Validation of XE991 use as a Selective Irreversible Blocker of Native Kv7 Channels in Smooth Muscle Myocytes

Abstract

A concern was recently raised about the selectivity of XE991 and its use as a Kv7 channel blocker in single cell electrophysiology and in vitro functional assays. In the present study, we evaluated the effects of XE991 on different components of the endogenous delayed rectifier potassium currents recorded in freshly isolated guinea pig airway myocytes using the perforated-patch clamp technique and a 5 s voltage step protocol. Application of XE991 (10 μM) reversibly inhibited a transient component of the total current by 27% at voltages positive to −4 mV, whereas it irreversibly inhibited 71% of sustained non-inactivating currents at membrane voltages positive to −30 mV. Comparison of the activation properties of both components (reversibly inhibited transient component and irreversibly inhibited sustained component) revealed voltages of the half activation (V0.5) of +6 mV for the transient component and −31 mV for the irreversibly inhibited sustained current component. These values are in good agreement with the V0.5 values reported for cloned Kv1/Kv2 family channels and cloned Kv7 family channels, respectively. Endogenous Kv7 currents measured in smooth muscle cells of different origins also have V0.5 values ∼−30 mV and resting membrane voltages are typically −60 to −45 mV. Our findings suggest that only the sustained component of the total delayed rectifier potassium current that is irreversibly blocked by XE991 can be attributed to Kv7 current. In functional in vitro models, such as pressurized arteries or lung slices, irreversible contraction of the smooth muscle cells by XE991, which is associated with membrane depolarization and activation of voltage gated calcium channels, can be attributed to the inhibition of Kv7 channels.