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


CONCLUSIONS
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.
Hajdú
Péter
P
Cell Biophysics Research Group of the Hungarian Academy of Sciences, Department of Biophysics and Cell Biology, University of Debrecen, Hungary. hajdup@jaguar.dote.hu
Ulens
Chris
C
Panyi
György
G
Tytgat
Jan
J
D020816:Amino Acid Motifs; D000700:Analgesics / Q000494:pharmacology*; D000818:Animals; D000928:Antidepressive Agents / Q000494:pharmacology*; D011431:Dextropropoxyphene / Q000031:analogs & derivatives* / Q000494:pharmacology*; D005260:Female; D005473:Fluoxetine / Q000494:pharmacology*; D007473:Ion Channels / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D024683:Potassium Channels, Tandem Pore Domain / Q000187:drug effects* / Q000235:genetics; D012964:Sodium / Q000378:metabolism; D014158:Transcription, Genetic; D014982:Xenopus laevis
D000700:Analgesics; D000928:Antidepressive Agents; D007473:Ion Channels; D024683:Potassium Channels, Tandem Pore Domain; D005473:Fluoxetine; D012964:Sodium; C002756:norpropoxyphene; D011431:Dextropropoxyphene