Functional and biochemical characterization of the human potassium channel Kv1.5 with a transplanted carboxyl-terminal epitope in stable mammalian cell lines

Biochim Biophys Acta. 1993 Nov 21;1153(1):111-21. doi: 10.1016/0005-2736(93)90282-5.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • CHO Cells
  • Cloning, Molecular
  • Cricetinae
  • Cricetulus
  • Epitopes / chemistry
  • Epitopes / metabolism
  • Gene Expression
  • Humans
  • Immunoblotting
  • Immunohistochemistry
  • Membrane Potentials
  • Molecular Sequence Data
  • Potassium Channels / chemistry
  • Potassium Channels / genetics*
  • Potassium Channels / metabolism
  • Transfection

Substances

  • Epitopes
  • Potassium Channels