In vivo monitoring of the potassium channel KcsA in Streptomyces lividans hyphae using immuno-electron microscopy and energy-filtering transmission electron microscopy.
The previous discovery of the Streptomyces lividans kcsA gene and its overexpression followed by the functional reconstitution of the purified gene product has resulted in new strategies to explore this channel protein in vitro. KcsA has evolved as a general model to investigate the structure/function relationship of ion channel proteins. Using specific antibodies raised against a domain of KcsA lacking membrane-spanning regions, KcsA has now been localized within numerous separated clusters between the outer face of the cytoplasm and the cell envelope in substrate hyphae of the S. lividans wild-type strain but not in a designed chromosomal disruption mutant DeltaK, lacking a functional kcsA gene. Previous findings had revealed that caesium ions led to a block of KcsA channel activity within S. lividans protoplasts fused to giant vesicles. As caesium can be scored by electron energy loss spectroscopy better than potassium, this technique was applied to hyphae that had been briefly exposed to caesium instead of potassium ions. Caesium was found preferentially at the cell envelope. Compared to the DeltaK mutant, the relative level of caesium was approximately 30 % enhanced in the wild-type. This is attributed to the presence of KcsA channels. Additional visualization by electron spectroscopic imaging supported this conclusion. The data presented are believed to represent the first demonstration of in vivo monitoring of KcsA in its original host.
Hegermann
Jan
J
FB Biologie/Chemie, Universität Osnabrück, Barbarastr. 11, D-49069 Osnabrück, Germany.
Overbeck
Jens
J
FB Biologie/Chemie, Universität Osnabrück, Barbarastr. 11, D-49069 Osnabrück, Germany.
Schrempf
Hildgund
H
FB Biologie/Chemie, Universität Osnabrück, Barbarastr. 11, D-49069 Osnabrück, Germany.
D002586:Cesium / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D025301:Hyphae / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D046032:Microscopy, Energy-Filtering Transmission Electron; D016253:Microscopy, Immunoelectron; D009154:Mutation; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D048170:Streptomyces lividans / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure*
D015221:Potassium Channels; D002586:Cesium