The pore and gate regions of voltage-gated cation channels have been often targeted with drugs acting as channel modulators. In contrast, the voltage-sensing domain (VSD) was practically not exploited for therapeutic purposes, although it is the target of various toxins. We recently designed unique diphenylamine carboxylates that are powerful Kv7.2 voltage-gated K + channel openers or blockers. Here we show that a unique Kv7.2 channel opener, NH29, acts as a nontoxin gating modifier. NH29 increases Kv7.2 currents, thereby producing a hyperpolarizing shift of the activation curve and slowing both activation and deactivation kinetics. In neurons, the opener depresses evoked spike discharges. NH29 dampens hippocampal glutamate and GABA release, thereby inhibiting excitatory and inhibitory postsynaptic currents. Mutagenesis and modeling data suggest that in Kv7.2, NH29 docks to the external groove formed by the interface of helices S1, S2, and S4 in a way that stabilizes the interaction between two conserved charged residues in S2 and S4, known to interact electrostatically, in the open state of Kv channels. Results indicate that NH29 may operate via a voltagesensor trapping mechanism similar to that suggested for scorpion and sea-anemone toxins. Reflecting the promiscuous nature of the VSD, NH29 is also a potent blocker of TRPV1 channels, a feature similar to that of tarantula toxins. Our data provide a structural framework for designing unique gating-modifiers targeted to the VSD of voltage-gated cation channels and used for the treatment of hyperexcitability disorders.oltage-sensitive cation channels play crucial roles in brain and cardiac excitability. These channels are endowed with two main transmembrane modules, a voltage-sensing domain (VSD) and a pore domain. Mutations of ion channel genes in humans lead to severe inherited neurological, cardiovascular, or metabolic disorders, called "channelopathies" (1). So far, the medicinal toolbox has focused on the pore domain and its gate in an attempt to cure ion channel-related dysfunctions by channel blockers or openers (2).In contrast, the VSD of voltage-gated cation channels was virtually not exploited for therapeutic purposes. VSDs are found in voltage-dependent cation channels and other voltage-regulated proteins (3). In voltage-gated cation channels, the linker S4-S5 of the VSD serves as an electromechanical coupling device, which opens the channel pore. VSDs have also been recently characterized in voltage-regulated proteins that lack associated ion channel pores (4-6). A voltage-sensitive phosphatase, Ci-VSP, has a VSD that is coupled to a phosphatase domain (4). In the human voltage-activated proton channel (Hv1), the VSD itself functions as a proton channel (5-7).Crystallographic studies of voltage-gated K + channels (Kv) have described the VSD architecture in its open-state conformation. It forms a module of four membrane-spanning segments (S1-S4) with the S3b helix and the charge-bearing S4 helix forming a helix-turn-helix structure, termed the "paddle ...

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