Inactivation Gating Research Articles

An improved method for the cost-effective expression and purification of large quantities of KcsA

KcsA, the bacterial K+ channel from Streptomyces lividans, is the prototypical model system to study the functional and structural correlations of the pore domain of eukaryotic voltage-gated K+ channels (Kv channels). It contains all the molecular elements responsible for ion conduction, activation, deactivation and inactivation gating [1]. KcsA’s structural simplicity makes it highly amenable for structural studies. Therefore, it is methodological advantageous to produce large amounts of functional and properly folded KcsA in a cost-effective manner. In the present study, we show an optimized protocol for the over-expression and purification of large amounts of high-quality, fully functional and crystallizable KcsA using inexpensive detergents, which significantly lowered the cost of the purification process.

Generation Mechanism of Alternans in Luo–Rudy Model

Electrical alternans is the alternating amplitude from beat to beat in the action potential of the cardiac cell. It has been associated with ventricular arrhythmias in many clinical studies; however, its dynamical mechanisms remain unknown. The reason is that we do not have realistic network models of the heart system. Recently, Yazawa clarified the network structure of the heart and the central nerve system in the crustacean heart. In this study, we construct a simple model of the heart system based on Yazawa’s experimental data. Using this model, we clarify that two parameters (the conductance of sodium ions and free concentration of potassium ions in the extracellular compartment) play the key roles of generating alternans. In particular, we clarify that the inactivation gate of the time-independent potassium channel is the most important parameter. Moreover, interaction between the membrane potential and potassium ionic currents is significant for generating alternate rhythms. This result indicates that if the muscle cell has problems such as channelopathies, there is great risk of generating alternans.

Cloning and molecular characterization of a putative voltage-gated sodium channel gene in the crayfish.

Voltage-gated sodium channel genes and associated proteins have been cloned and studied in many mammalian and invertebrate species. However, there is no data available about the sodium channel gene(s) in the crayfish, although the animal has frequently been used as a model to investigate various aspects of neural cellular and circuit function. In the present work, by using RNA extracts from crayfish abdominal ganglia samples, the complete open reading frame of a putative sodium channel gene has firstly been cloned and molecular properties of the associated peptide have been analyzed. The open reading frame of the gene has a length of 5793 bp that encodes for the synthesis of a peptide, with 1930 amino acids, that is 82% similar to the α-peptide of a sodium channel in a neighboring species, Cancer borealis. The transmembrane topology analysis of the crayfish peptide indicated a pattern of four folding domains with several transmembrane segments, as observed in other known voltage-gated sodium channels. Upon analysis of the obtained sequence, functional regions of the putative sodium channel responsible for the selectivity filter, inactivation gate, voltage sensor, and phosphorylation have been predicted. The expression level of the putative sodium channel gene, as defined by a qPCR method, was measured and found to be the highest in nervous tissue.

BK channel inactivation gates daytime excitability in the circadian clock.

Inactivation is an intrinsic property of several voltage-dependent ion channels, closing the conduction pathway during membrane depolarization and dynamically regulating neuronal activity. BK K+ channels undergo N-type inactivation via their β2 subunit, but the physiological significance is not clear. Here, we report that inactivating BK currents predominate during the day in the suprachiasmatic nucleus, the brain's intrinsic clock circuit, reducing steady-state current levels. At night inactivation is diminished, resulting in larger BK currents. Loss of β2 eliminates inactivation, abolishing the diurnal variation in both BK current magnitude and SCN firing, and disrupting behavioural rhythmicity. Selective restoration of inactivation via the β2 N-terminal ‘ball-and-chain' domain rescues BK current levels and firing rate, unexpectedly contributing to the subthreshold membrane properties that shift SCN neurons into the daytime ‘upstate'. Our study reveals the clock employs inactivation gating as a biophysical switch to set the diurnal variation in suprachiasmatic nucleus excitability that underlies circadian rhythm.

Editorial: Recent Advances in Voltage-Gated Sodium Channels, their Pharmacology and Related Diseases.

Editorial: Recent Advances in Voltage-Gated Sodium Channels, their Pharmacology and Related Diseases.

Computational Modeling of Sodium Channel Inactivation

Fast inactivation of the voltage-gated sodium (NaV) channel is crucial in the regulation of the membrane potential in neural and muscle cells. A “hinged lid” mechanism has been proposed to explain the rapid inactivation, in which the inactivation gate (a motif comprised of the intracellular linker between domain III and IV) occludes the channel pore. Interestingly, recent cross-link data suggest a close functional coupling between the inactivation gate and domain IV voltage sensor (DIV VS). However, there is no complete structure available for a eukaryotic NaV channel to support such coupling and reveal the related inactivation mechanism. In the present study, an atomic structure of a eukaryotic NaV channel, including both the transmembrane domain and the inactivation gate, was built by homology modeling and molecular dynamics refinement. The model suggests that the inactivation gate consists of a flexible linker with three short alpha helices. Despite its high flexibility, the inactivation gate mostly interacts with the central pore and DIV VS, a result that agrees with cross-link experimental data. Novel hydrophobic interactions between DIV VS and the inactivation gate have been identified as being key in the stabilization of the inactivated state.

Dynamic Interplay of Calmodulin and Fibroblast Growth Factor Homologous Factors in Regulating NA Channels

In recent years, the ubiquitous Ca2+-binding protein calmodulin (CaM), and the non-canonical intracellular fibroblast growth factor homologous factors (FHF) have emerged as two potent modulators of voltage-gated sodium channels with profound implications for the excitability of neuronal, cardiac, and skeletal muscle cells. Curiously, both regulatory molecules associate with the Na channel through a well-conserved channel carboxy-terminus with CaM preassociating to a canonical IQ motif and FHF interacting with the closely apposed preIQ - EF hand surface. Even so, the interaction of these auxiliary proteins is thought to evoke distinct and independent channel regulatory effects. CaM interaction with the Na channel confers a rapid Ca2+-dependent feedback regulation (Cell 157(7):1657) while FHF appear to alter various voltage-dependent properties of the canonical channel fast inactivation gate (Cell. Mol. Life Sci. 59:1067). Here, we demonstrate a novel role of FHF to allosterically suppress Ca2+/CaM-dependent regulation of skeletal muscle Na channels (NaV1.4). In the absence of FHF, the wild-type NaV1.4 channels exhibit rapid and robust Ca2+-dependent inhibition mediated by a resident CaM. However, co-expression of FHF1 entirely abolishes Ca2+-dependent inhibition of these channels. Similarly, co-expression of various isoforms of FHF (FHF2-4) with NaV1.4 results in a graded suppression of Ca2+/CaM regulation. These results argue that Ca2+-regulation of Na channels can be potently tuned by differential expression of various FHF isoforms, as are often associated with various pathophysiological conditions. Overall, the dynamic interplay of CaM and FHF identified here vastly enriches the modulatory landscape of Na channels and highlights the sophisticated mechanisms by which cytosolic proteins can tune cellular excitability.

Biophysical Characterization of the Honeybee's DSC1 Ortholog Highlights a New Voltage Dependant Calcium Channel Subfamily

The bilaterian voltage-gated sodium channel (Nav) would have evolved from the voltage-gated calcium channel (Cav). This hypothesis is supported by the existence of channels such as the Drosophila sodium channel 1 (DSC1) which features a DEEA selectivity sequence, an intermediate between the selectivity sequence of Cav and Nav channels.Initially, the DSC1 channel was classified as a calcium permeable sodium channel. Further phylogenetic investigations classified DSC1 and its homologs as Nav2 channels since their sequence would be more similar to known Nav channels although insect proteins investigated were more permeable to calcium than sodium.Here, we report the cloning, the molecular and biophysical characterization of the honeybee (Apis mellifera) DSC1 ortholog. The cloning of this channel revealed several sequence variations. When expressed in Xenopus oocytes, the channel exhibits slow activation and inactivation kinetics reminiscent of Cav channels. Furthermore, the channel is not inhibited by tetrodotoxin but is blocked by cadmium. Our results also show that the channel is calcium selective and is permeable to barium as well as marginally permeable to lithium. Magnesium and sodium did not yield measurable currents.Based on these properties, the existence of the DEEA selectivity filter and of an inactivation gate motif, we propose to classify DSC1 homologs as Cav4 rather than Nav2. Indeed, channels which feature the DEEA selectivity sequence are likely to feature the same properties as the honeybee's channel i.e. calcium as the most permeable physiological cation, slow activation and inactivation kinetics and small single channel conductance. Furthermore, we speculate that Cav channels might have evolved toward ancestral Nav channels in response to pressure to acquire an inactivation mechanism as Cav4 are calcium selective, but feature the inactivation gate motif which is characteristic of Nav channels.

Ginsenoside Rg3 Activates Human EAG Family of K+ Channels via Allosteric Modification of Gating

The human ether-a-go-go (EAG) family of voltage-gated K+ channels, including hERG, hEAG, and hELK exhibit variable biophysical and physiological properties. Ginsenoside Rg3 (Rg3), a steroid glycoside is a potent hERG channel activator. Here we characterize the mechanisms of action of Rg3 on three EAG-family representative channels, including hERG1, hEAG1 and hELK1 heterologously expressed in Xenopus laevis oocytes. Rg3 enhanced hERG1 channel current via a negative shift in the half-point for the voltage dependence of activation (V0.5act) that saturated at −14 mV, and a profound slowing in the rate of deactivation (EC50 = 242 nM). Rg3 had no effect on the rate of activation or the voltage dependence of hERG1 inactivation gating. For hEAG1 channels, Rg3 induced a −28 mV maxiumal shift in V0.5act (EC50 = 1.18 µM) and accelerated the rate of activation (∼20-fold faster). The maximum shift in V0.5act for hELK1 channels was much greater (>-100 mV; EC50 = 176 nM). The onset of Rg3 effects upon extracellular application was very fast and rapidly reversible upon washout for all 3 channel types, indicating this large molecule (MW = 785) binds to an extracellular region. Rg3 at 3 µM had no effect on Kv1.5 channels and only affects other Kv channels at much higher concentrations. Understanding the mechanisms of action of Rg3 may facilitate the development of more potent and selective gensenosides for therapeutic use.

Molecular Locations of Gates in Potassium Channels

Understanding how ion channels open and close their pores is crucial for understanding their physiological roles. We used intracellular quaternary ammonium blockers to locate the voltage and calcium-dependent gates in MthK potassium channels from Methanobacterium thermoautotrophicum with electrophysiology, stopped-flow spectrofluorometry, and X-ray crystallography. Blockers bind in an aqueous cavity between two putative gates, an intracellular gate and the selectivity filter. Thus, these blockers directly probe gate location: an intracellular gate will prevent binding when closed, whereas a selectivity filter gate will always allow binding.A kinetic single-channel analysis of tetrabutylammonium block of MthK channels combined with X-ray crystallographic analysis of the pore with tetrabutylantimony unequivocally determined that the voltage-dependent gate, like the C-type inactivation gate in eukaryotic channels, is located at the selectivity filter. State-dependent binding kinetics suggests that MthK gating with voltage also leads to conformational changes within the cavity and intracellular pore entrance.For locating the calcium gate, we employed a Tl+ flux assay using a stopped-flow spectrofluorometer. MthK channel activity was estimated from the rate of a MthK-containing liposome-trapped fluorophore quenching due to Tl+ influx through the channels. The high-affinity blocker tetrapentylammonium, applied prior to channel activation using a sequential mixing protocol, was able to fully block closed channels, indicating that the blocker can reach its binding site in closed channels. Given that the blocker binding site is in the cytoplasmic access below the selectivity filter, these results suggest that there is also a calcium-dependent gate at the selectivity filter in MthK.

Supernormal Conduction and Suppression of Spatially Discordant Alternans of Cardiac Action Potentials.

Spatially discordant alternans (DA) of action potential durations (APD) is thought to be more pro-arrhythmic than concordant alternans. Super normal conduction (SNC) has been reported to suppress formation of DA. An increase in conduction velocity (CV) as activation rate increases, i.e., a negative CV restitution, is widely considered as hallmark of SNC. Our aim in this study is to show that it is not an increase in CV for faster rates that prevents formation of DA, rather, it is the ratio of the CV for the short relative to the long activation that is critical in DA suppression. To illustrate this subtlety, we simulated this phenomenon using two approaches; (1) by using the standard, i.e., S1S2 protocol to quantify restitution and disabling the slow inactivation gate j of the sodium current (INa), and (2) by using the dynamic, i.e., S1S1 protocol for quantification of restitution and increasing INa at different cycle lengths (CL). Even though both approaches produced similar CV restitution curves, DA was suppressed only during the first approach, where the CV of the short of the long-short action potential (AP) pattern was selectively increased. These results show that negative CV restitution, which is considered characteristic of SNC, per se, is not causal in suppressing DA, rather, the critical factor is a change in the ratio of the velocities of the short and the long APs.

Controlling spiral wave and spatiotemporal chaos in cardiac tissues by slowing sodium channel activation and inactivation

Much evidence shows that the appearance and instability of the spiral wave in cardiac tissue can be linked to a kind of heart disease. Therefore there needs a method of controlling spiral wave more safely and effectively. The intelligent modification of specific ion channel to achieve desired control is the future direction of gene therapy in heart disease. The key question that has to be answered is which ion channel is the best candidate for controlling spiral wave. Modern biological technology has been able to make the mutation of sodium channel gene to change its relaxation time constant. In this paper, we adopt the Luo-Rudy phase I model to investigate how to regulate the relaxation time constant of sodium channel gate to control spiral wave and spatiotemporal chaos in cardiac tissues. We suggest a control strategy which slows down the rate of sodium current activation and inactivation by increasing the relaxation time constant of the sodium activation gate by up to times while its fast inactivation gate is clamped to 0.77. Numerical simulation results show that a gradual increase of will cause the activation gate of sodium current to reach maximum more slowly, and its amplitude is gradually reduced, so that the amplitude and duration of the action potential of cardiomyocyte are gradually reduced. When the factor is large enough, the spiral wave and spatiotemporal chaos cannot propagate in the medium except planar wave with low frequency. The reason is that the excitabilities of medium and wave speed significantly decrease. Therefore, the spiral waves and spatiotemporal chaos can be effectively eliminated when the control time is properly selected and the factor is large enough. Spiral wave and spatiotemporal chaos disappear mainly due to conduction obstacle. In some cases, spiral wave can disappear through the transition from spiral wave to target wave or tip retraction. Spatiotemporal chaos disappears after spatiotemporal chaos has evolved into meandering spiral wave. When the parameters are chosen properly, the phenomenon that spiral wave evolves into a self-sustained target wave is also observed. The corresponding target wave source is the pair of spiral waves with opposite rotation directions. These results can provide useful information for gene therapy in heart disease.

Physiology and Pathophysiology of Sodium Channel Inactivation.

Voltage-gated sodium channels are present in different tissues within the human body, predominantly nerve, muscle, and heart. The sodium channel is composed of four similar domains, each containing six transmembrane segments. Each domain can be functionally organized into a voltage-sensing region and a pore region. The sodium channel may exist in resting, activated, fast inactivated, or slow inactivated states. Upon depolarization, when the channel opens, the fast inactivation gate is in its open state. Within the time frame of milliseconds, this gate closes and blocks the channel pore from conducting any more sodium ions. Repetitive or continuous stimulations of sodium channels result in a rate-dependent decrease of sodium current. This process may continue until the channel fully shuts down. This collapse is known as slow inactivation. This chapter reviews what is known to date regarding, sodium channel inactivation with a focus on various mutations within each NaV subtypeand with clinical implications.

Modulation of the voltage-gated potassium channel Kv2.1 by the anti-tumor alkylphospholipid perifosine.

The aim of the present study was to assess the effects of perifosine-a third generation alkylphospholipid analog with anti-tumor properties-on the activity of Kv2.1 channels. The whole-cell patch clamp technique was applied to follow the modulatory effect of perifosine on Kv2.1 channels expressed in HEK293 cells. Obtained data provide evidence that perifosine application decreases the whole cell Kv2.1 currents in a concentration-independent manner. Perifosine induces a hyperpolarizing shift in the voltage dependence of Kv2.1 channels inactivation without altering the voltage dependence of channels activation. The kinetics of Kv2.1 closed-state inactivation was accelerated by perifosine, with no significant effects on the recovery rate from inactivation. Taken together, these results show that perifosine modified the Kv2.1 inactivation gating resulting in a decrease of the current amplitude. These data will help to elucidate the mechanism of action of this promising anti-cancer drug on ion channels and their possible implications.

Modulation of Kv2.1 channels inactivation by curcumin.

The aim of the present study was to assess the effects of curcumin on the voltage-dependent Kv2.1 potassium channel. The whole-cell patch-clamp technique was used to explore the regulation of Kv2.1 channels expressed in HEK293 cells by curcumin. Curcumin reduced the Kv2.1 currents; the inhibition occurred with a slow time course and was partially reversible. Curcumin did not alter the kinetics and voltage dependence of activation; however, the kinetics of open- and closed-state inactivation was accelerated by curcumin along with a hyperpolarizing shift in the voltage dependence of inactivation. Curcumin inhibition of Kv2.1 current was not use-dependent. Overall, our data suggest that curcumin inhibits Kv2.1 channels by modulating the inactivation gating, which would be expected to impact cellular physiology.

A novel mutation from gene splicing of a voltage-gated sodium channel in a marine copepod and its potential effect on channel function

The saxitoxins (STX), a group of potent neurotoxins produced by some marine algae (dinoflagellates and cyanobacteria), block voltage-gated sodium channels, inhibiting nerve-signal transmission in consumers of STX-bearing prey. Populations of grazers (clams and copepods) persistently exposed to the STX-bearing dinoflagellate Alexandrium fundyense are less susceptible to STX than naïve ones. Adaptation to STX in clams is linked to a point mutation at the STX-binding site in the sodium channel, which dramatically lowers the sensitivity to the toxin (STX resistance). The present study tested if a similar mechanism of STX resistance occurs in the copepod Acartia hudsonica. Our cloning and sequencing results indicate that two full-length cDNA variants (AhNaV1 and AhNaV2) of the sodium channel exist in A. hudsonica, which result from alternative splicing of the single coding gene. Both variants have identical nucleotide sequences except that AhNav1 (the putative mutant isoform) contains a three-amino-acid (GRD) insertion and a single adjacent aa-substitution (A to V) close to the inactivation gate on the cytoplasmic linker between domains III and IV of the sodium channel. All individuals express both AhNaV1 and AhNaV2 in varying proportions. The functional consequences of the mutation were studied by inserting the three-amino acid codons into a rat (rNav1.2) sodium channel expressed in both Xenopus oocytes and HEK cells. Currents carried by construct rNav1.2 bearing the GRD insertion did not inactivate as completely, and recovered faster from inactivation than rNav1.2. These two rNav1.2 constructs were, however, equally sensitive to STX, suggesting that the GRD variation does not confer STX resistance on the rat sequence of Nav1.2. These results render unlikely the hypothesis that this novel mutation is responsible for the adaptation (via resistance) of A. hudsonica to STX-bearing prey.

Heterologous expression of NaV1.9 chimeras in various cell systems.

SCN11A encodes the voltage-gated sodium channel NaV1.9, which deviates most strongly from the other eight NaV channels expressed in mammals. It is characterized by resistance to the prototypic NaV channel blocker tetrodotoxin and exhibits slow activation and inactivation gating. Its expression in dorsal root ganglia neurons suggests a role in motor or pain signaling functions as also recently demonstrated by the occurrence of various mutations in human SCN11A leading to altered pain sensation syndromes. The systematic investigation of human NaV1.9, however, is severely hampered because of very poor heterologous expression in host cells. Using patch-clamp and two-electrode voltage-clamp methods, we show that this limitation is caused by the C-terminal structure of NaV1.9. A chimera of NaV1.9 harboring the C terminus of NaV1.4 yields functional expression not only in neuronal cells but also in non-excitable cells, such as HEK 293T or Xenopus oocytes. The major functional difference of the chimeric channel with respect to NaV1.9 is an accelerated activation and inactivation. Since the entire transmembrane domain is preserved, it is suited for studying pharmacological properties of the channel and the functional impact of disease-causing mutations. Moreover, we demonstrate how mutation S360Y makes NaV1.9 channels sensitive to tetrodotoxin and saxitoxin and that the unusual slow open-state inactivation of NaV1.9 is also mediated by the IFM (isoleucine-phenylalanine-methionine) inactivation motif located in the linker connecting domains III and IV.

The Link between Inactivation and High-Affinity Block of hERG1 Channels.

Block of human ether-à-go-go-related gene 1 (hERG1) K(+) channels by many drugs delays cardiac repolarization, prolongs QT interval, and is associated with an increased risk of cardiac arrhythmia. Preferential block of hERG1 channels in an inactivated state has been assumed because inactivation deficient mutant channels can exhibit dramatically reduced drug sensitivity. Here we reexamine the link between inactivation gating and potency of channel block using concatenated hERG1 tetramers containing a variable number (0-4) of subunits harboring a point mutation (S620T or S631A) that disrupts inactivation. Concatenated hERG1 tetramers containing four wild-type subunits exhibited high-affinity block by cisapride, dofetilide, and MK-499, similar to wild-type channels formed from hERG1 monomers. A single S620T subunit within a tetramer was sufficient to fully disrupt inactivation gating, whereas S631A suppressed inactivation as a graded function of the number of mutant subunits present in a concatenated tetramer. Drug potency was positively correlated to the number of S620T subunits contained within a tetramer but unrelated to mutation-induced disruption of channel inactivation. Introduction of a second point mutation (Y652W) into S620T hERG1 partially rescued drug sensitivity. The potency of cisapride was not altered for tetramers containing 0 to 3 S631A subunits, whereas the potency of dofetilide was a graded function of the number of S631A subunits contained within a tetramer. Together these findings indicate that S620T or S631A substitutions can allosterically disrupt drug binding by a mechanism that is independent of their effects on inactivation gating.

Voltage gating by molecular subunits of Na+ and K+ ion channels: higher-dimensional cubic kinetics, rate constants, and temperature.

The structural similarity between the primary molecules of voltage-gated Na and K channels (alpha subunits) and activation gating in the Hodgkin-Huxley model is brought into full agreement by increasing the model's sodium kinetics to fourth order (m(3) → m(4)). Both structures then virtually imply activation gating by four independent subprocesses acting in parallel. The kinetics coalesce in four-dimensional (4D) cubic diagrams (16 states, 32 reversible transitions) that show the structure to be highly failure resistant against significant partial loss of gating function. Rate constants, as fitted in phase plot data of retinal ganglion cell excitation, reflect the molecular nature of the gating transitions. Additional dimensions (6D cubic diagrams) accommodate kinetically coupled sodium inactivation and gating processes associated with beta subunits. The gating transitions of coupled sodium inactivation appear to be thermodynamically irreversible; response to dielectric surface charges (capacitive displacement) provides a potential energy source for those transitions and yields highly energy-efficient excitation. A comparison of temperature responses of the squid giant axon (apparently Arrhenius) and mammalian channel gating yields kinetic Q10 = 2.2 for alpha unit gating, whose transitions are rate-limiting at mammalian temperatures; beta unit kinetic Q10 = 14 reproduces the observed non-Arrhenius deviation of mammalian gating at low temperatures; the Q10 of sodium inactivation gating matches the rate-limiting component of activation gating at all temperatures. The model kinetics reproduce the physiologically large frequency range for repetitive firing in ganglion cells and the physiologically observed strong temperature dependence of recovery from inactivation.

Dynamics of the KcsA Selectivity Filter Probed using Intrinsic Tyrosine Fluorescence

KcsA is activated by intracellular protons through a large conformational change in the inner helical-bundle gate. The selectivity filter and surrounding structures play a crucial role in ion conduction as an inactivation gate. To monitor the conformational dynamics of the selectivity filter under various experimental conditions we engineered a KcsA construct devoid of tryptophan and tyrosine residues with the exception of residue Y78, locatd in the middle of the selectivity filter. Fluorescence lifetimes indicate that the microenvironment polarity of Y78-KcsA decreases upon gating, irrespective of the type of ion (K+, Rb+, NH4+, Cs+) present in the selectivity filter. Further, time-resolved anisotropy measurements indicate that the dynamics associated with the segmental mobility of the selectivity filter is significantly restricted, and this gating-driven motional restriction is more pronounced in High K+ and Rb+ conditions. Red Edge Excitation Shift (REES) measurements of Y78-KcsA show slow water relaxation dynamics under conditions that stabilize conductive and non-conductive filter conformations, suggesting the presence of bound/restricted water molecules in both filter conformations. Interestingly, opening the lower gate appears to further restrict the water molecules in the vicinity of the selectivity filter. Overall, our results show that the conformational dynamics associated with the selectivity filters of collapsed (Low K+) and open-inactivated KcsA are significantly different despite showing essentially identical conformations as derived from crystal structures. This highlights the importance of hydration dynamics in gating of K+ channels.