Roles of surface residues of intracellular domains of heag potassium channels.
Ether-a-go-go potassium channels have large intracellular regions containing 'Per-Ant-Sim' (PAS) and cyclic nucleotide binding (cNBD) domains at the N- and C-termini, respectively. In heag1 and heag2 channels, recent studies have suggested that the N- and C-terminal domains interact, and affect activation properties. Here, we have studied the effect of mutations of residues on the surfaces of PAS and cNBD domains. For this, we introduced alanine and lysine mutations in heag1 channels, and recorded currents by two-electrode voltage clamp. In both the PAS domain and the cNBD domain, contiguous areas of conserved residues on the surfaces of these domains were found which affected the activation kinetics of the channel. Next, we investigated possible effects of mutations on domain interactions of PAS and cNBD proteins in heag2 by co-expressing these domain proteins followed by analysis with native gels and western blotting. We found oligomeric association between these domains. Mutations F30A and A609K (on the surfaces of the PAS and cNBD domains, respectively) affected oligomeric compositions of these domains when proteins for PAS and cNBD domains were expressed together. Taken together, the data suggest that the PAS and cNBD domains form interacting oligomers that have roles in channel function.
Stevens
Louisa
L
Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.
Ju
Min
M
Wray
Dennis
D
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D051638:Ether-A-Go-Go Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials; D008958:Models, Molecular; D020125:Mutation, Missense; D018408:Patch-Clamp Techniques; D013997:Time Factors; D014982:Xenopus laevis
D051638:Ether-A-Go-Go Potassium Channels; C496884:KCNH1 protein, human; C485729:KCNH5 protein, human