| 20357183 |
Dou Y, Balse E, Dehghani Zadeh A, Wang T, Goonesekara CL, Noble GP, Eldstrom J, Steele DF, Hatem SN, Fedida D: Normal targeting of a tagged Kv1.5 channel acutely transfected into fresh adult cardiac myocytes by a biolistic method. Am J Physiol Cell Physiol. 2010 Mar 31.
The transfection of cardiac myocytes is difficult and so most of the data regarding the regulation of trafficking and targeting of cardiac ion channels has 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 hours post-bombardment. Ventricular myocytes transfected with a tagged Kv1.5 exhibit an increased sustained current component that is ~40% sensitive to 100 muM 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 N- and C-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. |
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