Up-regulation of voltage-gated sodium channels by peptides mimicking S4-S5 linkers reveals a variation of the ligand-receptor mechanism

Abstract

prokaryotic na V channels are tetramers and eukaryotic na V channels consist of a single subunit containing four domains. Each monomer/domain contains six transmembrane segments (S1-S6), S1-S4 being the voltage-sensor domain and S5-S6 the pore domain. A crystal structure of Na V Ms, a prokaryotic na V channel, suggests that the S4-S5 linker (S4-S5 L) interacts with the C-terminus of S6 (S6 t) to stabilize the gate in the open state. However, in several voltage-gated potassium channels, using specific S4-S5 L-mimicking peptides, we previously demonstrated that S4-S5 L /S6 t interaction stabilizes the gate in the closed state. Here, we used the same strategy on another prokaryotic Na V channel, Na V Sp1, to test whether equivalent peptides stabilize the channel in the open or closed state. A na V Sp1-specific S4-S5 L peptide, containing the residues supposed to interact with S6 t according to the na V Ms structure, induced both an increase in Na V Sp1 current density and a negative shift in the activation curve, consistent with S4-S5 L stabilizing the open state. Using this approach on a human na V channel, hNa V 1.4, and testing 12 hNa V 1.4 S4-S5 L peptides, we identified four activating S4-S5 L peptides. These results suggest that, in eukaryotic Na V channels, the S4-S5 L of DI, DII and DIII domains allosterically modulate the activation gate and stabilize its open state. Voltage-gated sodium channels (Na V) are crucial in excitable as well as non-excitable cells and mutations in Na V 1.x-subunits have been associated with muscular, neuronal and cardiac channelopathies in human 1. Voltage-gated potassium (K V) channels and prokaryotic Na V channels are tetramers of subunits containing six transmembrane segments (S1 to S6). Each of the four subunits consists of one voltage-sensor domain (S1 to S4) and a pore domain (S5-S6). The four pore domains tetramerize to form a single pore module, which is regulated by the four voltage sensor domains. The arrangement of eukaryotic Na V channels is similar, with one major difference: the channel is made of a single subunit containing four homologous domains, rather than four identical subunits. Each domain in eukaryotic Na V channels is structurally equivalent to one subunit in K V or prokaryotic Na V channels, and consists of six transmembrane segments (S1 to S6). Despite intensive work on the voltage-gating of K V and Na V channels, we still lack a clear picture describing the coupling between S4 voltage-sensor movement and S6 pore gating. Both structural and functional studies identified the linker between S4 and S5 (named S4-S5 L) and the C-terminus of S6 (named S6 T), as major actors in this coupling 2-21. Different coupling mechanisms have been suggested. The crystal structure of K V 1.2, and more recently the cryo-electron microscopy and crystal structures of both eukaryotic and prokaryotic Na V channels suggested that the four S4-S5 L form a mechanical lever or a constriction ring intimately interacting with S6 T when

Druggability of Voltage-Gated Sodium Channels—Exploring Old and New Drug Receptor Sites

“…In TRPV channels, vanilloid ligands such as capsaicin and resiniferatoxin (RTX) bind to the vanilloid pocket formed by residues from S3 and S4 in the VSD, the S4-S5 linker, and S5 and S6 of the pore, and displace a phopsphatidylinositol lipid that normally occupies this pocket in the closed state to activate the channel ( Gao et al, 2016 ; Zhang et al, 2021 ) ( Figure 6C ). Recently, synthetic peptides mimicking the S4-S5 linkers have been shown to stabilize the open state of human Na V 1.4 ( Malak et al, 2020 ), highlighting an innovative strategy for ligand design that modulate the pore by targeting the electromechanical coupling of the channel.…”

New Challenges Resulting From the Loss of Function of Nav1.4 in Neuromuscular Diseases

“…However, the activity and selectivity of calliotoxin have not been tested among Na v channels and the boosting effect of this peptide on muscle force may be also due to activation of neuronal Na v channels. Although not natural, 16-amino acids-long peptides corresponding to DI, DII and DIIIS4S5 intracellular linkers increase Na + current density and shift hNa v 1.4 activation towards hyperpolarization by allosterically modulating the activation gate and stabilizing the open state ( Malak et al, 2020 ). These synthetic peptides add to the natural toxin arsenal towards identifying compounds able to selectively boost Na v 1.4 activity.…”