Domain Of Sodium Channel Research Articles

Novel Mutations Mapping to the Fourth Sodium Channel Domain of Nav1.7 Result in Variable Clinical Manifestations of Primary Erythromelalgia

We identified and clinically investigated two patients with primary erythromelalgia mutations (PEM), which are the first reported to map to the fourth domain of Nav1.7 (DIV). The identified mutations (A1746G and W1538R) were cloned and transfected to cell cultures followed by electrophysiological analysis in whole-cell configuration. The investigated patients presented with PEM, while age of onset was very different (3 vs. 61 years of age). Electrophysiological characterization revealed that the early onset A1746G mutation leads to a marked hyperpolarizing shift in voltage dependence of steady-state activation, larger window currents, faster activation kinetics (time-to-peak current) and recovery from steady-state inactivation compared to wild-type Nav1.7, indicating a pronounced gain-of-function. Furthermore, we found a hyperpolarizing shift in voltage dependence of slow inactivation, which is another feature commonly found in Nav1.7 mutations associated with PEM. In silico neuron simulation revealed reduced firing thresholds and increased repetitive firing, both indicating hyperexcitability. The late-onset W1538R mutation also revealed gain-of-function properties, although to a lesser extent. Our findings demonstrate that mutations encoding for DIV of Nav1.7 can not only be linked to congenital insensitivity to pain or paroxysmal extreme pain disorder but can also be causative of PEM, if voltage dependency of channel activation is affected. This supports the view that the degree of biophysical property changes caused by a mutation may have an impact on age of clinical manifestation of PEM. In summary, these findings extent the genotype–phenotype correlation profile for SCN9A and highlight a new region of Nav1.7 that is implicated in PEM.Electronic supplementary materialThe online version of this article (doi:10.1007/s12017-012-8216-8) contains supplementary material, which is available to authorized users.

Modeling P-Loops Domain of Sodium Channel: Homology with Potassium Channels and Interaction with Ligands

A large body of experimental data on Na + channels is available, but the interpretation of these data in structural terms is difficult in the absence of a high-resolution structure. Essentially different electrophysiological and pharmacological properties of Na + and K + channels and poor identity of their sequences obstruct homology modeling of Na + channels. In this work, we built the P-loops model of the Na + channel, in which the pore helices are arranged exactly as in the MthK bacterial K + channel. The conformation of the selectivity-filter region, which includes residues in positions −2 through +4 from the DEKA locus, was shaped around rigid molecules of saxitoxin and tetrodotoxin that are known to form multiple contacts with this region. Intensive Monte Carlo minimization that started from the MthK-like conformation produced practically identical saxitoxin- and tetrodotoxin-based models. The latter was tested to explain a wide range of experimental data that were not used at the model building stage. The docking of tetrodotoxin analogs unambiguously predicted their optimal orientation and the interaction energy that correlates with the experimental activity. The docking of μ-conotoxin produced a binding model consistent with experimentally known toxin-channel contacts. Monte Carlo-minimized energy profiles of tetramethylammonium pulled through the selectivity-filter region explain the paradoxical experimental data that this organic cation permeates via the DEAA but not the AAAA mutant of the DEKA locus. The model is also consistent with earlier proposed concepts on the Na + channel selectivity as well as Ca 2+ selectivity of the EEEE mutant of the DEKA locus. Thus, the model integrates available experimental data on the Na + channel P-loops domain, and suggests that it is more similar to K + channels than was believed before.

A novel LQT3 mutation implicates the human cardiac sodium channel domain IVS6 in inactivation kinetics.

The Long QT3 syndrome is associated with mutations in the cardiac sodium channel gene SCN5A. The aim of the present study was the identification and functional characterization of a mutation in a family with the long QT3 syndrome. The human cardiac sodium channel gene SCN5A was screened for mutations by single-stranded conformation polymorphism. The functional consequences of mutant sodium channels were characterized after expressing mutant and wild-type cRNAs in Xenopus oocytes by two-electrode voltage clamp measurements. SCN5A screening revealed an A-->G substitution at codon 1768, close to the C-terminal end of domain IVS6, which changes an isoleucine to a valine. Functional expression of mutant I1768V-channels in Xenopus oocytes showed that the voltage-dependence and slope factors of activation and inactivation were unchanged compared to wild-type channels. No difference in persistent TTX-sensitive current could be detected between wild-type and I1768V channels, a channel feature often increased in LQT3 mutants. However, I1768V mutant channels recovered faster from inactivation (2.4 times) than wild-type channels and displayed less slow inactivation. We postulate that severe destabilization of the inactivated state leads to increased arrhythmogenesis and QT prolongation in I1768V mutation carriers in the absence of a persistent inward sodium current.

Nodal sodium channel domain integrity depends on the conformation of the paranodal junction, not on the presence of transverse bands.

Our understanding of the role that axoglial interactions play in node of Ranvier formation and maintenance remains incomplete. Previous studies of CNS myelinated fibers of CGT-null mice showed abnormalities in the arrangement of paranodal myelin loops and absence of a conspicuous component of the paranodal junction, the ridge-like intercellular transverse bands. Axolemmal sodium channel domains were largely preserved at nodes of Ranvier but displayed some abnormalities in form. Using a combination of freeze-fracture and immunocytochemical methods, we have found additional evidence documenting abnormalities in the size, shape, and location of axolemmal sodium channel clusters in CGT-null mice as well as evidence that these nodal abnormalities are complementary to the organization of paranodal myelin loops, despite the absence of transverse bands. We conclude that the differentiated form of the nodal axolemma and the distribution of axolemmal sodium channels depend on the conformation of paranodal axoglial contacts but not on the presence of transverse bands at the sites of contact.

Simultaneous measurement of spectroscopic and physiological signals from a planar bilayer system: detecting voltage-dependent movement of a membrane-incorporated peptide.

We developed an experimental system that can measure spectroscopic and physiological signals simultaneously from ion channels in a planar lipid bilayer, to study the relationship between the structure and function of the ion channels. While the membrane potential was clamped, fluorescent emission and ionic currents were measured simultaneously. The fluorescent emissions from a planar bilayer constructed in a specially designed chamber were monitored exclusively, and the signal intensity was measured with a photon-counting system. The intensity of fluorescence and spectral shape were measured successfully from the planar bilayer, with a high signal-to-noise ratio. The system can measure the intensity of fluorescence from a restricted area of the planar bilayer, with a diameter of 70 micrometer and a focal depth of 15 micrometer. The low background signal was achieved by optimizing the optical system. More than 95% of the measured fluorescence comes from the planar lipid bilayer. A 22-mer peptide with a sequence identical to that of the S4 segment of the electric eel sodium channel domain IV was synthesized and fluorescence-labeled. This peptide formed a voltage-dependent ion channel in a planar bilayer. The changes in the intensity of the fluorescence accompanying ionic currents generated by a voltage clamp suggest that voltage gating involves the insertion of the N-terminal of the peptide into the membrane. The electrical and optical signals were measured with a gate time of 10 ms. This measurement enabled the detection of movement of the membrane-incorporated peptides with channel opening.

Molecular Studies of Voltage-gated Potassium Channels

The cloning and characterization of the voltage-activated Shaker potassium channel gene in Drosophila have led to the identification of structural elements involved in potassium channel gating. As found for the voltage-activated sodium channel, the S4 segment, located in the conserved core of the protein, plays a central role in voltage-dependent activation. Potassium channels appear to be formed by the assembly of several polypeptides into multisubunit channels. This is directly analogous to the proposed folding of the four internally homologous pseudosubunits of sodium and calcium channels. The amino- and carboxy-terminal regions of Shaker channels are specialized for, and appear to interact in, inactivation gating. This interaction probably includes interaction between subunits, as may be said for the role in inactivation gating of the junction between the carboxyl terminus of the third domain and amino terminus of the fourth domain of sodium channel (Vassilev et al. 1988). The capacity for coassembly in potassium channels extends not only to the alternatively spliced products of the same gene, but also to the products of different genes. Heteromultimeric channels that are formed in this way have kinetic and pharmacological properties that differ from homomultimers of their constituents and, as such, broaden the functional diversity of channels that can be produced by any given number of compatible potassium channel genes.