Insulin stimulation of rat ventricular K+ currents depends on the integrity of the cytoskeleton

1. The effect of insulin on K+ currents was studied with enzymatically disp ersed ventricular myocytes from insulin-deficient (type I) diabetic rats. D iabetic conditions were induced by a single intravenous injection of strept ozotocin (100 mg kg(-1)) given 8-13 days before the experiments. Measuremen ts of plasma glucose and insulin levels confirmed the diabetic status of th e animals. 2. A Ca2+-independent transient outward K+ current, I-t, and a slowly inact ivating, quasi steady-state current, I-ss which are depressed in diabetic m yocytes, could be restored by exposure to 1, 10 or 100 nM insulin. This was only observed after a delay of 5-6 h, although an insulin exposure of only 1 h was sufficient to initiate its stimulatory action on I-t and I-ss. The stimulatory effect of insulin on these K+ currents was prevented by 2 mu M cycloheximide, which in itself had no direct effect on these currents. 3. Disruption of the actin microfilament network with 1 mu M cytochalasin D (CD) also prevented the stimulatory effect of 100 nM insulin on both I-t a nd I-ss. Since CD was added 1 h after insulin, inhibitory effects on insuli n signalling were ruled out. Adding CD (1. mu M) 5-9 h after insulin, when currents were already augmented, had no effect (up to 50 min exposure). Inc ubating control cells for 6-10 h with 1 mu M CD had no effect on any of the currents measured. 4. Stabilization of the actin network by pre-exposure to 2.5 mu m phalloidi n restored the stimulatory effect of insulin, in the continued presence of CD, ruling out any effects of CD on components other than the cytoskeleton. 5. The stimulatory effect of insulin was also prevented by incubating cells with insulin in the presence of the microtubule-disrupting agent colchicin e (5 mu M) 6. These results suggest that the insulin-mediated augmentation of K+ curre nts in diabetic myocytes requires protein synthesis, possibly of K+ channel s, as well as an intact cytoskeleton. The possibility that newly formed cha nnels translocate to the plasma membrane in a process dependent on differen t elements of the cytoskeleton is discussed.