FIGURE 4.

Site-directed mutagenesis of the lateral portals. Panel A shows the location of Glu⁵¹ (red spheres) and Asp³³¹ (blue spheres) in our homology model of the hP2X4R. The left schematic shows the receptor viewed parallel to the membrane. The lateral portal is shown at a higher magnification in the right schematic. Here, the protein, as shown in the left schematic, is slanted 30° toward the viewer to give a view down into the portal entrance. Panel B shows ATP-gated (30 μm) current and fluorescence traces of D331S mutant receptors recorded from two different cells in the presence and absence of IVM (10 μm). In the left traces, the current was evoked in the absence of IVM. In the right traces, the cell was preincubated in IVM for 5 min before re-applying ATP. Currents evoked in the presence of IVM deactivated slower and had reduced Pf%. Average values of Pf% are shown in Panel C. Panel D shows ATP-gated current and fluorescence traces of two different cells expressing the E51Q mutant receptor evoked either in the absence (left traces) and presence (right traces) of IVM. Panel E shows the effect of 3 μm IVM on the kinetics of currents evoked by ATP (3 μm). Current was first tested in the absence of IVM (black). Then IVM was applied for 5 min, and ATP was reapplied to evoke modified current (red).