FRET with multiply labeled HERG K(+) channels as a reporter of the in vivo coarse architecture of the cytoplasmic domains.
The intracellular N-terminus of human ether-a-go-go-related gene (HERG) potassium channels constitutes a key determinant of activation and deactivation characteristics and is necessary for hormone-induced modifications of gating properties. However, the general organization of the long amino and carboxy HERG terminals remains unknown. In this study we performed fluorescence resonance energy transfer (FRET) microscopy with a library of fluorescent HERG fusion proteins obtained combining site-directed and transposon-based random insertion of GFP variants into multiple sites of HERG. Determinations of FRET efficiencies with functional HERG channels labeled in different combinations localize the fluorophores, introduced in the amino and carboxy ends, in two quadratic planes of 7.8 and 8.6 nm lateral size, showing a vertical separation of nearly 8 nm without major angular torsion between the planes. Similar analysis using labels at positions 345 and 905 of the amino and carboxy terminals, located them slightly above the planes delimited by the amino and carboxy end labels, respectively. Our data also indicate an almost vertical arrangement of the fluorophores introduced in the NH(2) and COOH ends and at position 905, but a near 45 degrees angular rotation between the planes delimited by these labels and the 345-located fluorophores. Systematic triangulation using interfluorophore distances coming from multiply labeled channels provides an initial constraint on the overall in vivo arrangement of the HERG cytoplasmic domains, suggesting that the C-linker/CNBD region of HERG hangs centrally below the transmembrane core, with the initial portion of the amino terminus around its top and side surfaces directed towards the gating machinery.
Miranda
Pablo
P
Departamento de Bioquímica y Biología Molecular, Edificio Santiago Gascón, Campus del Cristo, Universidad de Oviedo. E-33006. Oviedo, Asturias, Spain.
Manso
Diego G
DG
Barros
Francisco
F
Carretero
Luis
L
Hughes
Thomas E
TE
Alonso-Ron
Carlos
C
Domínguez
Pedro
P
de la Peña
Pilar
P
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism*; D000072237:ERG1 Potassium Channel; D004594:Electrophysiology; D051638:Ether-A-Go-Go Potassium Channels / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D018408:Patch-Clamp Techniques; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D011993:Recombinant Fusion Proteins