Received for publication October 25, 2005.
Revised December 15, 2005.
Accepted for publication December 15, 2005.

KN-93, a Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor, is a Direct Extracellular Blocker of Voltage-Gated Potassium Channels

Abstract

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 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 µM KN-93 was independent of CaMK II since 1 µM KN-92, an inactive analog of KN-93, caused similar inhibition of currents. In addition, dialysis of cells with 10 µM 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, while enhancement of inactivation induced by mutation of threonine 462 to cysteine (T462C) increased the potency of KN-93 by four-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.

Key words: Block, CaMK II, Electrophysiology, KN-93, Potassium, Voltage-Gated Channel

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