Verapamil block of large-conductance Ca-activated K channels in rat aorticmyocytes

The effects of verapamil on the large conductance Ca-activated K (BK) chann el from rat aortic smooth muscle cells were examined at the single channel level. Micromolar concentrations of verapamil produced a reversible flicker ing block of the BK channel activity. Kinetic analysis showed that verapami l decreased markedly the time constants of the open states, without any sig nificant change in the rime constants of the closed states. The appearance of an additional closed state specifically, a nonconducting, open-blocked s tate - was also observed, whose time constant would reflect the mean reside nce time of verapamil on the channel. These observations are indicative of a state-dependent, open channel block mechanism. Dedicated kinetic (group) analysis confirmed the state-dependent block exerted by verapamil, D600 (ga llopamil), the methoxy derivative of verapamil, was also tested and found t o exert a similar type of block, but with a higher affinity than verapamil, The permanently charged and membrane impermeant verapamil analogue D890 wa s used to address other important features of verapamil block, such as the sidedness of action and the location of the binding site on the channel pro tein. D890 induced a flickering block of BK channels similar to that observ ed with verapamil only when applied to the internal side of the membrane, i ndicating that D890 binds to a site accessible from the cytoplasmic side. F inally, the voltage dependence of D890 block was assessed. The experimental data fitted with a Langmuir equation incorporating the Woodhull model for charged blockers confirms that the D890-binding site is accessed from the i nternal mouth of the BK channel, and locates it approximately 40% of the me mbrane voltage drop along the permeation pathway.