Abstract:Voltage-gated sodium channels (VGSCs) are heteromeric protein complexes that initiate action potentials in excitable cells. The voltage-gated sodium channel accessory subunit, Navβ1, allosterically modulates the α subunit pore structure upon binding. To date, the molecular determinants of the interface remain unknown. We made use of sequence, knowledge and structure-based methods to identify residues critical to the association of the α and β1 Nav1.4 subunits. The Navβ1 point mutant C43A disrupted the modulati… Show more
“…The myelin P0 molecules assemble as tetramers in cis , and then the tetramers associate in trans to form tight intercellular contacts [ 94 ]. It may be significant that myelin P0 is a distant paralogue of the Na v β subunits, and its Ig domain has previously been used to model both the β1 and β3 Ig domains [ 23 , 25 , 74 , 95 ]. Figure 6.…”
Section: The Na
V
β Subunits As Cell-adhesion Molementioning
Voltage-gated sodium (Nav) channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are a major focus of research in neurobiology, structural biology, membrane biology and pharmacology. Mutations in Nav channels are implicated in a wide variety of inherited pathologies, including cardiac conduction diseases, myotonic conditions, epilepsy and chronic pain syndromes. Drugs active against Nav channels are used as local anaesthetics, anti-arrhythmics, analgesics and anti-convulsants. The Nav channels are composed of a pore-forming α subunit and associated β subunits. The β subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules. They modulate multiple aspects of Nav channel behaviour and play critical roles in controlling neuronal excitability. The recently published atomic resolution structures of the human β3 and β4 subunit Ig domains open a new chapter in the study of these molecules. In particular, the discovery that β3 subunits form trimers suggests that Nav channel oligomerization may contribute to the functional properties of some β subunits.
“…The myelin P0 molecules assemble as tetramers in cis , and then the tetramers associate in trans to form tight intercellular contacts [ 94 ]. It may be significant that myelin P0 is a distant paralogue of the Na v β subunits, and its Ig domain has previously been used to model both the β1 and β3 Ig domains [ 23 , 25 , 74 , 95 ]. Figure 6.…”
Section: The Na
V
β Subunits As Cell-adhesion Molementioning
Voltage-gated sodium (Nav) channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are a major focus of research in neurobiology, structural biology, membrane biology and pharmacology. Mutations in Nav channels are implicated in a wide variety of inherited pathologies, including cardiac conduction diseases, myotonic conditions, epilepsy and chronic pain syndromes. Drugs active against Nav channels are used as local anaesthetics, anti-arrhythmics, analgesics and anti-convulsants. The Nav channels are composed of a pore-forming α subunit and associated β subunits. The β subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules. They modulate multiple aspects of Nav channel behaviour and play critical roles in controlling neuronal excitability. The recently published atomic resolution structures of the human β3 and β4 subunit Ig domains open a new chapter in the study of these molecules. In particular, the discovery that β3 subunits form trimers suggests that Nav channel oligomerization may contribute to the functional properties of some β subunits.
“…Disruption of this disulfide bond, via the β1 C43A mutation, was suggested to have an impact on the interaction with Nav1.4 (Islas et al . ).…”
Section: Introductionmentioning
confidence: 97%
“…The pore loop of DIV (S5-S6DIV) in particular is involved in this interaction, while the transmembrane part of β1 seems to connect to one of the voltage-sensing domains . Disruption of this disulfide bond, via the β1 C43A mutation, was suggested to have an impact on the interaction with Nav1.4 (Islas et al 2013).…”
Voltage-gated sodium channels are key players in neuronal excitability and pain signalling. Precise gating of these channels is crucial as even small functional alterations can lead to pathological phenotypes such as pain or heart failure. Mechanical stress has been shown to affect sodium channel activation and inactivation. This suggests that stabilising components are necessary to ensure precise channel gating in living organisms. Here, we show that mechanical shear stress affects voltage dependence of activation and fast inactivation of the Nav1.7 channel. Co-expression of the β1 subunit, however, protects both gating modes of Nav1.7 against mechanical shear stress. Using molecular dynamics simulation, homology modelling and site-directed mutagenesis, we identify an intramolecular disulfide bond of β1 (Cys21-Cys43) which is partially involved in this process: the β1-C43A mutant prevents mechanical modulation of voltage dependence of activation, but not of fast inactivation. Our data emphasise the unique role of segment 6 of domain IV for sodium channel fast inactivation and confirm previous reports that the intracellular process of fast inactivation can be modified by interfering with the extracellular end of segment 6 of domain IV. Thus, our data suggest that physiological gating of Nav1.7 may be protected against mechanical stress in a living organism by assembly with the β1 subunit.
“…In more explicit terms the Na + channel gating, in presence of the β1 subunit, changes from slow to fast mode at different extents in practically every isoform except in Na v 1.5, which predominates in cardiac myocytes and exhibits fast gating on its own [10] . In stark contrast, the skeletal muscle isoform Na v 1.4 requires the co-expression of β1 to reconstitute the native fast Na + currents [4,11] .…”
Section: Introductionmentioning
confidence: 99%
“…Homology models of the α subunit for the wild type isoforms Na v 1.4 and β1 subunit had already been used in our laboratory to assist the experimental work [11,28,31] . Here we combined computed protein structure prediction, site-directed mutagenesis (SDM) and electrophysiological studies to investigate the possible function of relevant amino acids, involved in the inactivation process.…”
The molecular structure modeling of the β1 subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4) was carried out in the twilight zone of very low homology. Structural significance can per se be confounded with random sequence similarities. Hence, we combined (i) not automated computational modeling of weakly homologous 3D templates, some with interfaces to analogous structures to the pore-bearing Nav1.4 α subunit with (ii) site-directed mutagenesis (SDM), as well as (iii) electrophysiological experiments to study the structure and function of the β1 subunit. Despite the distant phylogenic relationships, we found a 3D-template to identify two adjacent amino acids leading to the long-awaited loss of function (inactivation) of Nav1.4 channels. This mutant type (T109A, N110A, herein called TANA) was expressed and tested on cells of hamster ovary (CHO). The present electrophysiological results showed that the double alanine substitution TANA disrupted channel inactivation as if the β1 subunit would not be in complex with the α subunit. Exhaustive and unbiased sampling of “all β proteins” (Ig-like, Ig) resulted in a plethora of 3D templates which were compared to the target secondary structure prediction. The location of TANA was made possible thanks to another “all β protein” structure in complex with an irreversible bound protein as well as a reversible protein–protein interface (our “Rosetta Stone” effect). This finding coincides with our electrophysiological data (disrupted β1-like voltage dependence) and it is safe to utter that the Nav1.4 α/β1 interface is likely to be of reversible nature.
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