Functional expression of GFP-linked human heart sodium channel (hH1) and subcellular localization of the a subunit in HEK293 cells and dog cardiac myocytes.
Recent evidence suggests that biosynthesis of the human heart Na+ channel (hH1) protein is rapidly modulated by sympathetic interventions. However, data regarding the intracellular processing of hH1 in vivo are lacking. In this study we sought to establish a model that would allow us to study the subcellular localization of hH1 protein. Such a model could eventually help us to better understand the trafficking of hH1 in vivo and its potential role in cardiac conduction. We labeled the C-terminus of hH1 with the green fluorescent protein (GFP) and compared the expression of this construct (hH1-GFP) and hH1 in transfected HEK293 cells. Fusion of GFP to hH1 did not alter its electrophysiological properties. Confocal microscopy revealed that hH1-GFP was highly expressed in intracellular membrane structures. Immuno-electronmicrographs showed that transfection of hH1-GFP and hH1 induced proliferation of three types of endoplasmic reticulum (ER) membranes to accommodate the heterologously expressed proteins. Labeling with specific markers for the ER and the Golgi apparatus indicated that the intracellular channels are almost exclusively retained within the ER. Immunocytochemical labeling of the Na+ channel in dog cardiomyocytes showed strong fluorescence in the perinuclear region of the cells, a result consistent with our findings in HEK293 cells. We propose that the ER may serve as a reservoir for the cardiac Na+ channels and that the transport from the ER to the Golgi apparatus is among the rate-limiting steps for sarcolemmal expression of Na+ channels.
Zimmer
T
T
Institute of Physiology II, Friedrich Schiller University Jena, Teichgraben 8, 07740 Jena, Germany. tzim@mti-n.uni-jena.de
Biskup
C
C
Dugarmaa
S
S
Vogel
F
F
Steinbis
M
M
Böhle
T
T
Wu
Y S
YS
Dumaine
R
R
Benndorf
K
K
D000818:Animals; D002478:Cells, Cultured; D004285:Dogs; D004594:Electrophysiology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D008954:Models, Biological; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D021122:Protein Subunits; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
D008164:Luminescent Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels; D049452:Green Fluorescent Proteins