Péter Hajdú1, Chris Ulens, György Panyi, Jan Tytgat. 1. Cell Biophysics Research Group of the Hungarian Academy of Sciences, Department of Biophysics and Cell Biology, University of Debrecen, Hungary. hajdup@jaguar.dote.hu
Abstract
OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: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.
Authors: Louise E Kennard; Justin R Chumbley; Kishani M Ranatunga; Stephanie J Armstrong; Emma L Veale; Alistair Mathie Journal: Br J Pharmacol Date: 2005-03 Impact factor: 8.739
Authors: Diego Fernández-Fernández; Alba Cadaveira-Mosquera; Lola Rueda-Ruzafa; Salvador Herrera-Pérez; Emma L Veale; Antonio Reboreda; Alistair Mathie; J Antonio Lamas Journal: PLoS One Date: 2018-06-21 Impact factor: 3.240