Normal targeting of a tagged Kv1.5 channel acutely transfected into fresh adult cardiac myocytes by a biolistic method.
The transfection of cardiac myocytes is difficult, and so most of the data regarding the regulation of trafficking and targeting of cardiac ion channels have been obtained using heterologous expression systems. Here we apply the fast biolistic transfection procedure to adult cardiomyocytes to show that biolistically introduced exogenous voltage-gated potassium channel, Kv1.5, is functional and, like endogenous Kv1.5, localizes to the intercalated disc, where it is expressed at the surface of that structure. Transfection efficiency averages 28.2 +/- 5.7% of surviving myocytes at 24 h postbombardment. Ventricular myocytes transfected with a tagged Kv1.5 exhibit an increased sustained current component that is approximately 40% sensitive to 100 microM 4-aminopyridine and which is absent in myocytes transfected with a fluorescent protein-encoding construct alone. Kv1.5 deletion mutations known to reduce the surface expression of the channel in heterologous cells similarly reduce the surface expression in transfected ventricular myocytes, although targeting to the intercalated disc per se is generally unaffected by both NH(2)- and COOH-terminal deletion mutants. Expressed current levels in wild-type Kv1.5, Kv1.5DeltaSH3(1), Kv1.5DeltaN209, and Kv1.5DeltaN135 mutants were well correlated with apparent surface expression of the channel at the intercalated disc. Our results conclusively demonstrate functionality of channels present at the intercalated disc in native myocytes and identify determinants of trafficking and surface targeting in intact cells. Clearly, biolistic transfection of adult cardiac myocytes will be a valuable method to study the regulation of surface expression of channels in their native environment.
Dou
Ying
Y
Department of Anesthesiology, University of British Columbia, Vancouver, BC, Canada.
Balse
Elise
E
Dehghani Zadeh
Alireza
A
Wang
Tiantian
T
Goonasekara
Charitha L
CL
Noble
Geoffrey P
GP
Eldstrom
Jodene
J
Steele
David F
DF
Hatem
Stéphane N
SN
Fedida
David
D
D015761:4-Aminopyridine / Q000494:pharmacology; D000818:Animals; D019470:Biolistics; D002470:Cell Survival; D002478:Cells, Cultured; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007365:Intercellular Junctions / Q000378:metabolism; D051666:Kv1.5 Potassium Channel / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008055:Lipids; D008297:Male; D008564:Membrane Potentials; D009154:Mutation; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism*; D026902:Potassium Channel Blockers / Q000494:pharmacology; D021381:Protein Transport; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection / Q000379:methods*
C496981:KCNA5 protein, human; D051666:Kv1.5 Potassium Channel; D008055:Lipids; C086724:Lipofectamine; D026902:Potassium Channel Blockers; D011993:Recombinant Fusion Proteins; D015761:4-Aminopyridine; D011188:Potassium