Somatic gene transfer of tagged K+ channel fragments to probe trafficking and electrical function in epithelial cells and cardiac myocytes

N. Neyroud, I. Deschênes, M. Akao, H. B. Nuss, E. Marbán

Research output: Contribution to journalArticle

Abstract

To evaluate the roles of the C-termini of K+ channels in subcellular targeting and protein-protein interactions, we created fusion constructs of the cell-surface antigen CD8 and the C-termini of Kv4.3, Kv1.4 and KvLQT1. Using a Cre-lox recombination system, we made 3 adenoviruses containing a fusion of the N-terminal-and transmembrane segments of CD8 with the C-termini of each of the 3 K+ channels. Expression in polarized Opossum Kidney (OK) epithelial cells led to localization of CD8-Kv4.3 and CD8-Kv1.4 into the apical and basolateral membranes, while CD8-KvLQT1 remained in the endoplasmic reticulum (ER), even when coexpressed with MinK. When expressed in rat cardiac myocytes in culture, all the 3 constructs were diffusely targeted to the surface membrane. The ER retention of CD8-KvLQT1 in OK cells but not in cardiomyocytes thus reveals functional differences in trafficking between these two cell types. To probe functional roles of C-termini, we studied K+ currents in cardiac myocytes expressing CD8-Kv4.3. Patch-clamp recordings of transient outward current revealed a hyperpolarizing shift of steady-state inactivation, implying that CD8-Kv4.3 may be disrupting the interaction of Kv4.x channels with one or more as-yet-undefined regulatory subunits. Thus, expression of tagged ion-channel fragments represents a novel, generalizable approach that may help to elucidate assembly, localization and function of these important signaling proteins.

Original languageEnglish (US)
Pages (from-to)133-144
Number of pages12
JournalJournal of Membrane Biology
Volume190
Issue number2
DOIs
StatePublished - Nov 15 2002

Keywords

  • C-terminus
  • Cardiac myocytes
  • Cell targeting
  • Epithelial cells
  • Gene transfer
  • K current

ASJC Scopus subject areas

  • Biophysics
  • Physiology
  • Cell Biology

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