Inhibitory effect of the recombinant Phoneutria nigriventer Tx1 toxin on voltage-gated sodium channels

Phoneutria nigriventer toxin Tx1 (PnTx1, also referred to in the literature as Tx1) exerts inhibitory effect on neuronal (Na_V1.2) sodium channels in a way dependent on the holding potential, and competes with μ-conotoxins but not with tetrodotoxin for their binding sites. In the present study we investigated the electrophysiological properties of the recombinant toxin (rPnTx1), which has the complete amino acid sequence of the natural toxin with 3 additional residues: AM on the N-terminal and G on the C-terminal. At the concentration of 1.5 μM, the recombinant toxin inhibits Na⁺ currents of dorsal root ganglia neurons (38.4 ± 6.1% inhibition at −80 mV holding potential) and tetrodotoxin-resistant Na⁺ currents (26.2 ± 4.9% at the same holding potential). At −50 mV holding potential the inhibition of the total current reached 71.3 ± 2.3% with 1.5 μM rPnTx1. The selectivity of rPnTx1 was investigated on ten different isoforms of voltage-gated sodium channels expressed in Xenopus oocytes. The order of potency for rPnTx1 was: rNa_V1.2 > rNa_V1.7 ≈ rNa_V1.4 ≥ rNa_V1.3 > mNa_V1.6 ≥ hNa_V1.8. No effect was seen on hNa_V1.5 and on the arthropods isoforms (DmNa_V1, BGNa_V1.1a and VdNa_V1). The IC₅₀ for Na_V1.2 was 33.7 ± 2.9 nM with a maximum inhibition of 83.3 ± 1.9%. The toxin did not alter the voltage-dependence of channel gating and was effective on Na_V1.2 channels devoid of inactivation. It was ineffective on neuronal calcium channels. We conclude that rPnTx1 has a promising selectivity, and that it may be a valuable model to achieve pharmacological activities of interest for the treatment of channelopathies and neuropathic pain.     

Sensitivity of cloned muscle,heart and neuronal voltage-gated sodium channels to block by polyamines: A possible basis for modulation of excitability in vivo

Spermidine and spermine, are endogenous polyamines (PAs) that regulate cell growth and modulate the activity of numerous ion channel proteins. In particular, intracellular PAs are potent blockers of many different cation channels and are responsible for strong suppression of outward K⁺ current, a phenomenon known as inward rectification characteristic of a major class of K_IR K⁺ channels. We previously described block of heterologously expressed voltage-gated Na⁺ channels (Na_V) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal Na_V channels. In this study, we compared the sensitivity of four different cloned mammalian Na_V isoforms to PAs to investigate whether PA block is a common feature of Na_V channel pharmacology. We find that outward Na⁺ current of muscle (Na_V1.4), heart (Na_V1.5), and neuronal (Na_V1.2, Na_V1.7) Na_V isoforms is blocked by PAs, suggesting that PA metabolism may be linked to modulation of action potential firing in numerous excitable tissues. Interestingly, the cardiac Na_V1.5 channel is more sensitive to PA block than other isoforms. Our results also indicate that rapid binding of PAs to blocking sites in the Na_V1.4 channel is restricted to access from the cytoplasmic side of the channel, but plasma membrane transport pathways for PA uptake may contribute to long-term Na_V channel modulation. PAs may also play a role in drug interactions since spermine attenuates the use-dependent effect of the lidocaine, a typical local anesthetic and anti-arrhythmic drug.     

Sensitivity of cloned muscle,heart and neuronal voltage-gated sodium channels to block by polyamines

Spermidine and spermine, are endogenous polyamines (PAs) that regulate cell growth and modulate the activity of numerous ion channel proteins. In particular, intracellular PAs are potent blockers of many different cation channels and are responsible for strong suppression of outward K⁺ current, a phenomenon known as inward rectification characteristic of a major class of K_IR K⁺ channels. We previously described block of heterologously expressed voltage-gated Na⁺ channels (Na_V) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal Na_V channels. In this study, we compared the sensitivity of four different cloned mammalian Na_V isoforms to PAs to investigate whether PA block is a common feature of Na_V channel pharmacology. We find that outward Na⁺ current of muscle (Na_V1.4), heart (Na_V1.5), and neuronal (Na_V1.2, Na_V1.7) Na_V isoforms is blocked by PAs, suggesting that PA metabolism may be linked to modulation of action potential firing in numerous excitable tissues. Interestingly, the cardiac Na_V1.5 channel is more sensitive to PA block than other isoforms. Our results also indicate that rapid binding of PAs to blocking sites in the Na_V1.4 channel is restricted to access from the cytoplasmic side of the channel, but plasma membrane transport pathways for PA uptake may contribute to long-term Na_V channel modulation. PAs may also play a role in drug interactions since spermine attenuates the use-dependent effect of the lidocaine, a typical local anesthetic and anti-arrhythmic drug.     

Mechanisms of a Human Skeletal Myotonia Produced by Mutation in the C-Terminus of NaV1.4: Is Ca2+ Regulation Defective?

Mutations in the cytoplasmic tail (CT) of voltage gated sodium channels cause a spectrum of inherited diseases of cellular excitability, yet to date only one mutation in the CT of the human skeletal muscle voltage gated sodium channel (hNa_V1.4_F1705I) has been linked to cold aggravated myotonia. The functional effects of altered regulation of hNa_V1.4_F1705I are incompletely understood. The location of the hNa_V1.4_F1705I in the CT prompted us to examine the role of Ca²⁺ and calmodulin (CaM) regulation in the manifestations of myotonia. To study Na channel related mechanisms of myotonia we exploited the differences in rat and human Na_V1.4 channel regulation by Ca²⁺ and CaM. hNa_V1.4_F1705I inactivation gating is Ca²⁺-sensitive compared to wild type hNa_V1.4 which is Ca²⁺ insensitive and the mutant channel exhibits a depolarizing shift of the V_1/2 of inactivation with CaM over expression. In contrast the same mutation in the rNa_V1.4 channel background (rNa_V1.4_F1698I) eliminates Ca²⁺ sensitivity of gating without affecting the CaM over expression induced hyperpolarizing shift in steady-state inactivation. The differences in the Ca²⁺ sensitivity of gating between wild type and mutant human and rat Na_V1.4 channels are in part mediated by a divergence in the amino acid sequence in the EF hand like (EFL) region of the CT. Thus the composition of the EFL region contributes to the species differences in Ca²⁺/CaM regulation of the mutant channels that produce myotonia. The myotonia mutation F1705I slows I_Na decay in a Ca²⁺-sensitive fashion. The combination of the altered voltage dependence and kinetics of I_Na decay contribute to the myotonic phenotype and may involve the Ca²⁺-sensing apparatus in the CT of Na_V1.4.     

State-Dependent Inhibition of Inactivation-Deficient CaV1.2 and CaV2.3 Channels By Mibefradil

The structural determinants of mibefradil inhibition were analyzed using wild-type and inactivation-modified Ca_V1.2 (α1C) and Ca_V2.3 (α1E) channels. Mibefradil inhibition of peak Ba²⁺ currents was dose- and voltage-dependent. An increase of holding potentials from −80 to −100 mV significantly shifted dose-response curves toward higher mibefradil concentrations, namely from a concentration of 108 ± 21 μm (n= 7) to 288 ± 17 μm (n= 3) for inhibition of half of the Ca_v1.2 currents (IC ₅₀) and from IC ₅₀= 8 ± 2 μm (n= 9) to 33 ± 7 μm (n= 4) for Ca_V2.3 currents. In the presence of mibefradil, Ca_V1.2 and Ca_V2.3 experienced significant use-dependent inhibition (0.1 to 1 Hz) and slower recovery from inactivation suggesting mibefradil could promote transition(s) to an absorbing inactivated state. In order to investigate the relationship between inactivation and drug sensitivity, mibefradil inhibition was studied in inactivation-altered Ca_V1.2 and Ca_V2.3 mutants. Mibefradil significantly delayed the onset of channel recovery from inactivation in CEEE (Repeat I + part of the I–II linker from Ca_V1.2 in the Ca_V2.3 host channel), in EC(AID)EEE (part of the I–II linker from Ca_V1.2 in the Ca_V2.3 host channel) as well as in Ca_V1.2 E462R, and Ca_V2.3 R378E (point mutation in the β-subunit binding motif) channels. Mibefradil inhibited the faster inactivating chimera EC(IS1-6)EEE with an IC ₅₀= 7 ± 1 μm (n= 3), whereas the slower inactivating chimeras EC(AID)EEE and CEEE were, respectively, inhibited with IC ₅₀= 41 ± 5 μm (n= 4) and IC ₅₀= 68 ± 9 μm (n= 5). Dose-response curves were superimposable for the faster EC(IS1-6)EEE and Ca_V2.3, whereas intermediate-inactivating channel kinetics (CEEE, Ca_V1.2 E462R, and Ca_V1.2 E462K) were inhibited by similar concentrations of mibefradil with IC ₅₀≈ 55–75 μm. The slower Ca_V1.2 wild-type and Ca_V1.2 Q473K channels responded to higher doses of mibefradil with IC ₅₀≈ 100–120 μm. Mibefradil was also found to significantly speed up the inactivation kinetics of slower channels (Ca_V1.2, CEEE) with little effect on the inactivation kinetics of faster-inactivating channels (Ca_V2.3). A open-channel block model for mibefradil interaction with high-voltage-activated Ca²⁺ channels is discussed and shown to qualitatively account for our observations. Hence, our data agree reasonably well with a ``receptor guarded mechanism' where fast inactivation kinetics efficiently trap mibefradil into the channel. Received: 14 March 2001/Revised: 25 June 2001     

Structural basis of cytoplasmic NaV1.5 and NaV1.4 regulation

Voltage-gated sodium channels (Na_Vs) are membrane proteins responsible for the rapid upstroke of the action potential in excitable cells. There are nine human voltage-sensitive Na_V1 isoforms that, in addition to their sequence differences, differ in tissue distribution and specific function. This review focuses on isoforms Na_V1.4 and Na_V1.5, which are primarily expressed in skeletal and cardiac muscle cells, respectively. The determination of the structures of several eukaryotic Na_Vs by single-particle cryo-electron microscopy (cryo-EM) has brought new perspective to the study of the channels. Alignment of the cryo-EM structure of the transmembrane channel pore with x-ray crystallographic structures of the cytoplasmic domains illustrates the complementary nature of the techniques and highlights the intricate cellular mechanisms that modulate these channels. Here, we review structural insights into the cytoplasmic C-terminal regulation of Na_V1.4 and Na_V1.5 with special attention to Ca²⁺ sensing by calmodulin, implications for disease, and putative channel dimerization.     

Modulation of muscle rNav1.4 Na+ channel isoform by arachidonic acid and its non‐metabolized analog

Arachidonic acid (AA) and its metabolic products are important second messengers which exert many biological actions, including modulation of various ion channels. However, the blockage of muscle Na⁺ channel isoforms by AA has not been examined in detail. Here, we investigated the modulating effects of AA on muscle rNa_V1.4 isoforms expressed in human embryonic kidney 293 cells. The results revealed that AA has both activation and inhibitory effects on rNa_V1.4 currents depending on the depolarizing potential: AA increased the rNa_V1.4 current evoked by a depolarization of ?30 or ?40 mV, but significantly decreased the rNa_V1.4 current evoked by a depolarization of membrane potential over ?10 mV. At concentrations of 1–500 µM, the inhibitory effect on the rNa_V1.4 current induced by AA was dose‐dependent and reversible. In addition to modulating the amplitude of the rNa_V1.4 current, AA significantly modulated the steady‐state activation and inactivation properties of rNa_V1.4 channels. Furthermore, treatment with AA resulted in a fairly slow recovery of the rNa_V1.4 channel from inactivation; however, the inhibitory effect of AA was not changed by repetitive pulses or by changing frequency. The effect of AA on rNa_V1.4 currents was completely mimicked by ETYA, the non‐metabolized analog of AA. Our data demonstrated that AA, but not the metabolic products of AA, can voltage‐dependent modulate rNa_V1.4 currents. J. Cell. Physiol. 219: 173–182, 2009. © 2008 Wiley‐Liss, Inc.     

Block of Persistent Late Na+ Currents by Antidepressant Sertraline and Paroxetine

Antidepressants, such as traditional tricyclic antidepressants (TCAs), are the first-line treatment for various pain syndromes. Available evidence indicates that TCAs may target Na⁺ channels for their analgesic action. In this report, we examined the effects of contemporary antidepressants sertraline and paroxetine on (1) neuronal Na⁺ channels expressed in GH₃ cells and (2) muscle rNav1.4 Na⁺ channels heterologously expressed in Hek293t cells. Our results showed that both antidepressants blocked Na⁺ channels in a highly state-dependent manner. The 50% inhibitory concentrations (IC₅₀) for sertraline and paroxetine ranged ∼18–28 μm for resting block and ∼2–8 μm for inactivated block of neuronal and rNav1.4 Na⁺ channels. Surprisingly, the IC₅₀ values for both drugs were about 0.6–0.7 μm for the open channel block of persistent late Na⁺ currents generated through inactivation-deficient rNav1.4 mutant Na⁺ channels. For comparison, the open channel block in neuronal hNav1.7 counterparts yielded IC₅₀ values around 0.3–0.4 μm for both drugs. Receptor mapping using fast inactivation-deficient rNav1.4-F1579A/K mutants with reduced affinities toward local anesthetics (LAs) and TCAs indicated that the F1579 residue is not involved in the binding of sertraline and paroxetine. Thus, sertraline and paroxetine are potent open channel blockers that target persistent late Na⁺ currents preferentially, but their block is not mediated via the phenylalanine residue at the known LA/TCA receptor site.     

Structural and functional insights into the inhibition of human voltage-gated sodium channels by μ-conotoxin KIIIA disulfide isomers

μ-Conotoxins are components of cone snail venom, well-known for their analgesic activity through potent inhibition of voltage-gated sodium channel (Na_V) subtypes, including Na_V1.7. These small, disulfide-rich peptides are typically stabilized by three disulfide bonds arranged in a ‘native’ CysI-CysIV, CysII-CysV, CysIII-CysVI pattern of disulfide connectivity. However, μ-conotoxin KIIIA, the smallest and most studied μ-conotoxin with inhibitory activity at Na_V1.7, forms two distinct disulfide bond isomers during thermodynamic oxidative folding, including Isomer 1 (CysI-CysV, CysII-CysIV, CysIII-CysVI) and Isomer 2 (CysI-CysVI, CysII-CysIV, CysIII-CysV), but not the native μ-conotoxin arrangement. To date, there has been no study on the structure and activity of KIIIA comprising the native μ-conotoxin disulfide bond arrangement. Here, we evaluated the synthesis, potency, sodium channel subtype selectivity, and 3D structure of the three isomers of KIIIA. Using a regioselective disulfide bond-forming strategy, we synthetically produced the three μ-conotoxin KIIIA isomers displaying distinct bioactivity and Na_V subtype selectivity across human Na_V channel subtypes 1.2, 1.4, and 1.7. We show that Isomer 1 inhibits Na_V subtypes with a rank order of potency of Na_V1.4 > 1.2 > 1.7 and Isomer 2 in the order of Na_V1.4≈1.2 > 1.7, while the native isomer inhibited Na_V1.4 > 1.7≈1.2. The three KIIIA isomers were further evaluated by NMR solution structure analysis and molecular docking with hNa_V1.2. Our study highlights the importance of investigating alternate disulfide isomers, as disulfide connectivity affects not only the overall structure of the peptides but also the potency and subtype selectivity of μ-conotoxins targeting therapeutically relevant Na_V subtypes.     

Of cilia,titin, and neurosteroids

Missense mutations at arginine residues in the S4 voltage-sensor domains of Na_V1.4 are an established cause of hypokalemic periodic paralysis, an inherited disorder of skeletal muscle involving recurrent episodes of weakness in conjunction with low serum K⁺. Expression studies in oocytes have revealed anomalous, hyperpolarization-activated gating pore currents in mutant channels. This aberrant gating pore conductance creates a small inward current at the resting potential that is thought to contribute to susceptibility to depolarization in low K⁺ during attacks of weakness. A critical component of this hypothesis is the magnitude of the gating pore conductance relative to other conductances that are active at the resting potential in mammalian muscle: large enough to favor episodes of paradoxical depolarization in low K⁺, yet not so large as to permanently depolarize the fiber. To improve the estimate of the specific conductance for the gating pore in affected muscle, we sequentially measured Na⁺ current through the channel pore, gating pore current, and gating charge displacement in oocytes expressing R669H, R672G, or wild-type Na_V1.4 channels. The relative conductance of the gating pore to that of the pore domain pathway for Na⁺ was 0.03%, which implies a specific conductance in muscle from heterozygous patients of ∼10 µS/cm² or 1% of the total resting conductance.Unexpectedly, our data also revealed a substantial decoupling between gating charge displacement and peak Na⁺ current for both R669H and R672G mutant channels. This decoupling predicts a reduced Na⁺ current density in affected muscle, consistent with the observations that the maximal dV/dt and peak amplitude of the action potential are reduced in fibers from patients with R672G and in a knock-in mouse model of R669H. The defective coupling between gating charge displacement and channel activation identifies a previously unappreciated mechanism that contributes to the reduced excitability of affected fibers seen with these mutations and possibly with other R/X mutations of S4 of Na_V, Ca_V, and K_V channels associated with human disease.     

Structural Pharmacology of Voltage-Gated Sodium Channels

Voltage-gated sodium (Na_V) channels initiate and propagate action potentials in excitable tissues to mediate key physiological processes including heart contraction and nervous system function. Accordingly, Na_V channels are major targets for drugs, toxins and disease-causing mutations. Recent breakthroughs in cryo-electron microscopy have led to the visualization of human Na_V1.1, Na_V1.2, Na_V1.4, Na_V1.5 and Na_V1.7 channel subtypes at high-resolution. These landmark studies have greatly advanced our structural understanding of channel architecture, ion selectivity, voltage-sensing, electromechanical coupling, fast inactivation, and the molecular basis underlying Na_V channelopathies. Na_V channel structures have also been increasingly determined in complex with toxin and small molecule modulators that target either the pore module or voltage sensor domains. These structural studies have provided new insights into the mechanisms of pharmacological action and opportunities for subtype-selective Na_V channel drug design. This review will highlight the structural pharmacology of human Na_V channels as well as the potential use of engineered and chimeric channels in future drug discovery efforts.     

cAMP/PKA Pathways and S56 Phosphorylation Are Involved in AA/PGE2-Induced Increases in rNaV1.4 Current

Arachidonic acid (AA) and its metabolites are important second messengers for ion channel modulation. The effects of extracellular application of AA and its non-metabolized analogue on muscle rNa_V1.4 Na⁺ current has been studied, but little is known about the effects of intracellular application of AA on this channel isoform. Here, we report that intracellular application of AA significantly augmented the rNa_V1.4 current peak without modulating the steady-state activation and inactivation properties of the rNa_V1.4 channel. These results differed from the effects of extracellular application of AA on rNa_V1.4 current. The effects of intracellular AA were mimicked by prostaglandin E₂ but not eicosatetraynoic acid (ETYA), the non-metabolized analogue of AA, and were eliminated by treatment with cyclooxygenase inhibitors, flufenamic acid, or indomethacin. AA/PGE₂-induced activation of rNa_V1.4 channels was mimicked by a cAMP analogue (db-cAMP) and eliminated by a PKA inhibitor, PKAi. Furthermore, inhibition of EP2 and EP4 (PGE₂ receptors) with AH6809 and AH23848 reduced the intracellular AA/PGE₂-induced increase of rNa_V1.4 current. Two mutated channels, rNa_V1.4S56A and rNa_V1.4T21A, were designed to investigate the role of predicted phosphorylation sites in the AA/PGE₂–mediated regulation of rNa_V1.4 currents. In rNa_V1.4S56A, the effects of intracellular db-cAMP, AA, and PGE₂ were significantly reduced. The results of the present study suggest that intracellular AA augments rNa_V1.4 current by PGE₂/EP receptor-mediated activation of the cAMP/PKA pathway, and that the S56 residue on the channel protein is important for this process.     

Arachidonic acid inhibition of L-type calcium (CaV1.3b) channels varies with accessory CaVβ subunits

Arachidonic acid (AA) inhibits the activity of several different voltage-gated Ca²⁺ channels by an unknown mechanism at an unknown site. The Ca²⁺ channel pore-forming subunit (Ca_Vα₁) is a candidate for the site of AA inhibition because T-type Ca²⁺ channels, which do not require accessory subunits for expression, are inhibited by AA. Here, we report the unanticipated role of accessory Ca_Vβ subunits on the inhibition of Ca_V1.3b L-type (L-) current by AA. Whole cell Ba²⁺ currents were measured from recombinant channels expressed in human embryonic kidney 293 cells at a test potential of −10 mV from a holding potential of −90 mV. A one-minute exposure to 10 µM AA inhibited currents with β_1b, β₃, or β₄ 58, 51, or 44%, respectively, but with β_2a only 31%. At a more depolarized holding potential of −60 mV, currents were inhibited to a lesser degree. These data are best explained by a simple model where AA stabilizes Ca_V1.3b in a deep closed-channel conformation, resulting in current inhibition. Consistent with this hypothesis, inhibition by AA occurred in the absence of test pulses, indicating that channels do not need to open to become inhibited. AA had no effect on the voltage dependence of holding potential–dependent inactivation or on recovery from inactivation regardless of Ca_Vβ subunit. Unexpectedly, kinetic analysis revealed evidence for two populations of L-channels that exhibit willing and reluctant gating previously described for Ca_V2 channels. AA preferentially inhibited reluctant gating channels, revealing the accelerated kinetics of willing channels. Additionally, we discovered that the palmitoyl groups of β_2a interfere with inhibition by AA. Our novel findings that the Ca_Vβ subunit alters kinetic changes and magnitude of inhibition by AA suggest that Ca_Vβ expression may regulate how AA modulates Ca²⁺-dependent processes that rely on L-channels, such as gene expression, enzyme activation, secretion, and membrane excitability.     

Interleukin-1β inhibits voltage-gated sodium currents in a time- and dose-dependent manner in cortical neurons

Interleukin-1β (IL-1β) is a multifunctional proinflammatory cytokine that plays a key role in the injuries and diseases of the central nervous system (CNS). A voltage-gated Na⁺ channel is essential for the excitability and electrical properties of neurons. However, it is not known whether IL-1β directly affects the central Na⁺ channels. In the present study, we examined the effects of IL-1β on Na⁺ currents in cultured cortical neurons using patch-clamp recording. Our results showed that IL-1β suppressed Na⁺ currents through its receptor in a time- and dose-dependent manner, but did not alter the voltage-dependent activation and inactivation. PKC and then p38 MAPK were involved in this inhibition. The spike amplitude was also inhibited by IL-1β in the doses that decreased the Na⁺ currents. Our findings revealed the inhibition of chronic IL-1β treatment on voltage-gated Na⁺ channels in the CNS, and showed that the action potential (AP) amplitude was reduced by IL-1β due to a decrease of Na⁺ currents.     

Electrophysiological characterization of Tityus obscurus β toxin 1 (To1) on Na+-channel isoforms

To1, previously named Tc49b, is a peptide neurotoxin isolated from venom of the scorpion Tityus obscurus that is responsible for lethal human poisoning cases in the Brazilian Amazonian region. Previously, To1 was shown to be lethal to mice and to change Na⁺ permeation in cerebellum granular neurons from rat brain. In addition, To1 did not affect Shaker B K⁺ channels. Based on sequence similarities, To1 was described as a β-toxin. In the present work, To1 was purified from T. obscurus venom and submitted to an electrophysiological characterization in human and invertebrate Na_V channels. The analysis of the electrophysiological experiments reveal that To1 enhances the open probability at more negative potentials of human Na_V 1.3 and 1.6, of the insect channel BgNa_V1 and of arachnid VdNa_V1 channel. In addition, To1 reduces the peak of Na⁺ currents in some of the Na_Vs tested. These results support the classification of the To1 as a β-toxin. A structure and functional comparison to other β-toxins that share sequence similarity to To1 is also presented.     

Molecular Dynamics Study of Binding of μ-Conotoxin GIIIA to the Voltage-Gated Sodium Channel Nav1.4

Homology models of mammalian voltage-gated sodium (Na_V) channels based on the crystal structures of the bacterial counterparts are needed to interpret the functional data on sodium channels and understand how they operate. Such models would also be invaluable in structure-based design of therapeutics for diseases involving sodium channels such as chronic pain and heart diseases. Here we construct a homology model for the pore domain of the Na_V1.4 channel and use the functional data for the binding of µ-conotoxin GIIIA to Na_V1.4 to validate the model. The initial poses for the Na_V1.4–GIIIA complex are obtained using the HADDOCK protein docking program, which are then refined in molecular dynamics simulations. The binding mode for the final complex is shown to be in broad agreement with the available mutagenesis data. The standard binding free energy, determined from the potential of mean force calculations, is also in good agreement with the experimental value. Because the pore domains of Na_V1 channels are highly homologous, the model constructed for Na_V1.4 will provide an excellent template for other Na_V1 channels.     

Positive inotropic action of NMDA receptor antagonist (+)-MK801 in rat heart

(+)-MK801, a noncompetitive NMDA receptor antagonist, was reported to exhibit anticonvulsive and neuroprotective activities during the postischemic period. Intravenous administration of (+)-MK801 produced tachycardia in rats, but bradycardia in pigs. We examined the mechanical and electrophysiological effects of (+)-MK801 on rat cardiac tissues. (+)-MK801 dose-dependently increased (3–100 µM) twitch tension in rat atria and ventricular strips. The spontaneous beating rate in rat right atria, however, was dose-dependently decreased by (+)-MK801. The inotropic effect of (+)-MK801 was affected neither by ₁-antagonist (1 µM prazosin) nor by ₁-adrenoceptor antagonist (3 µM atenolol), but significantly by a transient outward K⁺ channel blocker (3 mM 4-aminopyridine). (+)-MK801 did not cause any significant change of intracellular cAMP content. Electrophysiological study in rat ventricular cells revealed that (+)-MK801 concentration-dependently prolonged the action potential duration with a concomitant decrease in the maximum rate of the action potential upstroke (V_max) and an increase in the recovery time constant of V_max. Voltage clamp study showed that (+)-MK801 (3 µM) reduced inward Na⁺ current (I_Na), along with a slowing of its recovery from inactivation and a slight negative shift of its voltage-dependent steady-state inactivation curves. At a much higher concentration (30 µM), (+)-MK801 slightly reduced the amplitude of L-type calcium inward current (I_Ca), although the voltage dependence of its steady-state inactivation was unaffected. For the potassium currents in rat ventricular cells, 3 µM of (+)-MK801 reduced the peak transient outward current (I_to), steady-state outward current (I_ss) and inward current through K₁ channels. The inhibition of I_to was associated with a prominent negative shift in the voltage dependence of its steady-state inactivation curve. The outward current through K₁ channels was unaffected. These results indicate that (+)-MK801 may be a strong I_Na and I_to blocker with some I_Ca blocking activity. The inhibition of I_to and other K⁺ efflux would prolong action potential duration, produce positive inotropic action and contribute to the negative chronotropic effect of (+)-MK801.     

Is ZD7288 a selective blocker of hyperpolarization-activated cyclic nucleotide-gated channel currents?

ZD7288 has been widely used as a tool in the study of hyperpolarization-activated cyclic nucleotide-gated channels (HCN channels), and to test the relationships between HCN channels and heart and brain function. ZD7288 is widely considered a selective blocker of HCN currents. Here we show that ZD7288 inhibits not only HCN channel currents, but also Na⁺ currents in DRG neurons and ZD7288 was confirmed to inhibit Na⁺ current in HEK293 cells transfected with Na_v1.4 plasmids. Thus our findings challenge the view that ZD7288 is a selective blocker of HCN channels. Conclusions about the role of NCN channels in neuronal function should be re-evaluated if based exclusively on the effect of ZD7288.     

Is ZD7288 a selective blocker of hyperpolarization-activated cyclic nucleotide-gated channel currents?

ZD7288 has been widely used as a tool in the study of hyperpolarization-activated cyclic nucleotide-gated channels (HCN channels), and to test the relationships between HCN channels and heart and brain function. ZD7288 is widely considered a selective blocker of HCN currents. Here we show that ZD7288 inhibits not only HCN channel currents, but also Na⁺ currents in DRG neurons and ZD7288 was confirmed to inhibit Na⁺ current in HEK293 cells transfected with Na_v1.4 plasmids. Thus our findings challenge the view that ZD7288 is a selective blocker of HCN channels. Conclusions about the role of NCN channels in neuronal function should be re-evaluated if based exclusively on the effect of ZD7288.