Mechanism of Ivermectin Facilitation of Human P2X4 Receptor Channels

Abstract

Ivermectin (IVM), a widely used antiparasitic agent in human and veterinary medicine, was recently shown to augment macroscopic currents through rat P2X4 receptor channels (Khakh, B.S., W.R. Proctor, T.V. Dunwiddie, C. Labarca, and H.A. Lester. 1999. J. Neurosci. 19:7289–7299.). In the present study, the effects of IVM on the human P2X4 (hP2X4) receptor channel stably transfected in HEK293 cells were investigated by recording membrane currents using the patch clamp technique. In whole-cell recordings, IVM (≤10 μM) applied from outside the cell (but not from inside) increased the maximum current activated by ATP, and slowed the rate of current deactivation. These two phenomena likely result from the binding of IVM to separate sites. A higher affinity site (EC50 0.25 μM) 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 μM) slowed the rate of current deactivation, and increased the apparent affinity for ATP. In cell-attached patch recordings, P2X4 receptor channels exhibited complex kinetics, with multiple components in both the open and shut distributions. IVM (0.3 μM) 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 μM) 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.

Mouse Leydig cells express multiple P2X receptor subunits

“…The increase in current supports the idea that P2X 4 subunit is part of the receptor. Furthermore, the current decrease, which was not expected if only P2X 4 subunits were present, indicates that other subunits are involved in the receptor assembly [43]. Conversely, if P2X 4/7 heteromeric receptors were present, we should have observed an increase on the peak currents after 3 min of incubation with 3 μM ivermectin, a fact not seen in our results [4,19].…”

“…The higher-affinity binding site (EC 50 =0.25 µM) is able to increase the maximal ATPactivated currents without changing the ATP concentrationresponse relationship. The action at the lower affinity binding site (EC 50 =2 µM) results in a slower deactivation rate and enhancement of the ATP affinity [43]. Moreover, ivermectin does not act on P2X 2 , P2X 3 , P2X 2/3 , and P2X 7 receptors but does so on heteromeric channels containing the P2X 4 subunit [25,4,19].…”

P2X1and P2X5Subunits Form the Functional P2X Receptor in Mouse Cortical Astrocytes

“…In contrast, the selective positive modulator of P2X 4 receptors, ivermectin (Khakh et al, 1999a;Priel and Silberberg, 2004;Lalo et al, 2007) at 1-10 M, had no effect on ATP-induced currents in acutely isolated astrocytes (Fig. 6).…”

Involvement of P2X receptors in the NAD+-induced rise in [Ca2+]i in human monocytes

“…At present, the use of ivermectin seems to be the most appropriate tool to study P2X 4 receptor activation. Recently, single channel recordings of ATP-evoked currents through human P2X 4 expressed in HEK293 cells suggested that ivermectin increases maximal channel currents after binding to a high affinity site (pEC 50 =6.6) and may also bind to a low affinity site (pEC 50 =5.7) to increase the affinity of ATP by stabilizing the open channel conformation [44]. As ivermectin induced an increase in the NAD + -induced rise in [Ca 2+ ] i , we assume that P2X 4 receptors are involved in the NAD + response.…”