normalized and replotted. (D) Voltage dependency of EC50 values of the [ATP] response. Representative plots (filled circles) are derived from the data in B and C; the others (open circles) are from other oocytes. (E) Normalized G-V relationships are derived from the recording in Fig. 2. Tail current amplitudes at −60 mV were measured. Data were fitted with the two-state Boltzmann equation as described Materials and methods. (F and G) V1/2 values (F) and Z values (G) in various [ATP]. Representative plots (filled circles) were derived from the data in E; the others (open circles) are from other oocytes.
	Figure 4. Excised inside-out patch clamp recordings of the macroscopic current through P2X2 WT expressed in HEK293T cells with intensive perfusion of the bath (intracellular side) solution. (A) Macroscopic currents were recorded by inside-out patch clamp from HEK293T cell using the same voltage step protocols in Fig. 2 in the presence of the indicated [ATP]. Current traces recorded at 1 min (left) after excising the patch membrane and at 15 min (right) after an intensive perfusion by the blocker-free solution described in Materials and methods. (B) Dependence of the activation kinetics on voltage and [ATP].
	Figure 5. Kinetic analysis with a simple two-state model. (A) Two-state model for P2X channel gating. C represents a closed state, and O represents an open state. α and β represent the transition rates between the closed and open states. (B) Representative α and β at various [ATP] were plotted versus membrane potentials. α and β were calculated from the data in Figs. 2 and 3 using two equations: G/Gmax = β/(α + β) and τ = 1/(α + β). (C) α and β in B were replotted versus [ATP].
	Figure 6. Macroscopic current recordings through the G344A and G344P mutants evoked by step pulses during the steady state after the application of various [ATP]. Macroscopic currents were recorded as in Fig. 2 in the presence of the indicated [ATP]. Tail currents were recorded at −60 mV, and their enlarged images are shown in the insets. All current traces for each mutant were recorded from an identical oocyte and shown after subtracting data obtained in the absence of ATP.
	Figure 7. Analyses of the voltage-dependent gating of P2X2 mutants in the presence of various [ATP]. The data from the mutants were analyzed as in Fig. 2, and representative plots were derived from the data in Fig. 6. (A) Analyses of the activation kinetics of G344P at various [ATP]. (B) Normalized G-V relationships for the macroscopic currents through G344P. (C and D) V1/2 (C) and Z (D) values for G344P at various [ATP]. (E) Calculated α and β values for G344P in the presence of 300 μM ATP were plotted versus membrane potential. The data from the WT channel in 300 μM ATP were also plotted (gray symbols) for comparison. (F) Comparison of the EC50 values of the [ATP] response relationship for the WT channel and the mutants.
	Figure 8. Glycine scanning mutagenesis of the second TM helix of the G344A mutant. Amino acid sequence of the second TM helix of the P2X2 channel. Macroscopic currents were recorded as in Fig. 2. The pulse protocol used is shown at the bottom left. Current traces recorded in the presence of 10 μM ATP are shown at positions corresponding to the introduced glycine in the TM helix. Current traces are shown after subtracting data obtained in the absence of ATP. In the drawing of the helix, the positions of the amino acid residues that when mutated to glycine rescued the voltage-dependent activation phases are highlighted.
	Figure 9. Representative recordings and analyses of two glycine scanning mutants. (A and B) Macroscopic current recordings from G344A/I341G (A) and G344A/V343G (B) in the presence of two different [ATP]. G-V relationships for the two mutants at various [ATP] are shown at the bottom. The recordings and the data analyses were performed as in Fig. 2. (C) EC50 values of the [ATP] response relationship for the mutants. N.D., channels that showed no detectable current.
	Figure 10. Three-state, two-transition model of voltage- and [ATP]-dependent gating. (A) Simple three-state model consisting of an ATP-binding step and a gating step. C, the closed state with no bound ATP; CA, the closed state after ATP is bound; OA, the open state. kbind and kunbind represent the binding/unbinding rates for ATP binding to the channel. kon and koff represent the rates of the gating step. (B) kon and koff were calculated as described in Materials and methods from the data in Fig. 5 (B and C). Membrane potentials are shown in the figure.
	Figure 11. 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.

References [+] :

Barrera, Atomic force microscopy imaging demonstrates that P2X2 receptors are trimers but that P2X6 receptor subunits do not oligomerize. 2005, Pubmed