Sodium Channel Fast Inactivation Research Articles

Chemometric Models of Differential Amino Acids at the Navα and Navβ Interface of Mammalian Sodium Channel Isoforms.

(1) Background: voltage-gated sodium channels (Navs) are integral membrane proteins that allow the sodium ion flux into the excitable cells and initiate the action potential. They comprise an α (Navα) subunit that forms the channel pore and are coupled to one or more auxiliary β (Navβ) subunits that modulate the gating to a variable extent. (2) Methods: after performing homology in silico modeling for all nine isoforms (Nav1.1α to Nav1.9α), the Navα and Navβ protein-protein interaction (PPI) was analyzed chemometrically based on the primary and secondary structures as well as topological or spatial mapping. (3) Results: our findings reveal a unique isoform-specific correspondence between certain segments of the extracellular loops of the Navα subunits. Precisely, loop S5 in domain I forms part of the PPI and assists Navβ1 or Navβ3 on all nine mammalian isoforms. The implied molecular movements resemble macroscopic springs, all of which explains published voltage sensor effects on sodium channel fast inactivation in gating. (4) Conclusions: currently, the specific functions exerted by the Navβ1 or Navβ3 subunits on the modulation of Navα gating remain unknown. Our work determined functional interaction in the extracellular domains on theoretical grounds and we propose a schematic model of the gating mechanism of fast channel sodium current inactivation by educated guessing.

Effects of eslicarbazepine on slow inactivation processes of sodium channels in dentate gyrus granule cells.

Pharmacoresistance is a problem affecting ∼30% of chronic epilepsy patients. An understanding of the mechanisms of pharmacoresistance requires a precise understanding of how antiepileptic drugs interact with their targets in control and epileptic tissue. Although the effects of (S)-licarbazepine (S-Lic) on sodium channel fast inactivation are well understood and have revealed maintained activity in epileptic tissue, it is not known how slow inactivation processes are affected by S-Lic in epilepsy. We have used voltage clamp recordings in isolated dentate granule cells (DGCs) and cortical pyramidal neurons of control versus chronically epileptic rats (pilocarpine model of epilepsy) and in DGCs isolated from hippocampal specimens from temporal lobe epilepsy patients to examine S-Lic effects on sodium channel slow inactivation. S-Lic effects on entry into and recovery from slow inactivation were negligible, even at high concentrations of S-Lic (300 μmol/L). Much more pronounced S-Lic effects were observed on the voltage dependence of slow inactivation, with significant effects at 100 μmol/L S-Lic in DGCs from control and epileptic rats or temporal lobe epilepsy patients. For none of these effects of S-Lic could we observe significant differences either between sham-control and epileptic rats, or between human DGCs and the two animal groups. S-Lic was similarly effective in cortical pyramidal neurons from sham-control and epileptic rats. Finally, we show in expression systems that S-Lic effects on slow inactivation voltage dependence are only observed in Nav 1.2 and Nav 1.6 subunits, but not in Nav 1.1 and Nav 1.3 subunits. From these data, we conclude that a major mechanism of action of S-Lic is an effect on slow inactivation, primarily through effects on slow inactivation voltage dependence of Nav 1.2 and Nav 1.6 channels. Second, we demonstrate that this main effect of S-Lic is maintained in both experimental and human epilepsy and applies to principal neurons of different brain areas.

β1 subunit stabilises sodium channel Nav1.7 against mechanical stress.

The voltage-gated sodium channel Nav1.7 is a key player in neuronal excitability and pain signalling. In addition to voltage sensing, the channel is also modulated by mechanical stress. Using whole-cell patch-clamp experiments, we discovered that the sodium channel subunit β1 is able to prevent the impact of mechanical stress on Nav1.7. An intramolecular disulfide bond of β1 was identified to be essential for stabilisation of inactivation, but not activation, against mechanical stress using molecular dynamics simulations, homology modelling and site-directed mutagenesis. Our results highlight the role of segment 6 of domain IV in fast inactivation. We present a candidate mechanism for sodium channel stabilisation against mechanical stress, ensuring reliable channel functionality in living systems. 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.

Use of Genetically Encoded Photoactivable Cross-Linking Molecules to Probe NaV Channel Fast Inactivation

Fast inactivation in voltage-gated sodium channels requires efficient and rapidly reversible conformational changes in the cytoplasmic linker between domains III and IV (DIII-IV) and their putative receptor in the pore region of the channel. Introduced or acquired mutations in this complex lead to defective channel inactivation, and the resulting excessive sodium conductance can cause a myriad of pathophysiological disorders. We set out to better understand fast-inactivation by expressing voltage-gated sodium channels that carry genetically encoded orthogonal photocrosslinking (PC) amino acid molecules in the inactivation complex with the goal of capturing transient, but physiologically important complexes. PC molecules have widely been used to characterize protein-protein interactions through their ability to covalently bond with nearby molecules upon excitation by UV light. Conventional in-vivo use of PC molecules relies on their covalent attachment to introduced cysteine residues, which must be functionally tolerated and equally solvent accessible, and are limited by non-specific labelling of both target and off target proteins in the cellular environment. We have bypassed these limitations by utilizing the amber stop codon suppression system to genetically incorporate the PC unnatural amino acids (UAA) p-benzoyl-L-phenyl alanine and p-azido-L-phenylalanine into NaV1.5 channels in HEK cells. NaV1.5 channels carrying the UAA molecules showed robust expression at a number of sites in the DIII-IV linker with channel currents that displayed near normal activation, fast and steady state inactivation properties, verifying that the UAAs were well tolerated. Simultaneous patch clamp recording and UV irradiation of channels demonstrated that photo-activating the crosslinking UAA has effects on fast inactivation that are dependent upon the incorporation site and the state of the channel during UV exposure. This technique allows resolution of discrete, transient molecular conformations of the inactivation machinery that support sodium channel fast inactivation.

Flufenamic acid decreases neuronal excitability through modulation of voltage-gated sodium channel gating

The electrophysiological phenotype of individual neurons critically depends on the biophysical properties of the voltage-gated channels they express. Differences in sodium channel gating are instrumental in determining the different firing phenotypes of pyramidal cells and interneurons; moreover, sodium channel modulation represents an important mechanism of action for many widely used CNS drugs. Flufenamic acid (FFA) is a non-steroidal anti-inflammatory drug that has been long used as a blocker of calcium-dependent cationic conductances. Here we show that FFA inhibits voltage-gated sodium currents in hippocampal pyramidal neurons; this effect is dose-dependent with IC(50) = 189 μm. We used whole-cell and nucleated patch recordings to investigate the mechanisms of FFA modulation of TTX-sensitive voltage-gated sodium current. Our data show that flufenamic acid slows down the inactivation process of the sodium current, while shifting the inactivation curve ~10 mV toward more hyperpolarized potentials. The recovery from inactivation is also affected in a voltage-dependent way, resulting in slower recovery at hyperpolarized potentials. Recordings from acute slices demonstrate that FFA reduces repetitive- and abolishes burst-firing in CA1 pyramidal neurons. A computational model based on our data was employed to better understand the mechanisms of FFA action. Simulation data support the idea that FFA acts via a novel mechanism by reducing the voltage dependence of the sodium channel fast inactivation rates. These effects of FFA suggest that it may be an effective anti-epileptic drug.

Electrophysiologic techniques in critical illness-associated weakness

Neuromuscular disorders are increasingly recognized in the critically ill but conventional electrodiagnostic techniques often provide non-specific results or are hampered by local conditions that prevent adequate disease classification. Muscle fiber inexcitability is a common phenomenon in critical illness myopathy possibly secondary to disordered sodium channel fast inactivation and associated with loss of myosin staining. Direct muscle stimulation techniques, measuring evoked response amplitudes and comparison of nerve and muscle stimulated responses, are recognized methods of demonstrating this phenomenon. Other measures studied in this population include increased compound motor action potential duration, motor unit number estimates and mean step area of individual motor unit potentials during motor unit number estimate studies. An electrophysiologic approach to the study of patients with critical illness associated weakness is proposed.

Crucial role of sodium channel fast inactivation in muscle fibre inexcitability in a rat model of critical illness myopathy.

Critical illness myopathy is an acquired disorder in which skeletal muscle becomes electrically inexcitable. We previously demonstrated that inactivation of Na+ channels contributes to inexcitability of affected fibres in an animal model of critical illness myopathy in which denervated rat skeletal muscle is treated with corticosteroids (steroid denervated; SD). Our previous work, however, did not address the relative importance of membrane depolarization versus a shift in the voltage dependence of fast inactivation in causing inexcitability. It also remained unknown whether changes in the voltage dependence of activation or slow inactivation play a role in inexcitability. In the current study we found that a hyperpolarizing shift in the voltage dependence of fast inactivation of Na+ channels is the principal factor underlying inexcitability in SD fibres. Although depolarization tends to decrease excitability, it is insufficient to account for inexcitability in SD fibres since many normal and denervated fibres retain normal excitability when depolarized to the same resting potentials as affected SD fibres. Changes in the voltage dependence of activation and slow inactivation of Na+ channels were also observed in SD fibres; however, the changes appear to increase rather than decrease excitability. These results highlight the importance of the change in fast inactivation in causing inexcitability of SD fibres.

Movement of voltage sensor S4 in domain 4 is tightly coupled to sodium channel fast inactivation and gating charge immobilization.

The highly charged transmembrane segments in each of the four homologous domains (S4D1-S4D4) represent the principal voltage sensors for sodium channel gating. Hitherto, the existence of a functional specialization of the four voltage sensors with regard to the control of the different gating modes, i.e., activation, deactivation, and inactivation, is problematic, most likely due to a functional coupling between the different domains. However, recent experimental data indicate that the voltage sensor in domain 4 (S4D4) plays a unique role in sodium channel fast inactivation. The correlation of fast inactivation and the movement of the S4D4 voltage sensor in rat brain IIA sodium channels was examined by site-directed mutagenesis of the central arginine residues to histidine and by analysis of both ionic and gating currents using a high expression system in Xenopus oocytes and an optimized two-electrode voltage clamp. Mutation R1635H shifts the steady state inactivation to more hyperpolarizing potentials and drastically increases the recovery time constant, thereby indicating a stabilized inactivated state. In contrast, R1638H shifts the steady state inactivation to more depolarizing potentials and strongly increases the inactivation time constant, thereby suggesting a preferred open state occupancy. The double mutant R1635/1638H shows intermediate effects on inactivation. In contrast, the activation kinetics are not significantly influenced by any of the mutations. Gating current immobilization is markedly decreased in R1635H and R1635/1638H but only moderately in R1638H. The time courses of recovery from inactivation and immobilization correlate well in wild-type and mutant channels, suggesting an intimate coupling of these two processes that is maintained in the mutations. These results demonstrate that S4D4 is one of the immobilized voltage sensors during the manifestation of the inactivated state. Moreover, the presented data strongly suggest that S4D4 is involved in the control of fast inactivation.

Inactivation and secondary structure in the D4/S4-5 region of the SkM1 sodium channel.

The D4/S4-5 interhelical region plays a role in sodium channel fast inactivation. Examination of S4-5 primary structure in all domains suggests a possible amphipathic helical conformation in which a conserved group of small hydrophobic residues occupies one contiguous surface with a more variable complement of nonpolar and polar residues on the opposite face. We evaluated this potential structure by replacing each residue in D4/S4-5 of the rat SkM1 skeletal muscle sodium channel with substitutions having different side chain properties. Of the 63 mutations analyzed, 44 produced functional channels. P1473 was intolerant of substitutions. Nonpolar substitutions in the conserved hydrophobic region were functionally similar to wild type, while charged mutations in this region before P1473 were nonfunctional. Charged mutations at F1466, M1469, M1470, and A1474, located on the opposite surface of the predicted helix, produced functional channels with pronounced slowing of inactivation, shifted voltage dependence of steady-state inactivation, and increased rate of recovery from inactivation. The substituted-cysteine-accessibility method was used to probe accessibility at each position. Residues L1465, F1466, A1467, M1469, M1470, L1472, A1474, and F1476C were easily accessible for modification by sulfhydryl reagents; L1464, L1468, S1471, and L1475 were not accessible within the time frame of our measurements. Molecular dynamics simulations of residues A1458 to N1477 were then used to explore energetically favorable local structures. Based on mutagenesis, substituted-cysteine-accessibility method, and modeling results, we suggest a secondary structure for the D4/S4-5 region in which the peptide chain is alpha-helical proximal to P1473, bends at this residue, and may continue beyond this point as a random coil. In this configuration, the entire resultant loop is amphipathic; four residues on one surface could form part of the binding site for the inactivation particle.

Cardiac sodium channel mutations in patients with long QT syndrome, an inherited cardiac arrhythmia.

Long QT syndrome (LQT) is an inherited cardiac disorder that causes syncope, seizures and sudden death from ventricular tachyarrhythmias. We used single-strand conformation polymorphism (SSCP) and DNA sequence analyses to identify mutations in the cardiac sodium channel gene, SCN5A, in affected members of four LQT families. These mutations include two identical intragenic deletions and two missense mutations. These data suggest that SCN5A mutations cause LQT. The location and character of these mutations suggest that this form of LQT results from a delay in cardiac sodium channel fast inactivation or altered voltage-dependence of inactivation.