Allosteric control of gating and kinetics at P2X(4) receptor channels

Abstract

The CNS abundantly expresses P2X receptor channels for ATP; of these the most widespread in the brain is the P2X(4) channel. We show that ivermectin (IVM) is a specific positive allosteric effector of heterologously expressed P2X(4) and possibly of heteromeric P2X(4)/P2X(6) channels, but not of P2X(2), P2X(3), P2X(2)/P2X(3,) or P2X(7) channels. In the submicromolar range (EC(50,) approximately 250 nM) the action of IVM was rapid and reversible, resulting in increased amplitude and slowed deactivation of P2X(4) channel currents evoked by ATP. IVM also markedly increased the potency of ATP and that of the normally low-potency agonist alpha, beta-methylene-ATP in a use- and voltage-independent manner without changing the ion selectivity of P2X(4) channels. Therefore, IVM evokes a potent pharmacological gain-of-function phenotype that is specific for P2X(4) channels. We also tested whether IVM could modulate endogenously expressed P2X channels in the adult trigeminal mesencephalic nucleus and hippocampal CA1 neurons. Surprisingly, IVM produced no significant effect on the fast ATP-evoked inward currents in either type of neuron, despite the fact that IVM modulated P2X(4) channels heterologously expressed in embryonic hippocampal neurons. These results suggest that homomeric P2X(4) channels are not the primary subtype of P2X receptor in the adult trigeminal mesencephalic nucleus and in hippocampal CA1 neurons.

Fig. 1.

IVM potentiates P2X₄ but not P2X₂ or P2X₃ channel currents.A, Representative recordings of currents mediated by P2X₄ channels expressed in oocytes. Left trace, 100 μm ATP-evoked current before and after (right trace) addition of IVM (10 μm); IVM potentiates the amplitude as well as duration of the ATP-evoked current. B, Representative recordings for P2X₂ channels expressed in oocytes. Left trace, 100 μm ATP-evoked current before and after (right trace) addition of IVM (10 μm); IVM causes no change in either the holding current or ATP-evoked current in P2X₂-expressing cells. C, Summary of data from a number of cells (n > 5 for each) showing that IVM (10 μm) potentiates ATP-evoked currents at P2X₄ channels but not at P2X₂, P2X₃, and P2X₂/P2X₃channels. D, Summary of data from a number of cells (n > 5 for each) showing that suramin (30 μm) can block ATP-evoked currents at P2X₂, P2X₃, and P2X₂/P2X₃ channels but not at P2X₄ channels. Subsequent figures show a more pronounced potentiation of P2X₄ channels at lower IVM and/or ATP concentrations. The data are derived from measurements of peak ATP-evoked currents.

Fig. 2.

Properties of the IVM potentiation of P2X₄ channel currents. A, Time course of potentiation by IVM. IVM was added at t = 0, just after the first puff of ATP (3 μm). Potentiation by IVM occurs within 15 sec (time of the second ATP pulse) and is maximal by 30 sec. B, The oocyte was exposed once to ATP (3 μm) in the absence of IVM (t = −5 min). Fifteen seconds later, IVM was applied for 5 min. Att = 0, a second ATP application evoked a greater than fourfold larger ATP response. Thus the ATP-evoked current is potentiated in the absence of ATP, showing that IVM action is use-independent. After washout of IVM, the ATP-evoked current returns to baseline levels within 10–20 min. For A andB the period of IVM application is shown by thehorizontal bar, and the bold line is an exponential fit to the data. C, Concentration dependence of IVM action, tested with pulses of 1 μm ATP. IVM causes significant potentiation at concentrations ≥0.1 μm. The superimposed heavy line represents a fitted curve (Hill equation) with an EC₅₀ of 257 nm and a Hill coefficient of 1; note, however, that the IVM potentiation decreases at 10 μm. D. Concentration–effect relationship for ATP in the absence (●) and presence (○) of 3 μm IVM. IVM both increases the maximal current and decreases the EC₅₀ for ATP. E, Leak-subtracted current–voltage relationships for the ATP-evoked current in the absence (●) and presence (○) of 3 μmIVM; note that there is no shift in the reversal potential.F, Data from experiments like that shown inD. The ratio of the IVM potentiated current to that of the ATP-evoked current alone is plotted versus the membrane voltage. IVM can potentiate the ATP-evoked current at all membrane voltages tested; in addition, the potentiation is slightly greater at positive voltages. For this figure the data are plotted as mean ± SEM.

Fig. 3.

IVM affects P2X₄ deactivation kinetics but neither basal current nor membrane capacitance. A, Representative recording showing that IVM produces a large increase in P2X₄ channel current evoked by 3 μm ATP, although IVM produces no change in the holding current.B–D, Bar graphs showing summary of ATP-evoked current properties from cells recorded in the experiment shown in A. B, peak response; C, deactivation half-time;D, cell capacitance.

Fig. 4.

Effect of α,β-methylene-ATP on homomeric P2X₄ and heteromeric P2X₄/P2X₆ channels. A, P2X₄ channels: concentration–effect curves for α,β-methylene-ATP in the absence and presence of 3 μmIVM. Right graphs, Same data on an expanded current scale. B, Co-expression of P2X₄/P2X₆ channels: concentration–effect curves for α,β-methylene-ATP in the absence and presence of 3 μm IVM. Right graphs, Same data on an expanded current scale. Theasterisks indicate the threshold concentration for α,β-methylene-ATP (see Table 1).

Fig. 5.

IVM potentiates the high-selectivity (I₁) but not the low-selectivity (I₂) state of the P2X₄ channel.A, Representative 3 μm ATP-evoked current from an oocyte expressing P2X₄ channels; the solid bar indicates the period of ATP application. B, Representative recording of a 3 μm ATP-evoked current during exposure to IVM (3 μm). C, Bar graph summarizing data from experiments such as those illustrated inA and B from seven cells; onlyI₁ is potentiated. D, Table showing reversal potentials for control and IVM-potentiatedI₁ in both Na⁺ and NMDG⁺ solutions, as well as the ratio of NMDG⁺ to Na⁺ permeability (n = 5). The NMDG⁺ reversal potential for I₂ is typically −30 mV (Khakh et al., 1999).

Fig. 6.

IVM potentiates P2X₄ channel currents in a clonal mammalian cell line. Top panel, Amplitudes of ATP (10 μm)-evoked currents from a single HEK 293 cell; ATP was applied every 2 min and IVM (3 μm) was added for the period indicated. The sample traces at theright were elicited from this cell at the time points indicated. Bottom panel, Averaged data from four such cells tested with the same protocol.

Fig. 7.

Endogenous P2X channels in MNV and hippocampal CA1 neurons. A, Bright-field image of a brainstem slice containing the MNV. MNV neurons can be seen as round neurons. Scale bar, 10 μm. B, Image of the CA1 region in a hippocampal slice. CA1 pyramidal neurons can be seen extending a dendrite into the stratum radiatum. Scale bar, 10 μm.C, Representative ATP-evoked currents from an MNV neuron. D, Representative traces of ATP-evoked currents from a CA1 neuron. In both cases recordings in the presence of 2–5 μm IVM are also shown. The bar graphrepresents average values for MNV neurons (n = 3), whereas the scatter graph shows data for nonresponding CA1 neurons (○), the three responding neurons (▪;arrows), and the average for all 13 neurons (●).

Fig. 8.

P2X₄ channels expressed in embryonic hippocampal neurons. A, B, Bright-field (A) and fluorescence (B) image of embryonic hippocampal neurons transfected with plasmids for P2X₄ and EGFP. Note that the morphology of transfected and nontransected neurons is very similar, and this appearance is typical of healthy cells. The extended fluorescent cells in Bare glial cells. C, Top trace,Representative voltage-clamp currents recorded from an untransfected neuron showing no ATP-evoked currents (holding potential, −60 mV);bottom trace, large ATP-evoked currents in a transfected hippocampal neuron. D, Top trace,Representative current-clamp recording (resting potential, −52 mV) from an untransfected neuron showing no ATP-evoked change in membrane potential; bottom trace, large ATP-evoked depolarization in a transfected hippocampal neuron. Action potentials have been clipped. E, Representative traces from a transfected hippocampal neuron showing 10 μm ATP-evoked currents before and after 3 μm IVM application. F, Summary of experiments such as those illustrated in Efrom six neurons. Note that the size of the ATP-evoked current varies markedly between neurons, but in all neurons IVM potentiates the current.