Recombinant Kv channels at the membrane of Escherichia coli bind specifically agitoxin2.
Potassium voltage-gated channels (Kv) are considered as molecular targets in a number of serious neuronal, immune, and cardiac disorders. Search for efficient low-molecular weight modulators of Kv channel function provides a basis for the development of an appropriate therapy for various Kv-mediated diseases. We report here on a new bacterial cell-based system, which is suitable for study of interactions between ligands and ligand-binding sites of eukaryotic Kv1.3 and Kv1.1 channels. To create this system, high-level expression of KcsA-Kv1.3 and KcsA-Kv1.1 hybrid proteins (ligand-binding sites of Kv1.3 or Kv1.1 fused with prokaryotic KcsA potassium channel) was achieved in the plasma membrane of Escherichia coli. An efficient procedure of E. coli conversion to intact spheroplasts was developed. We demonstrate that fluorescently labeled agitoxin 2 binds specifically to high-affinity and lower-affinity sites of KcsA-Kv1.3 and KcsA-Kv1.1, respectively, at the membrane of spheroplasts. Number of binding sites per cell is estimated to be (1.0 +/- 0.6) x 10(5) and (0.3 +/- 0.2) x 10(5) for KcsA-Kv1.3- and KcsA-Kv1.1-presenting cells, respectively, that allows reliable detection of ligand-receptor interactions by confocal laser scanning microscopy. This bacterial cell-based system is intended for screening of ligands to membrane-embedded pharmaceutical targets.
Nekrasova
Oksana V
OV
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia. genchem@ibch.ru
Ignatova
Anastasia A
AA
Nazarova
Anna I
AI
Feofanov
Alexey V
AV
Korolkova
Yuliya V
YV
Boldyreva
Elena F
EF
Tagvei
Anna I
AI
Grishin
Eugene V
EV
Arseniev
Alexander S
AS
Kirpichnikov
Mikhail P
MP
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism*; D018613:Microscopy, Confocal; D010957:Plasmids / Q000235:genetics; D024642:Potassium Channels, Voltage-Gated / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012604:Scorpion Venoms / Q000378:metabolism*; D013104:Spheroplasts / Q000378:metabolism; D048170:Streptomyces lividans / Q000235:genetics
D017931:DNA Primers; D004269:DNA, Bacterial; D024642:Potassium Channels, Voltage-Gated; D012604:Scorpion Venoms; C087603:agitoxin 2