Evidence for an internal phenylalkylamine action on the voltage-gated potassium channel Kv1.3

We characterized the action of verapamil and N-methyl-verapamil on current through the delayed-rectifier potassium channel Kv1.3 mouse (mKv1.3). The whole-cell and inside-out configuration of the patch-clamp technique was used to examine the channel properties after injection of in vitro transcribed cRNA into rat basophilic leukemia cells. The action of verapamil on current through mKv1.3 channels could be separated into an acceleration of the rate of current decay during depolarizing pulses and a reduction of steady state peak current when applied either extracellularly or intracellularly. Both effects were greatly reduced when the membrane-impermeable N-methyl-verapamil was applied extracellularly, but it affected current through mKv1.3 channels similar to verapamil if applied to the intracellular side of the membrane. Mutations in the outer vestibule of the mKv1.3 channel did not change the ability of verapamil to accelerate the mKv1.3 current decay during depolarizing pulses, whereas the reduction of the steady state peak current by verapamil applied either extracellularly and intracellularly and by N-methyl-verapamil applied intracellularly was decreased approximately 25-fold in all three cases. Substances known to interact with an extracellular site of the channel (e.g., extracellularly applied tetraethylammonium or kaliotoxin) did not compete with extracellularly applied verapamil on blocking steady state peak current, whereas intracellularly applied tetraethylammonium, which is known to interact with an intracellular site of the channel, was able to reduce the effect of extracellularly applied verapamil on blocking steady state peak current, suggesting competition for a common binding site between verapamil and intracellularly applied tetraethylammonium. The results from the competition experiments as well as from the mutations in the outer vestibule of mKv1.3 are compatible with the idea that verapamil applied extracellularly moves through the membrane to reach its internal binding site on the mKv1.3 channel.