The voltage-gated Na ؉ (Nav) channel provides the basis for electrical excitability in the brain. This channel is regulated by a number of accessory proteins including fibroblast growth factor 14 (FGF14), a member of the intracellular FGF family. In addition to forming homodimers, FGF14 binds directly to the Nav1.6 channel C-tail, regulating channel gating and expression, properties that are required for intrinsic excitability in neurons. Seeking amino acid residues with unique roles at the protein-protein interaction interface (PPI) of FGF14⅐Nav1.6, we engineered model-guided mutations of FGF14 and validated their impact on the FGF14⅐Nav1.6 complex and the FGF14: FGF14 dimer formation using a luciferase assay. Divergence was found in the ␤-9 sheet of FGF14 where an alanine (Ala) mutation of Val-160 impaired binding to Nav1.6 but had no effect on FGF14:FGF14 dimer formation. . Altogether these studies indicate that the ␤-9 sheet and the N terminus of FGF14 are well positioned targets for drug development of PPI-based allosteric modulators of Nav channels.Voltage-gated sodium (Nav) 2 channels are responsible for the initiation and propagation of the action potential in excitable cells. Nine isoforms of Nav channels (Nav1.1-Nav1.9) have been characterized functionally, and evidence for a tenth one (Na x ) has been provided (1-12). Nav channels are differentially expressed in organs, with Nav1.1, -1.2, -1.3, and -1.6 found primarily in the central and peripheral nervous systems, Nav1.4 in the adult skeletal muscle, Nav1.5 in cardiac muscle, and Nav1.7, -1.8, and -1.9 primarily in the peripheral nervous system (3,4,7,12,13). With such widespread expression, it is not surprising that numerous diseases have been ascribed to mutations of specific Nav channel isoforms (4, 14). These include the Dravet syndrome and other types of epilepsy (15-17); pain-related syndromes, such as congenital insensitivity to pain (18, 19), primary erythromelalgia (20), and paroxysmal extreme pain disorder (21, 22); and cardiac arrhythmias with congenital long QT syndrome (LQTS) type 3 (23, 24); and Brugada syndrome (25). Furthermore, SNPs and/or copy variants within Nav channel genes have been associated recently with autism (Nav1.2) (26). Nav channels blockers are currently used in combined therapy for bipolar disorder (27, 28), depression (29, 30), and schizophrenia (31), extending the role of Nav channels to virtually all brain disorders both neurological and psychiatric (14,26,32). Their centrality in the pathophysiology of so many disruptive diseases has made Nav channels key pharmacological target sites for antiepileptic, analgesic, antiarrhythmic, and psychiatric drugs (11,14,33,34). Unfortunately, current Nav channel blockers lack specificity, as they are directed against molecular domains conserved across all Nav isoforms. As such, therapies based on these medications can result in severe side effects, such as Stevens-Johnson syndrome, blood dyscrasias, and ataxia (35). Although some success has been achieved in developing more ta...

Protein-protein interactions (PPI) offer unexploited opportunities for CNS drug discovery and neurochemical probe development. Here, we present ZL181, a novel peptidomimetic targeting the PPI interface of the voltage-gated Na channel Nav1.6 and its regulatory protein fibroblast growth factor 14 (FGF14). ZL181 binds to FGF14 and inhibits its interaction with the Nav1.6 channel C-tail. In HEK-Nav1.6 expressing cells, ZL181 acts synergistically with FGF14 to suppress Nav1.6 current density and to slow kinetics of fast inactivation, but antagonizes FGF14 modulation of steady-state inactivation that is regulated by the N-terminal tail of the protein. In medium spiny neurons in the nucleus accumbens, ZL181 suppresses excitability by a mechanism that is dependent upon expression of FGF14 and is consistent with a state-dependent inhibition of FGF14. Overall, ZL181 and derivatives could lay the ground for developing allosteric modulators of Nav channels that are of interest for a broad range of CNS disorders.

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Ethanol alters BK (slo1) channel function leading to perturbation of physiology and behavior. Site(s) and mechanism(s) of ethanol-BK channel interaction are unknown. We demonstrate that ethanol docks onto a water-accessible site that is strategically positioned between the slo1 calcium-sensors and gate. Ethanol only accesses this site in presence of calcium, the BK channel's physiological agonist. Within the site, ethanol hydrogen-bonds with K361. Moreover, substitutions that hamper hydrogen bond formation or prevent ethanol from accessing K361 abolish alcohol action without altering basal channel function. Alcohol interacting site dimensions are approximately 10.7 × 8.6 × 7.1 Å, accommodating effective (ethanol-heptanol) but not ineffective (octanol, nonanol) channel activators. This study presents: (i) to our knowledge, the first identification and characterization of an n-alkanol recognition site in a member of the voltage-gated TM6 channel superfamily; (ii) structural insights on ethanol allosteric interactions with ligand-gated ion channels; and (iii) a first step for designing agents that antagonize BK channel-mediated alcohol actions without perturbing basal channel function.is a psychoactive agent that has been overwhelmingly consumed by mankind across cultures and civilizations. Alcohol actions on central nervous system (CNS) physiology and behavior are largely independent of beverage type but due to ethanol itself (1). Ethanol alters cell excitability by modifying function of transmembrane (TM) ion channel proteins, including K + channels. These channels constitute the most heterogeneous and extensive group of ion channels, its members belonging to TM2, TM4, and TM6 protein superfamilies. Within this myriad of proteins, several K + channels have been shown to modify behavior in response to acute exposure to ethanol concentrations that reach the CNS and other excitable tissues during alcohol drinking (2-5). However, with the sole exception of the TM2, G protein-regulated inward rectifier K + (GIRK) channel (6), there is no structural information on ethanol-K + channel protein interacting sites currently available. Voltage/Ca 2+ -gated, large conductance K + channels (BK), which are members of the TM6 voltage-gated ion channel superfamily, constitute major mediators of alcohol actions in excitable tissues. Acute exposure to ethanol levels reached in CNS during alcohol intoxication alters BK-mediated currents and thus, elicits widespread and profound modifications in physiology and behavior. In rodent models, acute ethanol exposure leads to reduced vasopressin, oxytocin and growth hormone release with consequent perturbation in physiology and behavior (7), altered firing rates in nucleus accumbens (8) and dorsal root ganglia neurons (9), and alcohol-induced cerebral artery constriction (10, 11). Moreover, studies in both mammals and invertebrate models demonstrate that ethanol targeting of neuronal BK is involved in development of alcohol tolerance and dependence (12-16). Although the physiological and be...