On November 30, 2023, the team of Yan Ning of Tsinghua University and Shenzhen Academy of Medical Sciences published a speech entitled ".a structural atlas of druggable sites on na channelsThe review article will be:An overview of the latest advances in the structural pharmacology of Na channels, including the structural mapping of ligand-binding Na channels. These findings lay an important foundation for future drug development.
The voltage-gated sodium (Na) channel is a transmembrane protein that selectively inputs sodium ions upon membrane depolarization. NA channels are essential for the transmission of electrical signals in excitable cells, such as neurons and muscle cells, and are responsible for the generation and propagation of action potentials. The voltage-dependent activation of sodium currents was first recorded by Hodgkin and Huxley nearly 70 years ago, marking the discovery of action potentials. Hille investigated the ion-selectivity of the Na channel and proposed a "four-barrier, three-point model" to describe the selectivity of sodium ions. He also analyzed the binding patterns of local anesthetics to related NA-targeted drugs. Armstrong and Bezanilla measured gating currents using a high-resolution electrophysiological recording method and explored the process and determinants of rapid inactivation of NA channels. In the early 80s of the 20th century, Barchi and Catterall's group studied the biochemical properties of NA channels;Numa et al. reported the coding sequence of the poreogenic subunit (subunit) of the NA channel. Taken together, these milestones established an early concept of the function, working mechanism, and structural properties of NA channels.
Na channels belong to the voltage-gated ion channel (VGIC) superfamily. They typically include a core subunit, which is sufficient in itself for channel activity, and an auxiliary subunit (1-4), which helps in membrane localization and channel regulation. A subunit is a single polypeptide with a length of about 2000 residues, folded into 4 homologous repeats (repeats I-IV). Each repeat consists of six transmembrane helices (S1-S6), of which S5 and S6 in four repeats constitute the ion permeation pore domain (PD), and S1-S4 in each repeat constitute the flanking voltage induction domain (VSD). The sequences between S5 and S6 are assembled into extracellular loops (ECLs) and ion-selective filters (SFs). Four different residues, ASP Glu Lys Ala (DEKA), are located at the corresponding SF site for each replicate and are responsible for sodium selectivity. The III-IV linker contains the Ile Phe Met (IFM) motif, which is essential for the rapid inactivation of NA channels. This mutation of hydrophobic clusters can completely eliminate this rapid inactivation.
An overview of the structure and general working mechanism of the NA channel (Figure source).
There are 9 subtypes of the human NA subunit, respectively, na11-na1.9。na1.1-na1.3 and na16 is mainly expressed in the central nervous system. na1.4 and na15 are found in skeletal muscle and cardiac muscle, respectively. na1.7-na1.9 Mainly acts on the peripheral nervous system.
NA channels are key to the production of membrane excitability. Even small changes in voltage-dependent sodium current can have detrimental effects. Dysfunction or aberrant regulation of NA channels has been implicated in a variety of diseases, primarily with the tissue specificity of the channel. For example, in na1In addition to hundreds of nonsense mutations, many missense mutations have been found in the gene encoding SCN1A, making it the most susceptible risk factor for seizures. na1.5 is another hot spot for pathogenic mutations. Hundreds of mutations have been found in patients with heart disease, such as long QT syndrome type 3, Brugada syndrome, and fibrillation. Including na13 and na16-na1.Several subtypes of NA channels, including 9, have been implicated in pain perception. NA channels have been the primary drug targets for epilepsy, arrhythmias, psychiatric disorders, and pain disorders.
Structural mapping of ligand binding sites on NA channels (Figure source).
Dissecting the working and pathogenesis of NA channels and the mode of action (MOA) of NA-targeted drugs requires high-resolution structures in different functional states. The simplified working diagram of the NA channel consists of three main states: hibernating, active, and inactive. At resting membrane potential, the NA channel is non-conductive, the PD is closed, and the VSD is in a "downward" conformation. In response to membrane depolarization, the S4 helix is driven to the outer side of the cell to achieve an "up" conformation. The movement of the VSD causes the PD to open, a process known as electromechanical coupling. After activation, the channel is rapidly inactivated within 1 2 ms. During membrane hyperpolarization, the NA channel is released from the inactive state to the resting state, ready for the next duty cycle.
Breakthroughs in cryo-electron microscopy (cryo-EM) techniques and algorithms have provided unprecedented opportunities for near-atomic-resolution visualization of NA channels. In 2017, the first structure of eukaryotic NA channels, napas, named after the short subtype of the cockroach (Periplaneta americana), was published. A few months later, researchers reported a report on the use of electric eels (eena14) Cryo-EM structures isolated in 1-bound NA channels. These studies reveal the assembly principle and structural details of single-stranded Na channels that differ from homotetrameric VGICs. Surprisingly, the two structures were acquired in completely different states. NAPA has a constricted pore domain structure, with 4 VSDs showing a distinct upward conformation, while Eena 1The pore domain structure of 4 is relaxed at the intracellular gate, and all 4 VSDs are in a similar "upward" state. The most significant structural differences occur on short III-IV connectors. Based on structural differences, the researchers proposed a "gate wedge" allosteric blocking mechanism for the rapid inactivation of NA channels by IFM motifs.
Binding site on VSD (figure sourced).
In the years that followed, the researchers eventually captured human Na11、na1.2 and na14-na1.8 structures, most of which are complexed with auxiliary subunits and toxins drugs. The Catterall and Jiang groups reported rat Na1., respectively5 (rna1.5) And people na13 of the structure. Together, these structures provide the molecular basis for understanding the vast amount of experimental and clinical data accumulated over the past half-century. In this review, we will focus on recent advances in the structural pharmacology of NA channels, which provide important insights for future rational drug development.