Somatic gene transfer of tagged K+ channel fragments to probe trafficking and electrical function in epithelial cells and cardiac myocytes.
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 co-expressed 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.
Neyroud
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Institute of Molecular Cardiology, The Johns Hopkins University School of Medicine, 844 Ross Bldg., 720 N Rutland Avenue, Baltimore, MD 21205, USA.
Deschênes
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I
Akao
M
M
Nuss
H B
HB
Marbán
E
E
D000818:Animals; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology* / Q000502:physiology*; D018014:Gene Transfer Techniques; D006352:Heart Ventricles / Q000166:cytology; D051656:KCNQ Potassium Channels; D051657:KCNQ1 Potassium Channel; D007668:Kidney / Q000166:cytology / Q000502:physiology; D051665:Kv1.4 Potassium Channel; D032342:Muscle Cells / Q000166:cytology* / Q000502:physiology*; D009893:Opossums; D015221:Potassium Channels / Q000145:classification / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated; D051381:Rats; D011993:Recombinant Fusion Proteins; D016324:Sequence Tagged Sites; D051672:Shal Potassium Channels; D014161:Transduction, Genetic; D016276:Ventricular Function
D051656:KCNQ Potassium Channels; D051657:KCNQ1 Potassium Channel; C496979:Kcna4 protein, rat; C497023:Kcnd3 protein, rat; C496911:Kcnq1 protein, rat; D051665:Kv1.4 Potassium Channel; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011993:Recombinant Fusion Proteins; D051672:Shal Potassium Channels