External Ba2+block of Kv4.2 channels is enhanced in the closed-inactivated state

The effect of external barium ions on rat Kv4.2 channels expressed in HEK293 cells was investigated using whole cell, voltage-clamp recordings to determine its mechanism of action as well as its usefulness as a tool to probe the permeation pathway. Ba(2+) caused a concentration-dependent inhibition of current that was antagonized by increasing the external concentration of K(+) ([K(+)](o)), and the concentration and time dependence of the inhibition were well fitted by a model involving two binding sites aligned in series. Recovery from current inhibition was enhanced by increasing the intensity, duration, or frequency of depolarizing steps or by increasing [K(+)](o). These properties are consistent with the conclusion that Ba(2+) is a permeant ion that, by virtue of a stable interaction with a deep pore site, is able to block conduction. This blocking action was subsequently exploited to gain insights into the pore configuration in different channel states. In addition to blocking one or more states populated by brief depolarizing pulses to 80 mV, Ba(2+) blocked closed channels [the membrane voltage (V(m)) = -80 mV] and closed-inactivated channels (V(m) = -40 mV). Interestingly, the block of closed-inactivated channels was faster and more complete than for closed channels, which we interpret to mean that conformational changes underlying closed-state inactivation (CSI) enhance Ba(2+) binding and that the outer pore mouth remains patent during CSI. This provides the first direct evidence that an inactivation process involving a constriction of the outer pore mouth does not account for CSI in Kv4.2.