Calcium and calmodulin dependent protein kinase II (CaMKII) is physically associated to the Kv4.2 and Kv4.3 channels, and phosphorylation by the CaMKII modifies their current kinetics by slowing the rate of inactivation. Lipid microdomains have been shown to play an important role in both protein targeting and in controlling protein-protein interactions. In this work we studied this possible mechanism of channel-CaMKII association.

Methods: Ventricular myocyte membranes were isolated and centrifuged in a sucrose density gradient to isolate low and high density fractions. Samples were separated by SDS-PAGE and proteins identified by western blot. Caveolin-3 and Na/K ATPase were used as controls for low and high density membrane fractions respectively. The patch-clamp method was employed to record the effect of the CaMKII inhibitor KN-93 on the cardiac transient potassium current (Ito), in control and in the presence of cyclodextrin or colchicine.

Results: Sucrose density gradients revealed that Kv4.2, Kv4.3 and CaMKII are targeted to low density fractions, corresponding to lipid rafts as confirmed by caveolin-3. Ito current recordings show that KN-93 accelerates current inactivation kinetics in control conditions. In cells with lipid rafts eliminated by pretreatment with cyclodextrin current kinetics are already accelerated, and do not respond to CaMKII inhibition. In cells in which only caveolae have been internalized with colchicine, KN-93 accelerates current inactivation kinetics as in control conditions.

Conclusion: Kv4.2, Kv4.3 and CaMKII are clustered in non-caveolar lipid rafts in the membrane of rat cardiac myocytes. This work was supported by a MEC (SAF-2005-00906) and UPV/EHU (UPV05/147) grants.