Functional and biochemical characterization of the human potassium channel Kv1.5 with a transplanted carboxyl-terminal epitope in stable mammalian cell lines.
The role of the C-terminal domain of the hPCN1/Kv1.5 delayed rectifier K+ channel was investigated in transfected stable cell lines employing antipeptide and anti-epitope antibodies against hPCN1-cp, an epitope-fusion gene carrying additional sequences encoding a 32 amino acid C-terminal extension. Both wild-type and chimeric genes showed high levels of K+ channel expression. Detailed electrophysiologic characterization showed there to be no significant effect of the C-terminal extension on channel activity. Immunoblots of whole-cell and membrane preparations demonstrated primarily intact protein in which the C-terminal extension was not cleaved from the peptide chain. Two bands were visualized from cells transfected with either the wild-type or chimeric channels; the slower migrating band was a non-N-glycosylated form. The epitope-fusion method will be a useful adjunct to studying the role of functional domains in ion channels, and may provide a means for rapid affinity purification of channel protein.
Philipson
L H
LH
University of Chicago, Department of Medicine, IL 60637.
Malayev
A
A
Kuznetsov
A
A
Chang
C
C
Nelson
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D000939:Epitopes; D015221:Potassium Channels