P2X1 and P2X5 subunits form the functional P2X receptor in mouse cortical astrocytes

Abstract

ATP plays an important role in signal transduction between neuronal and glial circuits and within glial networks. Here we describe currents activated by ATP in astrocytes acutely isolated from cortical brain slices by non-enzymatic mechanical dissociation. Brain slices were prepared from transgenic mice that express enhanced green fluorescent protein under the control of the human glial fibrillary acidic protein promoter. Astrocytes were studied by whole-cell voltage clamp. Exogenous ATP evoked inward currents in 75 of 81 astrocytes. In the majority ( approximately 65%) of cells, ATP-induced responses comprising a fast and delayed component; in the remaining subpopulation of astrocytes, ATP triggered a smoother response with rapid peak and slowly decaying plateau phase. The fast component of the response was sensitive to low concentrations of ATP (with EC(50) of approximately 40 nm). All ATP-induced currents were blocked by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS); they were insensitive to ivermectin. Quantitative real-time PCR demonstrated strong expression of P2X(1) and P2X(5) receptor subunits and some expression of P2X(2) subunit mRNAs. The main properties of the ATP-induced response in cortical astrocytes (high sensitivity to ATP, biphasic kinetics, and sensitivity to PPADS) were very similar to those reported for P2X(1/5) heteromeric receptors studied previously in heterologous expression systems.

Figure 1.

Kinetics and concentration dependences of ATP-induced currents in cortical astrocytes. A, Examples of two types of ATP-induced currents, recorded from acutely isolated astrocytes. The left shows a type 1 response, observed in the majority of cells, and the right shows a type 2 response expressed in approximately one-third of all cells tested. B, ATP currents evoked by repetitive applications of the agonist show no apparent desensitization. Current traces have a complex kinetics; the peak of the response, the steady-state component, and the rebound inward current recorded during ATP washout are indicated on the graph. C, Increase in the extracellular Ca²⁺ concentration from 2.5 to 10 mm does not affect the ATP-induced currents. All recordings were made at a holding potential of −80 mV.

Figure 2.

Concentration dependence of ATP-induced currents in cortical astrocytes. A, Concentration dependence of peak and steady-state components of the type 1 ATP-induced response. Membrane currents recorded from a single cell in response to different ATP concentrations are shown on the top. The bottom shows the concentration–response curves constructed from nine similar experiments; current amplitudes were measured at the initial peak and at the end of the current, as indicated on the graph. B, Concentration dependence of the type 2 ATP-induced response. The family of membrane currents evoked by various ATP concentrations is shown on the top. The concentration–response curves constructed for the peak and steady-state of the ATP-induced currents (as indicated on the graph) recorded from eight cells are represented in the bottom. C, Membrane currents recorded from single astrocyte in response to different concentrations of αβmeATP are represented on the top; bottom shows the concentration–response curves for peak and steady-state currents; data were pooled from six experiments. All recordings were made at a holding potential of −80 mV.

Figure 3.

Voltage dependence of the ATP-induced currents. A and B show the voltage dependence of the type 1 (A) and type 2 (B) ATP-induced currents. The left panels demonstrate families of ion currents evoked by ATP at holding potentials varying between −80 and 40 mV. Current–voltage plots for ATP-induced currents are shown on the right (A, n = 6; B, n = 7). The amplitudes of currents were normalized to the value measured at −80 mV.

Figure 4.

Inhibition of ATP-induced currents by PPADS. A, PPADS sensitivity of type 1 ATP-induced responses. The left panel demonstrates the family of currents triggered by 3 μm ATP in control conditions and in the presence of various concentrations of PPADS, as indicated. Membrane currents are shown at two different timescales. The right panel shows the concentration dependence of the block for nine cells; the peak component of the response is considerably more sensitive to PPADS. B, PPADS sensitivity of type 2 ATP-induced responses. Currents recorded at various concentrations of PPADS are shown on the left, and the concentration dependence of inhibition for peak and steady-state components constructed for seven individual experiments is presented on the right. Similarly to currents shown in A, the peak component of the response was more sensitive to PPADS. Application of PPADS started 2 min before application of ATP. All recordings were made at a holding potential of −80 mV.

Figure 5.

Inhibition of ATP-induced currents by TNP-ATP. A, ATP-induced current measured for the cortical astrocytes in control conditions, in the presence of TNP-ATP, and after the washout. B, Mean data showing the percentage of ATP-induced current inhibition at three different concentrations of TNP-ATP; the number of experiments is shown on the graph. Application of TNP-ATP started 2 min before application of ATP. All recordings were made at a holding potential of −80 mV.

Figure 6.

Ivermectin does not affect ATP-induced currents. Type 1 (top) and type 2 ATP-induced responses recorded in control conditions and in the presence of the positive modulator of P2X₄ receptor ivermectin. The holding potential was −80 mV.

Figure 7.

Quantitative real-time PCR analysis to identify mRNAs for P2X receptor subunits and their relative expression levels. Samples of mRNA were prepared from total mouse cortex (a) or from FACS-sorted cortical astrocytes (b) of postnatal mice (P6) and were subjected to qRT- PCR. Data represent mRNA levels relative to ATP5B and are given in means ± SEM.