While PKA-mediated phosphorylation of Na⁺ and Ca²⁺ channels is well documented, data on the role of PKA in phosphorylating potassium channels is controversial. Some studies have shown both I_K1 and I_to to be increased by the catalytic subunit of PKA, while others have shown them to be decreased. We used H-89 and calyculin A (known inhibitors of PKA and PP1/PP2A, respectively) to determine whether the basal I_K1and I_to were modulated by phosphorylation in the absence of any additional stimulation. Rats were humanely killed and ventricular myocytes were isolated by collagenase and protease digestion. Cells were then voltage clamped in the perforated patch clamp configuration using amphotericin B (240 μg ml⁻¹). External solutions contained (mM): 140 NaCl, 5.4 KCl, 1.0 MgCl₂.6H₂O, 1.0 CaCl₂, 10.0 glucose, 10 Hepes, pH 7.3. Internal solutions contained (mM) 120 K glutamate, 20 KCl, 10 NaCl, 10.0 Hepes, pH 7.2. All recordings were made at room temperature. Currents were recorded either using 2 second descending voltage ramps from 80 to −120 mV from a holding potential of −40 mV or 5 mV step increments from −40 mV. The figure shows that 5-60 μM H-89 produced a concentration-dependent inhibition of basal I_to and I_K1 recorded during descending voltage ramps. The effects of H-89 on I_to were studied in more detail using square depolarising steps from −40 to 80mV. Again, H-89 reduced I_to in a concentration-dependent manner: I_to was reduced to 87.4 ± 6.1, 74.3 ± 3.0, 48.2 ± 2.8, 31.4 ± 5.4% at 10, 20, 40 and 60 μM H-89, respectively with an EC₅₀ of 38 μM. Double-pulse protocols used to investigate steady state activation/inactivation properties of I_to showed that the time-dependent recovery of I_to from voltage-dependent inactivation was slowed at 60 μM H-89 but not 10–20 μM (P<0.05): T½ (half time for recovery) was 113.3 ± 6.7 ms (n=7) in control and 313.3 ± 67.7 ms in 60 μM H-89 (n=3).