To investigate the still-undetermined role of the Ca2+-independent transient outward current (Ito1) on repolarization of the cardiac action potential, we used cell fusion to introduce Ito1 into guinea pig cardiomyocytes, which normally lack this current. This technique enables the rapid delivery of premade functional ion channels to cardiomyocytes within hours of isolation, thus eliminating the action potential alterations that complicate prolonged cell culture. Chinese hamster ovary (CHO) cells stably expressing Kv4.3 (CHO-Kv4. 3) were loaded with a fluorescent dye and fused to guinea pig cardiomyocytes using polyethylene glycol. As controls, nontransfected CHO cells were fused using the same protocol. Myocytes fused with CHO-Kv4.3 cells exhibited a robust Ito1 (16. 5+/-2.6 pA/pF at +40 mV; 37 degrees C; n=19), whereas controls had none. Ito1 accelerated the early repolarization velocity (r=-0.68; 3 ms after the overshoot) and progressively suppressed the voltage of the plateau phase (r=-0.90) with increasing Ito1 density. Reduction of the action potential duration to 50% repolarization (r=-0.76) and to 90% repolarization (r=-0.65) also correlated well with Ito1 density. Thus, Ito1 exerted a significant effect on the early repolarization phase and abbreviated action potential duration. Cell fusion is a valuable and generalizable technique to introduce preformed membrane proteins into native cells.