scholarly journals μ-Conotoxin Giiia Interactions with the Voltage-Gated Na+ Channel Predict a Clockwise Arrangement of the Domains

2000 ◽  
Vol 116 (5) ◽  
pp. 679-690 ◽  
Author(s):  
Samuel C. Dudley ◽  
Nancy Chang ◽  
Jon Hall ◽  
Gregory Lipkind ◽  
Harry A. Fozzard ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Na Channel ◽  
Ion Permeation ◽  
Coupling Energy ◽  
Adult Skeletal Muscle ◽  
Voltage Gated ◽  
Single Polypeptide Chain ◽  
Anesthetic Drugs ◽  
Domain Iii ◽  
Analytical Approaches

Voltage-gated Na+ channels underlie the electrical activity of most excitable cells, and these channels are the targets of many antiarrhythmic, anticonvulsant, and local anesthetic drugs. The channel pore is formed by a single polypeptide chain, containing four different, but homologous domains that are thought to arrange themselves circumferentially to form the ion permeation pathway. Although several structural models have been proposed, there has been no agreement concerning whether the four domains are arranged in a clockwise or a counterclockwise pattern around the pore, which is a fundamental question about the tertiary structure of the channel. We have probed the local architecture of the rat adult skeletal muscle Na+ channel (μ1) outer vestibule and selectivity filter using μ-conotoxin GIIIA (μ-CTX), a neurotoxin of known structure that binds in this region. Interactions between the pore-forming loops from three different domains and four toxin residues were distinguished by mutant cycle analysis. Three of these residues, Gln-14, Hydroxyproline-17 (Hyp-17), and Lys-16 are arranged approximately at right angles to each other in a plane above the critical Arg-13 that binds directly in the ion permeation pathway. Interaction points were identified between Hyp-17 and channel residue Met-1240 of domain III and between Lys-16 and Glu-403 of domain I and Asp-1532 of domain IV. These interactions were estimated to contribute −1.0 ± 0.1, −0.9 ± 0.3, and −1.4 ± 0.1 kcal/mol of coupling energy to the native toxin–channel complex, respectively. μ-CTX residues Gln-14 and Arg-1, both on the same side of the toxin molecule, interacted with Thr-759 of domain II. Three analytical approaches to the pattern of interactions predict that the channel domains most probably are arranged in a clockwise configuration around the pore as viewed from the extracellular surface.

scholarly journals Charged Residues between the Selectivity Filter and S6 Segments Contribute to the Permeation Phenotype of the Sodium Channel

1999 ◽  
Vol 115 (1) ◽  
pp. 81-92 ◽  
Author(s):  
Ronald A. Li ◽  
Patricio Vélez ◽  
Nipavan Chiamvimonvat ◽  
Gordon F. Tomaselli ◽  
Eduardo Marbán
Keyword(s):  
Single Channel ◽  
Structural Features ◽  
Selectivity Filter ◽  
Na Channel ◽  
Ion Permeation ◽  
Structural Topology ◽  
Functional Roles ◽  
Charged Residues ◽  
Domain Iii ◽  
And Function

The deep regions of the Na+ channel pore around the selectivity filter have been studied extensively; however, little is known about the adjacent linkers between the P loops and S6. The presence of conserved charged residues, including five in a row in domain III (D-III), hints that these linkers may play a role in permeation. To characterize the structural topology and function of these linkers, we neutralized the charged residues (from position 411 in D-I and its homologues in D-II, -III, and -IV to the putative start sites of S6) individually by cysteine substitution. Several cysteine mutants displayed enhanced sensitivities to Cd2+ block relative to wild-type and/or were modifiable by external sulfhydryl-specific methanethiosulfonate reagents when expressed in TSA-201 cells, indicating that these amino acids reside in the permeation pathway. While neutralization of positive charges did not alter single-channel conductance, negative charge neutralizations generally reduced conductance, suggesting that such charges facilitate ion permeation. The electrical distances for Cd2+ binding to these residues reveal a secondary “dip” into the membrane field of the linkers in domains II and IV. Our findings demonstrate significant functional roles and surprising structural features of these previously unexplored external charged residues.

scholarly journals The Position of the Fast-Inactivation Gate during Lidocaine Block of Voltage-gated Na+ Channels

1999 ◽  
Vol 113 (1) ◽  
pp. 7-16 ◽  
Author(s):  
Vasanth Vedantham ◽  
Stephen C. Cannon
Keyword(s):  
Sodium Channel ◽  
Crucial Role ◽  
Ionic Current ◽  
Na Channel ◽  
Dependent Manner ◽  
Na Channels ◽  
Fast Inactivation ◽  
Adult Skeletal Muscle ◽  
Α Subunit ◽  
Voltage Gated

Lidocaine produces voltage- and use-dependent inhibition of voltage-gated Na+ channels through preferential binding to channel conformations that are normally populated at depolarized potentials and by slowing the rate of Na+ channel repriming after depolarizations. It has been proposed that the fast-inactivation mechanism plays a crucial role in these processes. However, the precise role of fast inactivation in lidocaine action has been difficult to probe because gating of drug-bound channels does not involve changes in ionic current. For that reason, we employed a conformational marker for the fast-inactivation gate, the reactivity of a cysteine substituted at phenylalanine 1304 in the rat adult skeletal muscle sodium channel α subunit (rSkM1) with [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET), to determine the position of the fast-inactivation gate during lidocaine block. We found that lidocaine does not compete with fast-inactivation. Rather, it favors closure of the fast-inactivation gate in a voltage-dependent manner, causing a hyperpolarizing shift in the voltage dependence of site 1304 accessibility that parallels a shift in the steady state availability curve measured for ionic currents. More significantly, we found that the lidocaine-induced slowing of sodium channel repriming does not result from a slowing of recovery of the fast-inactivation gate, and thus that use-dependent block does not involve an accumulation of fast-inactivated channels. Based on these data, we propose a model in which transitions along the activation pathway, rather than transitions to inactivated states, play a crucial role in the mechanism of lidocaine action.

scholarly journals Statistical Approach to Incorporating Experimental Variability into a Mathematical Model of the Voltage-Gated Na+ Channel and Human Atrial Action Potential

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1516
Author(s):  
Daniel Gratz ◽  
Alexander J Winkle ◽  
Seth H Weinberg ◽  
Thomas J Hund
Keyword(s):  
Action Potential ◽  
Mathematical Models ◽  
Cardiac Myocyte ◽  
Population Level ◽  
Na Channel ◽  
Model Parameters ◽  
Healthy Population ◽  
Experimental Measurements ◽  
Voltage Gated ◽  
Experimental Variability

The voltage-gated Na+ channel Nav1.5 is critical for normal cardiac myocyte excitability. Mathematical models have been widely used to study Nav1.5 function and link to a range of cardiac arrhythmias. There is growing appreciation for the importance of incorporating physiological heterogeneity observed even in a healthy population into mathematical models of the cardiac action potential. Here, we apply methods from Bayesian statistics to capture the variability in experimental measurements on human atrial Nav1.5 across experimental protocols and labs. This variability was used to define a physiological distribution for model parameters in a novel model formulation of Nav1.5, which was then incorporated into an existing human atrial action potential model. Model validation was performed by comparing the simulated distribution of action potential upstroke velocity measurements to experimental measurements from several different sources. Going forward, we hope to apply this approach to other major atrial ion channels to create a comprehensive model of the human atrial AP. We anticipate that such a model will be useful for understanding excitability at the population level, including variable drug response and penetrance of variants linked to inherited cardiac arrhythmia syndromes.

scholarly journals Anti-EGFR antibody 528 binds to domain III of EGFR at a site shifted from the cetuximab epitope

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Koki Makabe ◽  
Takeshi Yokoyama ◽  
Shiro Uehara ◽  
Tomomi Uchikubo-Kamo ◽  
Mikako Shirouzu ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Growth Factor Receptor ◽  
Cancer Therapeutics ◽  
Antibody Engineering ◽  
Egfr Mutations ◽  
Particle Analysis ◽  
Extracellular Region ◽  
Domain Iii ◽  
A Site ◽  
Egfr Antibody

AbstractAntibodies have been widely used for cancer therapy owing to their ability to distinguish cancer cells by recognizing cancer-specific antigens. Epidermal growth factor receptor (EGFR) is a promising target for the cancer therapeutics, against which several antibody clones have been developed and brought into therapeutic use. Another antibody clone, 528, is an antagonistic anti-EGFR antibody, which has been the focus of our antibody engineering studies to develop cancer drugs. In this study, we explored the interaction of 528 with the extracellular region of EGFR (sEGFR) via binding analyses and structural studies. Dot blotting experiments with heat treated sEGFR and surface plasmon resonance binding experiments revealed that 528 recognizes the tertiary structure of sEGFR and exhibits competitive binding to sEGFR with EGF and cetuximab. Single particle analysis of the sEGFR–528 Fab complex via electron microscopy clearly showed the binding of 528 to domain III of sEGFR, the domain to which EGF and cetuximab bind, explaining its antagonistic activity. Comparison between the two-dimensional class average and the cetuximab/sEGFR crystal structure revealed that 528 binds to a site that is shifted from, rather than identical to, the cetuximab epitope, and may exclude known drug-resistant EGFR mutations.

scholarly journals Double mutant gating perturbation analysis predicts a high conformational stability of the domain IV S6 segment of the voltage-gated Na+ channel

2009 ◽  
Vol 9 (Suppl 2) ◽  
pp. A25
Author(s):  
René Cervenka ◽  
Touran Zarrabi ◽  
Péter Lukács ◽  
Xaver König ◽  
Karlheinz Hilber ◽  
...  
Keyword(s):  
Perturbation Analysis ◽  
Double Mutant ◽  
Conformational Stability ◽  
Na Channel ◽  
Voltage Gated

scholarly journals Ca 2+ /Calmodulin-Dependent Protein Kinase II–Based Regulation of Voltage-Gated Na + Channel in Cardiac Disease

Circulation ◽  
2012 ◽  
Vol 126 (17) ◽  
pp. 2084-2094 ◽  
Author(s):  
Olha M. Koval ◽  
Jedidiah S. Snyder ◽  
Roseanne M. Wolf ◽  
Ryan E. Pavlovicz ◽  
Patric Glynn ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cardiac Disease ◽  
Na Channel ◽  
Dependent Protein Kinase ◽  
Voltage Gated ◽  
Protein Kinase Ii ◽  
Dependent Protein

Simulation on the Physical Process of Neural Electromagnetic Signal Generation Based on a Simple but Functional Bionic Na+ Channel

2021 ◽  
Author(s):  
Fan Wang ◽  
Jingjing Xu ◽  
Yanbin Ge ◽  
Shengyong Xu ◽  
Yanjun Fu ◽  
...  
Keyword(s):  
Dynamic Equilibrium ◽  
Electromagnetic Signal ◽  
Na Channel ◽  
Physical Processes ◽  
Flux Transport ◽  
Ionic Flux ◽  
Concentration Difference ◽  
Voltage Gated ◽  
Electromagnetic Signals ◽  
The Impact

Abstract The physical processes occurring at open Na+ channels in neural fibers are essential for understanding the nature of neural signals and the mechanism by which the signals are generated and transmitted along nerves. However, there is less generally accepted description of these physical processes. We studied changes in the transmembrane ionic flux and the resulting two types of electromagnetic signals by simulating the Na+ transport across a bionic nanochannel model simplified from voltage-gated Na+ channels. Results show that the Na+ flux can reach a steady state in approximately 10 ns owing to the dynamic equilibrium of Na+ ions concentration difference between the both sides of membrane. After characterizing the spectrum and transmission of these two electromagnetic signals, the low-frequency transmembrane electric field is regarded as the physical quantity transmitting in waveguide-like lipid dielectric layer and triggering the neighboring voltage-gated channels. Factors influencing the Na+ flux transport are also studied. The impact of the Na+ concentration gradient is found higher than that of the initial transmembrane potential on the Na+ transport rate, and introducing the surface-negative charge in the upper third channel could increase the transmembrane Na+ current. This work can be further studied by improving the simulation model; however, the current work helps to better understand the electrical functions of voltage-gated ion channels in neural systems.

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