KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-( 4-chlorocinnamyl)-N-methylbenzylamine), a calcium/calmodulin-dependent protein kinase II inhibitor, is a direct extracellular blocker of voltage-gated potassium channels

The effect of Ca2+/calmodulin-dependent protein kinase II (CaMK II) on voltage- gated ion channels is widely studied through the use of specific CaMK II blockers such as 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN-93). The present study demonstrates that KN-93 is a direct extracellular blocker of a wide range of cloned Kv channels from a number of different subfamilies. In all channels tested, the effect of 1 mu M KN-93 was independent of CaMK II because1 mu M 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)N-methylbenzylamine, phosphate (KN-92), an inactive analog of KN-93, caused similar inhibition of currents. In addition, dialysis of cells with 10 mu M CaMK II inhibitory peptide fragment 281-301 (CIP) had no effect on current kinetics and did not prevent the inhibitory effect of KN-93. The IC50 for block of the Kv1.5 channel (used as an example to determine the nature of KN-93 block) was 307 +/- 12 nM. KN-93 blocked open channels with little voltage dependence that did not alter the V-1/2 of channel activation. Removal of P/C-type inactivation by mutation of arginine 487 to valine in the outer pore region of Kv1.5 (R487V) greatly reduced KN-93 block, whereas enhancement of inactivation induced by mutation of threonine 462 to cysteine (T462C) increased the potency of KN-93 by 4-fold. This suggested that KN-93 acted through promotion and stabilization of C-type inactivation. Importantly, KN-93 was ineffective as a blocker when applied intracellularly, suggesting that CaMK II-independent effects of KN-93 on Kv channels can be circumvented by intracellular application of KN-93.