Abstract

1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) is widely used to inhibit phospholipase C (PLC)-mediated signaling, but we and others have also reported a PLC-independent block of Kir3 channels in native cells. To elaborate on this major side effect, we examined the action of U73122 and 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrollidinedione (U73343), a structurally related but not PLC-inhibiting analog, on Kir1.1, Kir2.1, or Kir3.1/3.2 channels expressed in HEK293 cells. Both compounds (10 μM) displayed an unusual degree of selectivity for Kir3, superior even to that of tertiapin, which discriminates between Kir3 and Kir2 but also inhibits Kir1.1. Recordings from mutant Kir2 and Kir3 channels showed that U73122 is unlikely to block Kir3 by interfering with binding of phosphatidylinositol 4,5-bisphosphate, and U73122 did not seem to act like a pore blocker. U73122 and U73343 also unexpectedly suppressed Ca²⁺-activated K⁺ channels of the large-conductance type (MaxiK, BK) in a PLC-independent fashion. In single-channel recordings, both compounds significantly decreased open probability of BK channels and slowed their ultrafast gating (“flickering”) at very depolarized potentials. Alignment of the amino acid sequences of Kir3 and BK channels suggested that the highly selective effect of U73122/U73343 is mediated by a homologous domain within the long C-terminal ends. In fact, mutations in the C-terminal region of Kir2 and Kir3 channels significantly altered their sensitivity to the two compounds. Our data strongly caution against the use of U73122 when exploring signaling pathways involving Kir3 and BK channels. However, the apparent binding of U73122/U73343 to a common structural motif might be exploited to develop drugs selectively targeting Kir3 and BK channels.