Assess in vitro activity of opakalim (BHV-7000), a selective Kv7.2/7.3 channel activator, in Kv7.2/7.3 channels with a DEE-causing mutation.

To control the stoichiometry of Kv7.2/Kv7.3 subunit assembly, an expression construct was made where Kv7.2 and Kv7.3 subunits were concatenated. A construct was made to achieve one copy of Kv7.2 WT and one copy of Kv7.2 G281E. The concatenated construct, in addition to unconcatenated Kv7.2 WT/Kv7.3 WT subunits, were expressed in CHO-K1 cells and evaluated in the presence and absence of opakalim or the first-generation activator using whole-cell voltage-clamp electrophysiology.

The construct containing one copy of Kv7.2 G281E produced active channels with ½-maximal voltage of activation like unconcatenated Kv7.2 WT/Kv7.3 WT channels. When treated with opakalim or the first-generation activator, the voltage-dependence of activation for channels containing WT Kv7.2/Kv7.3 subunits or one copy of Kv7.2 G281E was shifted toward negative potentials to a similar degree. The potency of both drugs for potentiating channels containing one copy of Kv7.2 G281E was lower compared to channels containing only WT Kv7.2/Kv7.3 subunits.

Since the first-generation activator and opakalim activate Kv7.2 G281E-containing channels and the patient responded favorably to replacement of the first-generation activator with opakalim, therapeutic benefit likely arises from potentiation of both WT and Kv7.2 G281E-containing channels, underscoring the potential of opakalim for treatment of KCNQ2-DEE.