Drug- and mutagenesis-induced changes in the selectivity filter of a cardiac two-pore background K+ channel

As compared with voltage-gated K(+) channels (Kv-type), our knowledge of the structure-function and pharmacology of two-pore background K(+) channels is still very limited. Here we have used a drug- and mutagenesis-based approach to study the effect of the antidepressant fluoxetine (FL) and analgesic D-norpropoxyphene (NORP) on the cardiac two-pore background K(+) channel.

Whole-cell currents of the cTBAK-1 channel expressed in Xenopus laevis oocytes were investigated using conventional two-microelectrode voltage-clamp recording method combined with functional mutagenesis of the channel protein.

Both drugs inhibit cTBAK-1 current: FL proved to be a voltage-dependent pore-blocker, while NORP induced a change in the selectivity of cTBAK-1 giving rise to a shift in the reversal potential (E(rev)) toward more positive voltages due to an increased Na(+) permeability. Mutations were introduced into the selectivity filter of the first (Y105F) and the second (F211Y) pore to mimic the P-region of HERG (GFGN) and Kv1.1 (GYGD) channels. Point mutations in the channel resulted in two distinct phenotypes of cTBAK-1: the mutant Y105F channel lost its selectivity and was unaffected by NORP, in contrast to the F211Y mutant.

FL and NORP block the current of cTBAK-1 channels differently, the latter modified the selectivity of the channel pore. Our mutagenesis study revealed that NORP interacts with the selectivity filter of cTBAK-1. The significant role of the GYGD motif in this type of K(+) channels is emphasized.