Two electrode voltage-clamp recordings in Xenopus laevis oocytes

For this approach X. laevis oocytes were injected with P2X2 cRNA alone or with a mixture of cRNAs encoding for P2X2 and Fe65, and the ATP-elicited currents were compared in both cases. Essentially, the same recordings as the ones performed for HEK293 cells were performed on oocytes.

Sensivity to ATP, concentration-response curves.

Since we changed the studied system from HEK293 cells to X. laevis oocytes, we had first to verify the similar dependence on ATP concentration for the P2X2 receptor, both in the presence and absence of Fe65.

In line with what was observed in HEK293 cells, the currents obtained for P2X2 or P2X2-Fe65 receptors in oocytes, with the two electrode voltage-clamp technique, showed comparable basic properties. They presented similar current amplitude (Table 4.2), kinetics (Figure 4.22A) and ATP EC50 values: 10.2 ± 0.3 and 15.3 ± 1.2 µM with Hill slopes of 1.9 ± 0.1 and 1.9 ± 0.3, for P2X2 and P2X2-Fe65, respectively (n=3-15), (Figure 4.22B). Under our experimental set up, it is expected that a change in the EC50 would have to be close to one order of magnitude to be considered as significant.

Figure 4.22. Fe65 does not substantially change the current kinetics or the ATP sensitivity of P2X2 receptors. Two electrode voltage clamps on X. laevis oocytes expressing P2X₂ (black) or P2X₂ and Fe65 (red). (A) currents evoked by 10 µM ATP on oocytes expressing P2X₂ or P2X₂-Fe65 receptors. No apparent changes in current kinetics were observed upon co-expression. (B) concentration-response curves for ATP in oocytes expressing P2X2 or P2X2-Fe65 receptors.

Concentration-response curves were fitted to the data using IgorPro software. Values obtained for the EC₅₀s were 10.2 ± 0.3 and 15.3 ± 1.2 µM for P2X₂ and P2X₂-Fe65, respectively. Data are mean ± S.E.M. of 4 to 15 experiments from 2 different batches of oocytes.

Current-voltage relationship.

We performed current-voltage relation measurements with extracellular Na⁺ solutions for P2X2 and P2X2-Fe65 receptors expressed in X. laevis oocytes. As mentioned before, P2X2

receptors display a time- and activation-dependent increase in the permeability to bulky organic cations (Khakh et al., 1999; Virginio et al., 1999), and measurements of fluorescence resonance energy transfer (FRET) have revealed that restriction of cytosolic channel motions are associated with the impairment of pore dilation. This is a plausible scenario to investigate physiological consequences for the binding event of Fe65 to the C-terminus of P2X2.

Therefore, we measured selectivity changes in oocytes expressing P2X2 alone or in the presence of Fe65. We observed the shifts in the reversal potential of currents (Erev) recorded during prolonged exposure to ATP, in an extracellular solution containing the organic cation NMDG⁺ as sole permeant ion. A voltage ramp protocol consisting of four segments, 50 ms at -60mV, 50 ms at -90 mV, 250 ms ramp from -90 to 0 mV, 125 ms at -90 mV, was applied (Figure 4.23G). The first segment was used to determine the time course of

we observed a time-dependent shift in the reversal potential of the current as previously described for P2X2 receptors expressed in Xenopus oocytes (Eickhorst et al., 2002). This change was drastically reduced in oocytes expressing P2X2-Fe65 receptors (Figure 4.23B and D). Thus, the mean reversal potential (Erev) of the current at 1 second (I1) and 30 seconds (I2) after the start of ATP application changed from -72.2 ± 0.6 mV (at I1) to -56.5 ± 0.9 mV (at I2; n=47) for P2X2 receptors and from -74.7 ± 0.5 mV (at I1) to -67.8 ± 0.6 mV (at I2, n=48) for P2X2-Fe65 receptors (Table 4.2). The corresponding PNMDG/PNa permeability ratios changed from 0.057 ± 0.001 (at I1) to 0.107 ± 0.004 (at I2; n=47) and from 0.052 ± 0.001 (at I1) to 0.068 ± 0.002 (at I2, n=48) for P2X2 and P2X2-Fe65 receptors, respectively.

Thus a notable decrease in the permeation to NMDG⁺ in P2X2-Fe65 receptors with respect to P2X2 receptors is observed. The time course of the changes in Erev upon ATP application for the oocytes expressing P2X2 or P2X2-Fe65 receptors are depicted in figure 4.23B and D is shown in figure 4.23E.

These measurements show the first drastic physiological difference for P2X2 receptors in the presence and absence of Fe65. Importantly, these results were repeated in all of four batches of oocytes tested, supporting the reproducibility of the observed effect. In addition, P2X2 and P2X2-Fe65 receptors presented similar current amplitudes (Table 4.2), ruling out differences in the expression level as a confounding factor on the analysis. Representative currents to supramaximal concentrations of ATP (100 µM) in NMDG⁺ solution at a holding potential of -60 mV for P2X2 and P2X2-Fe65 receptors are shown in figure 4.23A and C, respectively.

Figure 4.23. The interaction with Fe65 hinders the time- and activation-dependent change in pore selectivity presented by P2X2 receptors. Two electrode voltage clamps on X. laevis oocytes expressing P2X₂ (black) or P2X₂ and Fe65 (red). (A) steady state current obtained upon activation of oocytes expressing the P2X₂ receptor with 100 µM ATP at a holding potential of -60 mV in NMDG⁺ containing solutions. (B) ATP was applied during 30 second-ramps (-90 to 0 mV; 250 ms), in NMDG⁺ extracellular solution. Time points 1 and 2 represent the current-voltage relationships taken after 1 and 30 seconds of ATP application, respectively. (C-D) same data as in (A-B) recorded for oocytes co-injected with P2X₂ and Fe65. (E) time-dependence of the E_rev shift for oocytes presented in (B) and (D), respectively. (F) correlation between the expression level of P2X₂ receptors and the permeability ratio at 30 seconds after the beginning of ATP application, for oocytes injected with P2X₂ cRNA alone (Black; n=34) or with P2X₂+Fe65 cRNA (red; n=41). (G) Voltage ramp protocol, it consists of four segments, 50 ms at -60mV, 50 ms at -90 mV, 250 ms ramp from -90 to 0 mV, 125 ms at -90. The squares represent the mean ± S.E.M values of permeability versus current.

In order to determine the specificity of the regulation of P2X2 receptor function by Fe65, we performed the same functional experiments by co-expressing Fe65 and P2X7

receptor subunits. Similarly to P2X2 receptors, P2X7 receptors undergo changes in permeability upon exposure to agonist (Surprenant et al., 1996; Khakh et al., 1999).

However, P2X7 subunits do not seem to interact with Fe65 as we observed by Y2H and by co-immunoprecipitation experiments (Figure 4.5 and 4.12-4.13A, respectively).

By repeatedly applying ramp pulses (Figure 4.23G), we observed a time-dependent shift in the reversal potential of P2X7 receptor expressed in Xenopus oocytes. Nevertheless, and in contrast to what we observed for P2X2 receptors, the changes were of the same magnitude in oocytes expressing P2X7-Fe65 receptors. Thus, the mean reversal potential (Erev) of the current at 1 second (I1) and 45 seconds (I2) after start of ATP application changed from -68.4

± 0.6 mV (at I1) to -48,1 ± 0.9 mV (at I2; n=25) and from -69.1 ± 0.7 mV (at I1) to -50.9 ± 1.4

Table 4.2. P2X2 receptors show time- and activation-dependent changes in ionic selectivity that are hindered upon coexpression with Fe65. Overall results obtained by two electrode voltage-clamp technique on P2X₂ and P2X₇ receptors after heterologous expression in Xenopus oocytes. Reversal potential (E_rev) was measured and P_NMDG/P_Na was calculated 1 second (I₁) and 30 (or 45) seconds (I₂) after the beginning of 100 µM (or 1 mM) ATP application in an extracellular solution containing NMDG⁺ as the sole permeant ion. I_Na refers to the current obtained when the extracellular solution contained Na⁺ as the sole permeant ion. The number of oocytes measured is represented by n.

The corresponding PNMDG/PNa permeability ratios changed from 0.065 ± 0.002 (at I1) to 0.141 ± 0.007 (at I2; n=25) and from 0.065 ± 0.002 (at I1) to 0.148 ± 0.006 (at I2, n=22) for P2X7 and P2X7-Fe65 receptors, respectively. Also, no significant changes were observed in current amplitude or in reversal potential when Na⁺ was the main permeant ion (Table 4.2).

These data reveal that the interaction of Fe65 leads to a change in the function of the P2X2 receptor, and underline the importance of the C-terminal domain for the regulation of permeability changes on P2X2 receptors. Due to the co-localization of P2X2 and Fe65 in the post-synaptic specialization, these findings represent indeed a novel mechanism by which anchoring proteins in the cytoplasmic periphery of the neuronal membrane could control the function of synaptic receptors.