fast inactivation
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2022 ◽  
Vol 12 ◽  
Author(s):  
Jennifer R. Deuis ◽  
Lotten Ragnarsson ◽  
Samuel D. Robinson ◽  
Zoltan Dekan ◽  
Lerena Chan ◽  
...  

Venoms from cone snails and arachnids are a rich source of peptide modulators of voltage-gated sodium (NaV) channels, however relatively few venom-derived peptides with activity at the mammalian NaV1.8 subtype have been isolated. Here, we describe the discovery and functional characterisation of β-theraphotoxin-Eo1a, a peptide from the venom of the Tanzanian black and olive baboon tarantula Encyocratella olivacea that modulates NaV1.8. Eo1a is a 37-residue peptide that increases NaV1.8 peak current (EC50 894 ± 146 nM) and causes a large hyperpolarising shift in both the voltage-dependence of activation (ΔV50–20.5 ± 1.2 mV) and steady-state fast inactivation (ΔV50–15.5 ± 1.8 mV). At a concentration of 10 μM, Eo1a has varying effects on the peak current and channel gating of NaV1.1–NaV1.7, although its activity is most pronounced at NaV1.8. Investigations into the binding site of Eo1a using NaV1.7/NaV1.8 chimeras revealed a critical contribution of the DII S3-S4 extracellular loop of NaV1.8 to toxin activity. Results from this work may form the basis for future studies that lead to the rational design of spider venom-derived peptides with improved potency and selectivity at NaV1.8.


2021 ◽  
Vol 12 ◽  
Author(s):  
Shuijiao Peng ◽  
Minzhi Chen ◽  
Zhen Xiao ◽  
Xin Xiao ◽  
Sen Luo ◽  
...  

Venomous animals have evolved to produce peptide toxins that modulate the activity of voltage-gated sodium (Nav) channels. These specific modulators are powerful probes for investigating the structural and functional features of Nav channels. Here, we report the isolation and characterization of δ-theraphotoxin-Gr4b (Gr4b), a novel peptide toxin from the venom of the spider Grammostola rosea. Gr4b contains 37-amino acid residues with six cysteines forming three disulfide bonds. Patch-clamp analysis confirmed that Gr4b markedly slows the fast inactivation of Nav1.9 and inhibits the currents of Nav1.4 and Nav1.7, but does not affect Nav1.8. It was also found that Gr4b significantly shifts the steady-state activation and inactivation curves of Nav1.9 to the depolarization direction and increases the window current, which is consistent with the change in the ramp current. Furthermore, analysis of Nav1.9/Nav1.8 chimeric channels revealed that Gr4b preferentially binds to the voltage-sensor of domain III (DIII VSD) and has additional interactions with the DIV VSD. The site-directed mutagenesis analysis indicated that N1139 and L1143 in DIII S3-S4 linker participate in toxin binding. In sum, this study reports a novel spider peptide toxin that may slow the fast inactivation of Nav1.9 by binding to the new neurotoxin receptor site-DIII VSD. Taken together, these findings provide insight into the functional role of the Nav channel DIII VSD in fast inactivation and activation.


2021 ◽  
Vol 12 ◽  
Author(s):  
Anastasia K. Zaytseva ◽  
Aleksandr S. Boitsov ◽  
Anna A. Kostareva ◽  
Boris S. Zhorov

Motion transmission from voltage sensors to inactivation gates is an important problem in the general physiology of ion channels. In a cryo-EM structure of channel hNav1.5, residues N1736 and R1739 in the extracellular loop IVP2-S6 approach glutamates E1225 and E1295, respectively, in the voltage-sensing domain III (VSD-III). ClinVar-reported variants E1230K, E1295K, and R1739W/Q and other variants in loops IVP2-S6, IIIS1-S2, and IIIS3-S4 are associated with cardiac arrhythmias, highlighting the interface between IVP2-S6 and VSD-III as a hot spot of disease mutations. Atomic mechanisms of the channel dysfunction caused by these mutations are unknown. Here, we generated mutants E1295R, R1739E, E1295R/R1739E, and N1736R, expressed them in HEK-293T cells, and explored biophysical properties. Mutation E1295R reduced steady-state fast inactivation and enhanced steady-state slow inactivation. In contrast, mutation R1739E slightly enhanced fast inactivation and attenuated slow inactivation. Characteristics of the double mutant E1295R/R1739E were rather similar to those of the wild-type channel. Mutation N1736R attenuated slow inactivation. Molecular modeling predicted salt bridging of R1739E with the outermost lysine in the activated voltage-sensing helix IIIS4. In contrast, the loss-of-function substitution E1295R repelled R1739, thus destabilizing the activated VSD-III in agreement with our data that E1295R caused a depolarizing shift of the G-V curve. In silico deactivation of VSD-III with constraint-maintained salt bridge E1295-R1739 resulted in the following changes: 1) contacts between IIIS4 and IVS5 were switched; 2) contacts of the linker-helix IIIS4-S5 with IVS5, IVS6, and fast inactivation tripeptide IFM were modified; 3) contacts of the IFM tripeptide with helices IVS5 and IVS6 were altered; 4) mobile loop IVP2-S6 shifted helix IVP2 that contributes to the slow inactivation gate and helix IVS6 that contributes to the fast inactivation gate. The likelihood of salt bridge E1295-R1739 in deactivated VSD-III is supported by Poisson–Boltzmann calculations and state-dependent energetics of loop IVP2-S6. Taken together, our results suggest that loop IVP2-S6 is involved in motion transmission from VSD-III to the inactivation gates.


Biomedicines ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 1141
Author(s):  
Chiung-Wei Huang ◽  
Pi-Chen Lin ◽  
Jian-Lin Chen ◽  
Ming-Jen Lee

Cannabidiol (CBD), one of the cannabinoids from the cannabis plant, can relieve the myotonia resulting from sodium channelopathy, which manifests as repetitive discharges of muscle membrane. We investigated the binding kinetics of CBD to Nav1.4 channels on the muscle membrane. The binding affinity of CBD to the channel was evaluated using whole-cell recording. The CDOCKER program was employed to model CBD docking onto the Nav1.4 channel to determine its binding sites. Our results revealed no differential inhibition of sodium current by CBD when the channels were in activation or fast inactivation status. However, differential inhibition was observed with a dose-dependent manner after a prolonged period of depolarization, leaving the channel in a slow-inactivated state. Moreover, CBD binds selectively to the slow-inactivated state with a significantly faster binding kinetics (>64,000 M−1 s−1) and a higher affinity (Kd of fast inactivation vs. slow-inactivation: >117.42 μM vs. 51.48 μM), compared to the fast inactivation state. Five proposed CBD binding sites in a bundle crossing region of the Nav1.4 channels pore was identified as Val793, Leu794, Phe797, and Cys759 in domain I/S6, and Ile1279 in domain II/S6. Our findings imply that CBD favorably binds to the Nav1.4 channel in its slow-inactivated state.


2021 ◽  
Author(s):  
Erva Bayraktar ◽  
Yuanyuan Liu ◽  
Ulrike B.S. Hedrich ◽  
Yildirim Sara ◽  
Holger Lerche ◽  
...  

Background and Purpose: Among genetic epilepsies, variants in sodium channel coding genes constitute a major subgroup. Variants in SCN8A, the coding gene for NaV1.6 channels, are characterized by a variety of symptoms including intractable epileptic seizures, psychomotor delay, progressive cognitive decline, and others such as autistic features, ataxia or dystonia. Standard anticonvulsant treatment has only limited impact on the course of disease. Experimental Approach: Personalized therapeutic regimens tailored to disease-causing pathophysiological mechanisms may offer the specificity required to overcome intractability. Toward this aim, we investigated in vitro in neuroblastoma cells the effects of S-Licarbazepine, a third-generation dibenzazepine and enhancer of slow inactivation of voltage gated sodium channels, on three gain-of-function NaV1.6 variants linked to representative phenotypes of mild epilepsy (G1475R), developmental and epileptic encephalopathy (M1760I) and intellectual disability without epilepsy (A1622D). Key Results: S-Licarbazepine strongly enhances the slow and — less pronounced — the fast inactivation of NaV1.6 wildtype channels. It acts similarly on all tested variants and irrespective of their particular biophysical dysfunction mechanism. Beyond that S–Licarbazepine has variant-specific effects including a partial reversal of pathologically slowed fast inactivation dynamics (A1622D, M1760I) and a trend to reduce the enhanced persistent Na+ current by A1622D variant channels. Conclusion and Implications: These data bring out that S-Licarbazepine not only owns substance-specific effects, but also holds variant-specific effects, which can variably contribute to functional compensation of distinct channel-specific biophysical properties and thereby highlighting the role of personalized approaches, which likely will be key to improved and successful treatment not only of SCN8A-related disease.


2021 ◽  
Vol 153 (5) ◽  
Author(s):  
Emilio Carbone

Using Nav1.3 and FGF14 KO mice, Martinez-Espinosa et al. provide new findings on how intracellular FGF14 proteins interfere with the endogenous fast inactivation gating and regulate the “long-term inactivation” of Nav1.3 channels that sets Nav channel availability and spike adaptation during sustained stimulation in adrenal chromaffin cells.


2021 ◽  
Vol 118 (11) ◽  
pp. e2100069118
Author(s):  
Zhangqiang Li ◽  
Xueqin Jin ◽  
Tong Wu ◽  
Xin Zhao ◽  
Weipeng Wang ◽  
...  

Nav1.5 is the primary voltage-gated Na+ (Nav) channel in the heart. Mutations of Nav1.5 are associated with various cardiac disorders exemplified by the type 3 long QT syndrome (LQT3) and Brugada syndrome (BrS). E1784K is a common mutation that has been found in both LQT3 and BrS patients. Here we present the cryo-EM structure of the human Nav1.5-E1784K variant at an overall resolution of 3.3 Å. The structure is nearly identical to that of the wild-type human Nav1.5 bound to quinidine. Structural mapping of 91- and 178-point mutations that are respectively associated with LQT3 and BrS reveals a unique distribution pattern for LQT3 mutations. Whereas the BrS mutations spread evenly on the structure, LQT3 mutations are clustered mainly to the segments in repeats III and IV that are involved in gating, voltage-sensing, and particularly inactivation. A mutational hotspot involving the fast inactivation segments is identified and can be mechanistically interpreted by our “door wedge” model for fast inactivation. The structural analysis presented here, with a focus on the impact of mutations on inactivation and late sodium current, establishes a structure-function relationship for the mechanistic understanding of Nav1.5 channelopathies.


2021 ◽  
Vol 153 (4) ◽  
Author(s):  
Pedro L. Martinez-Espinosa ◽  
Chengtao Yang ◽  
Xiao-Ming Xia ◽  
Christopher J. Lingle

Adrenal chromaffin cells (CCs) in rodents express rapidly inactivating, tetrodotoxin (TTX)-sensitive sodium channels. The resulting current has generally been attributed to Nav1.7, although a possible role for Nav1.3 has also been suggested. Nav channels in rat CCs rapidly inactivate via two independent pathways which differ in their time course of recovery. One subpopulation recovers with time constants similar to traditional fast inactivation and the other ∼10-fold slower, but both pathways can act within a single homogenous population of channels. Here, we use Nav1.3 KO mice to probe the properties and molecular components of Nav current in CCs. We find that the absence of Nav1.3 abolishes all Nav current in about half of CCs examined, while a small, fast inactivating Nav current is still observed in the rest. To probe possible molecular components underlying slow recovery from inactivation, we used mice null for fibroblast growth factor homology factor 14 (FGF14). In these cells, the slow component of recovery from fast inactivation is completely absent in most CCs, with no change in the time constant of fast recovery. The use dependence of Nav current reduction during trains of stimuli in WT cells is completely abolished in FGF14 KO mice, directly demonstrating a role for slow recovery from inactivation in determining Nav current availability. Our results indicate that FGF14-mediated inactivation is the major determinant defining use-dependent changes in Nav availability in CCs. These results establish that Nav1.3, like other Nav isoforms, can also partner with FGF subunits, strongly regulating Nav channel function.


2021 ◽  
Vol 153 (4) ◽  
Author(s):  
Pedro L. Martinez-Espinosa ◽  
Alan Neely ◽  
Jiuping Ding ◽  
Christopher J. Lingle

Voltage-dependent sodium (Nav) current in adrenal chromaffin cells (CCs) is rapidly inactivating and tetrodotoxin (TTX)–sensitive. The fractional availability of CC Nav current has been implicated in regulation of action potential (AP) frequency and the occurrence of slow-wave burst firing. Here, through recordings of Nav current in rat CCs, primarily in adrenal medullary slices, we describe unique inactivation properties of CC Nav inactivation that help define AP firing rates in CCs. The key feature of CC Nav current is that recovery from inactivation, even following brief (5 ms) inactivation steps, exhibits two exponential components of similar amplitude. Various paired pulse protocols show that entry into the fast and slower recovery processes result from largely independent competing inactivation pathways, each of which occurs with similar onset times at depolarizing potentials. Over voltages from −120 to −80 mV, faster recovery varies from ∼3 to 30 ms, while slower recovery varies from ∼50 to 400 ms. With strong depolarization (above −10 mV), the relative entry into slow or fast recovery pathways is similar and independent of voltage. Trains of short depolarizations favor recovery from fast recovery pathways and result in cumulative increases in the slow recovery fraction. Dual-pathway fast inactivation, by promoting use-dependent accumulation in slow recovery pathways, dynamically regulates Nav availability. Consistent with this finding, repetitive AP clamp waveforms at 1–10 Hz frequencies reduce Nav availability 80–90%, depending on holding potential. These results indicate that there are two distinct pathways of fast inactivation, one leading to conventional fast recovery and the other to slower recovery, which together are well-suited to mediate use-dependent changes in Nav availability.


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