Abstract
Voltage-gated sodium (NaV) channels are pore-forming transmembrane proteins that play essential roles in excitable cells, and they are key targets for antiepileptic, antiarrhythmic, and analgesic drugs. We implemented a heterobivalent design strategy to modulate the potency, selectivity, and binding kinetics of NaV channel ligands. We conjugated μ-conotoxin KIIIA, which occludes the pore of the NaV channels, to an analogue of huwentoxin-IV, a spider-venom peptide that allosterically modulates channel gating. Bioorthogonal hydrazide and copper-assisted azide–alkyne cycloaddition conjugation chemistries were employed to generate heterobivalent ligands using polyethylene glycol linkers spanning 40–120 Å. The ligand with an 80 Å linker had the most pronounced bivalent effects, with a significantly slower dissociation rate and 4–24-fold higher potency compared to those of the monovalent peptides for the human NaV1.4 channel. This study highlights the power of heterobivalent ligand design and expands the repertoire of pharmacological probes for exploring the function of NaV channels.
Highlights
Voltage-gated sodium (NaV) channels are fundamental for the generation and propagation of action potentials in excitable cells, and they are important therapeutic targets for antiepileptic, antiarrhythmic, and analgesic drugs.[1−3] Humans have nine NaV channel subtypes denoted NaV1.1−NaV1.9
NaV channels are large transmembrane proteins composed of a pore-forming α-subunit in complex with one or two auxiliary β-subunits that modulate their expression, localization, gating, kinetics, and pharmacology (Figure 1).[3,4]
The S1−S4 segments within each domain form a voltage-sensing domain (VSD), while the S5 and S6 segments from each domain come together in a circular fashion to form the central pore of the channel (Figure 1A).[1,2,4]
Summary
Voltage-gated sodium (NaV) channels are fundamental for the generation and propagation of action potentials in excitable cells, and they are important therapeutic targets for antiepileptic, antiarrhythmic, and analgesic drugs.[1−3] Humans have nine NaV channel subtypes denoted NaV1.1−NaV1.9. NaV1.1−NaV1.3 and NaV1.6 are expressed in both the central nervous system (CNS) and the peripheral nervous system (PNS), while NaV1.7−NaV1.9 are found primarily in peripheral sensory neurons.[2] NaV1.4 and NaV1.5 are predominantly located in skeletal and cardiac muscles, respectively, where they play critical roles in muscle contraction.[2]. NaV channels are large transmembrane proteins composed of a pore-forming α-subunit in complex with one or two auxiliary β-subunits that modulate their expression, localization, gating, kinetics, and pharmacology (Figure 1).[3,4] The α-subunit (∼260 kDa) folds into four homologous but nonidentical domains (denoted DI−DIV) joined by intracellular linkers, with each domain containing six transmembrane segments (S1−S6). The S1−S4 segments within each domain form a voltage-sensing domain (VSD), while the S5 and S6 segments from each domain come together in a circular fashion to form the central pore of the channel (Figure 1A).[1,2,4] The VSDs allow the channel to respond to changes in the membrane electrical potential, causing it to cycle (or “gate”) among three distinct states: a closed/resting state in which the channel can be activated by membrane depolarization, an open ion-conducting state, and a nonconducting inactivated state.[1,2,4]
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