Mechanism of ivermectin facilitation of human P2X4 receptor channels

Ivermectin (IVM), a widely used antiparasitic agent in human and veterinary medicine, was recently shown to augment macroscopic currents through rat P2X(4) receptor channels (Khakh, B.S., WR. Proctor, T. Dunwiddie, C. Labarca, and H.A. Lester. 1999. J. Neurosci. 19:7289-7299.). In the present study, the effects of IVM on the human P2X(4) (hP2X(4)) receptor channel stably transfected in HEK293 cells were investigated by recording membrane currents using the patch clamp technique. In whole-cell recordings, IVM (less than or equal to10 muM) applied front outside the cell (but not from inside) increased the maximum current activated by ATP, and slowed the rate Of CLIF rent deactivation. These two phenomena likely result front the bindin of IVM to separate sites. A higher affinity site (EC50 0.25 muM) increased the maximal current activated by saturating concentrations of ATP without significantly changing the rate of current deactivation or the EC50 and Hill slope of the ATP concentration-response relationship. A lower affinity site (EC50 2 muM) slowed the rate of current deactivation, and increased the apparent affinity for ATP. In cell-attached patch recordings, P2X(4) receptor channels exhibited complex kinetics, with multiple components in both the open and shut distributions. IVM (0.3 muM) increased the number of openings per burst, without significantly changing the mean open or mean shut time within a burst. At higher concentrations (1.5 muM) of IVM, two additional open time components of long duration were observed that gave rise to long-lasting bursts of channel activity Together, the results suggest that the binding of IVM to the higher affinity site increases current amplitude by reducing channel desensitization, whereas the binding of IVM to the lower affinity site slows the deactivation of the current predominantly by stabilizing the open conformation of the channel.