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1.  Binary architecture of the Nav1.2-β2 signaling complex 
eLife  null;5:e10960.
To investigate the mechanisms by which β-subunits influence Nav channel function, we solved the crystal structure of the β2 extracellular domain at 1.35Å. We combined these data with known bacterial Nav channel structural insights and novel functional studies to determine the interactions of specific residues in β2 with Nav1.2. We identified a flexible loop formed by 72Cys and 75Cys, a unique feature among the four β-subunit isoforms. Moreover, we found that 55Cys helps to determine the influence of β2 on Nav1.2 toxin susceptibility. Further mutagenesis combined with the use of spider toxins reveals that 55Cys forms a disulfide bond with 910Cys in the Nav1.2 domain II pore loop, thereby suggesting a 1:1 stoichiometry. Our results also provide clues as to which disulfide bonds are formed between adjacent Nav1.2 912/918Cys residues. The concepts emerging from this work will help to form a model reflecting the β-subunit location in a Nav channel complex.
eLife digest
Our bodies run on electricity. The brain, heart and some other organs depend on small electrical signals that are generated by ions moving through specialized protein complexes that sit in the membrane surrounding a cell. One of these channels is a ‘sodium channel’, through which positively charged sodium ions move. Tiny changes in the structure of the sodium channel can cause severe conditions such as epilepsy and heart arrhythmias, so it is crucial that we know how it works
Sodium channels consist of different protein building blocks (called α and β) and it was not known exactly how these come together to form the full channel complex. However, previous studies hinted at which parts of the β building block make contact with the α protein.
Now, Das, Gilchrist et al. have been able to visualize the three-dimensional structure of the β building block of the sodium channel in extremely high detail by using a technique called X-ray crystallography. The level of detail in the structure also allowed the amino acids that make up the β building block to be identified.
Das, Gilchrist et al. then altered some of the amino acids in the sodium channel, and treated frog cells containing the mutant channel with a spider toxin that binds between the α and β building blocks. This revealed the location and identity of the exact contact points between the proteins. In the future, a full three-dimensional structure showing the α and β subunits bound together would yield invaluable information on how they cooperate to form the sodium channel complex and give insights into mutations that cause cardiac arrhythmias and epilepsy.
PMCID: PMC4769172  PMID: 26894959
voltage-gated sodium channel; beta2 subunit; scn2b; spider toxin; X-ray structure; disulfide; E. coli; Xenopus
2.  Microhemorrhage is an Early Event in the Pulmonary Fibrotic Disease of PECAM-1 Deficient FVB/n Mice 
Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) deficient mice in the FVB/n strain exhibit fatal chronic pulmonary fibrotic disease. The illness occurs in the absence of a detectable pro-inflammatory event. PECAM-1 is vital to the stability of vascular permeability, leukocyte extravasation, clotting of platelets, and clearance of apoptotic cells. We show here that the spontaneous development of fibrotic disease in PECAM-1 deficient FVB/n mice is characterized by early loss of vascular integrity in pulmonary capillaries, resulting in spontaneous microbleeds. Hemosiderin-positive macrophages were found in interstitial spaces and bronchoalveolar lavage (BAL) fluid in relatively healthy animals. We also observed a gradually increasing presence of hemosiderin-positive macrophages and fibrin deposition in the advanced stages of disease, corresponding to the accumulation of collagen, IL-10 expression, and myofibroblasts expressing alpha smooth muscle actin (SMA). Together with the growing evidence that pulmonary microbleeds and coagulation play an active part in human pulmonary fibrosis, this data further supports our hypothesis that PECAM-1 expression is necessary for vascular barrier function control and regulation of homeostasis specifically, in the pulmonary environment.
PMCID: PMC4414406  PMID: 24972347
interstitial fibrosis; macrophage biology; pulmonary oedema
3.  Nav1.1 Modulation by a Novel Triazole Compound Attenuates Epileptic Seizures in Rodents 
ACS Chemical Biology  2014;9(5):1204-1212.
Here, we report the discovery of a novel anticonvulsant drug with a molecular organization based on the unique scaffold of rufinamide, an anti-epileptic compound used in a clinical setting to treat severe epilepsy disorders such as Lennox-Gastaut syndrome. Although accumulating evidence supports a working mechanism through voltage-gated sodium (Nav) channels, we found that a clinically relevant rufinamide concentration inhibits human (h)Nav1.1 activation, a distinct working mechanism among anticonvulsants and a feature worth exploring for treating a growing number of debilitating disorders involving hNav1.1. Subsequent structure–activity relationship experiments with related N-benzyl triazole compounds on four brain hNav channel isoforms revealed a novel drug variant that (1) shifts hNav1.1 opening to more depolarized voltages without further alterations in the gating properties of hNav1.1, hNav1.2, hNav1.3, and hNav1.6; (2) increases the threshold to action potential initiation in hippocampal neurons; and (3) greatly reduces the frequency of seizures in three animal models. Altogether, our results provide novel molecular insights into the rational development of Nav channel-targeting molecules based on the unique rufinamide scaffold, an outcome that may be exploited to design drugs for treating disorders involving particular Nav channel isoforms while limiting adverse effects.
PMCID: PMC4027953  PMID: 24635129
4.  A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a 
Nature communications  2014;5:4350.
β-Diguetoxin-Dc1a (Dc1a) is a toxin from the desert bush spider Diguetia canities that incapacitates insects at concentrations that are non-toxic to mammals. Dc1a promotes opening of German cockroach voltage-gated sodium (Nav) channels (BgNav1), whereas human Nav channels are insensitive. Here, by transplanting commonly targeted S3b-S4 paddle motifs within BgNav1 voltage sensors into Kv2.1, we find that Dc1a interacts with the domain II voltage sensor. In contrast, Dc1a has little effect on sodium currents mediated by PaNav1 channels from the American cockroach even though their domain II paddle motifs are identical. When exploring regions responsible for PaNav1 resistance to Dc1a, we identified two residues within the BgNav1 domain II S1–S2 loop that when mutated to their PaNav1 counterparts drastically reduce toxin susceptibility. Overall, our results reveal a distinct region within insect Nav channels that helps determine Dc1a sensitivity, aconcept that will be valuable for the design of insect-selective insecticides.
PMCID: PMC4115291  PMID: 25014760
voltage-gated sodium channel; voltage sensor; spider toxin; Dc1a; insect; cockroach
5.  Animal Toxins Can Alter the Function of Nav1.8 and Nav1.9 
Toxins  2012;4(8):620-632.
Human voltage-activated sodium (Nav) channels are adept at rapidly transmitting electrical signals across long distances in various excitable tissues. As such, they are amongst the most widely targeted ion channels by drugs and animal toxins. Of the nine isoforms, Nav1.8 and Nav1.9 are preferentially expressed in DRG neurons where they are thought to play an important role in pain signaling. Although the functional properties of Nav1.8 have been relatively well characterized, difficulties with expressing Nav1.9 in established heterologous systems limit our understanding of the gating properties and toxin pharmacology of this particular isoform. This review summarizes our current knowledge of the role of Nav1.8 and Nav1.9 in pain perception and elaborates on the approaches used to identify molecules capable of influencing their function.
PMCID: PMC3446747  PMID: 23012651
Nav1.8; Nav1.9; pain; animal toxins; voltage sensor; voltage-activated sodium channel
6.  Non-invasive Diagnosis of Early Pulmonary Disease in PECAM Deficient Mice Using Infrared Pulse Oximetry 
Pulse oximetry is a common tool for detecting reduced pulmonary function in human interstitial lung diseases. It has not previously been used in a mouse model of interstitial lung disease. Further, Platelet Endothelial Cell Adhesion Molecule deficient mice rarely show symptoms until disease is advanced.
Using blood oxygen saturation, different stages of disease could be identified in a non-invasive manner. These stages could be correlated to pathology. Collagen deposition, using Picrosirius Red, did correlate with blood oxygen saturation. These studies are the first to show the use of an infrared pulse oximetry system to analyze the progression of a fibrotic interstitial lung disease in a mouse model of the human diseases. Further, these studies show that an early alveolar damage/enlargement event precedes the fibrosis in this mouse model, a stage that represents the best targets for disease analysis and prevention. This stage does not have extensive collagen deposition. Most importantly, targeting this earliest stage of disease for therapeutic intervention may lead to novel treatment for human disease.
PMCID: PMC2753694  PMID: 19646434
PECAM; lung; fibrosis; blood; oxygen

Results 1-6 (6)