Molecular action of lidocaine on the voltage sensors of sodium channels

Block of sodium ionic current by lidocaine is associated with alteration of the gating charge-voltage (Q-V) relationship characterized by a 38% reduction in maximal gating charge (Q(max)) and by the appearance of additional gating charge at negative test potentials. We investigated the molecular basis of the lidocaine-induced reduction in cardiac Na channel-gating charge by sequentially neutralizing basic residues in each of the voltage sensors (S4 segments) in the four domains of the human heart Na channel (hH1a). By determining the relative reduction in the Q(max) of each mutant channel modified by lidocaine we identified those S4 segments that contributed to a reduction in gating charge. No interaction of lidocaine was found with the voltage sensors in domains I or II. The largest inhibition of charge movement was found for the S4 of domain III consistent with lidocaine completely inhibiting its movement. Protection experiments with intracellular MTSET (a charged sulfhydryl reagent) in a Na channel with the fourth outermost arginine in the S4 of domain III mutated to a cysteine demonstrated that lidocaine stabilized the S4 in domain III in a depolarized configuration. Lidocaine also partially inhibited movement of the S4 in domain IV, but lidocaine's most dramatic effect was to alter the voltage-dependent charge movement of the S4 in domain IV such that it accounted for the appearance of additional gating charge at potentials near -100 mV. These findings suggest that lidocaine's actions on Na channel gating charge result from allosteric coupling of the binding site(s) of lidocaine to the voltage sensors formed by the S4 segments in domains III and IV.     

Molecular properties of sodium and calcium channels

Voltage-gated sodium and calcium channels are responsible for inward movement of sodium and calcium during electrical signals in cell membranes. Their principal subunits are members of a gene family and can function as voltage-gated ion channels by themselves. They are expressed in association with one or more auxiliary subunits which increase functional expression and modify the functional properties of the principal subunits. Structural elements which are required for voltage-dependent activation, selective ion conductance, and inactivation have been identified, and their mechanisms of action are being explored through mutagenesis, expression in heterologous cells, and functional analysis. These experiments reveal that these two channels are built on a common structural theme with variations appropriate for functional specialization of each channel type.     

Block of Brain Sodium Channels by Peptide Mimetics of the Isoleucine,Phenylalanine, and Methionine (IFM) Motif from the Inactivation Gate

Inactivation of sodium channels is thought to be mediated by an inactivation gate formed by the intracellular loop connecting domains III and IV. A hydrophobic motif containing the amino acid sequence isoleucine, phenylalanine, and methionine (IFM) is required for the inactivation process. Peptides containing the IFM motif, when applied to the cytoplasmic side of these channels, produce two types of block: fast block, which resembles the inactivation process, and slow, use-dependent block stimulated by strong depolarizing pulses. Fast block by the peptide ac-KIFMK-NH₂, measured on sodium channels whose inactivation was slowed by the α-scorpion toxin from Leiurus quinquestriatus (LqTx), was reversed with a time constant of 0.9 ms upon repolarization. In contrast, control and LqTx-modified sodium channels were slower to recover from use-dependent block. For fast block, linear peptides of three to six amino acid residues containing the IFM motif and two positive charges were more effective than peptides with one positive charge, whereas uncharged IFM peptides were ineffective. Substitution of the IFM residues in the peptide ac-KIFMK-NH₂ with smaller, less hydrophobic residues prevented fast block. The positively charged tripeptide IFM-NH₂ did not cause appreciable fast block, but the divalent cation IFM-NH(CH₂)₂NH₂ was as effective as the pentapeptide ac-KIFMK-NH₂. The constrained peptide cyclic KIFMK containing two positive charges did not cause fast block. These results indicate that the position of the positive charges is unimportant, but flexibility or conformation of the IFM-containing peptide is important to allow fast block. Slow, use-dependent block was observed with IFM-containing peptides of three to six residues having one or two positive charges, but not with dipeptides or phenylalanine-amide. In contrast to its lack of fast block, cyclic KIFMK was an effective use-dependent blocker. Substitutions of amino acid residues in the tripeptide IFM-NH₂ showed that large hydrophobic residues are preferred in all three positions for slow, use-dependent block. However, substitution of the large hydrophobic residue diphenylalanine or the constrained residues phenylglycine or tetrahydroisoquinoline for phe decreased potency, suggesting that this phe residue must be able to enter a restricted hydrophobic pocket during the binding of IFM peptides. Together, the results on fast block and slow, use-dependent block indicate that IFM peptides form two distinct complexes of different stability and structural specificity with receptor site(s) on the sodium channel. It is proposed that fast block represents binding of these peptides to the inactivation gate receptor, while slow, use-dependent block represents deeper binding of the IFM peptides in the pore.     

Activation and closed-state inactivation mechanisms of the human voltage-gated KV4 channel complexes

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External barium affects the gating of KCNQ1 potassium channels and produces a pore block via two discrete sites

The pore properties and the reciprocal interactions between permeant ions and the gating of KCNQ channels are poorly understood. Here we used external barium to investigate the permeation characteristics of homomeric KCNQ1 channels. We assessed the Ba(2+) binding kinetics and the concentration and voltage dependence of Ba(2+) steady-state block. Our results indicate that extracellular Ba(2+) exerts a series of complex effects, including a voltage-dependent pore blockade as well as unique gating alterations. External barium interacts with the permeation pathway of KCNQ1 at two discrete and nonsequential sites. (a) A slow deep Ba(2+) site that occludes the channel pore and could be simulated by a model of voltage-dependent block. (b) A fast superficial Ba(2+) site that barely contributes to channel block and mostly affects channel gating by shifting rightward the voltage dependence of activation, slowing activation, speeding up deactivation kinetics, and inhibiting channel inactivation. A model of voltage-dependent block cannot predict the complex impact of Ba(2+) on channel gating in low external K(+) solutions. Ba(2+) binding to this superficial site likely modifies the gating transitions states of KCNQ1. Both sites appear to reside in the permeation pathway as high external K(+) attenuates Ba(2+) inhibition of channel conductance and abolishes its impact on channel gating. Our data suggest that despite the high degree of homology of the pore region among the various K(+) channels, KCNQ1 channels display significant structural and functional uniqueness.     

Selective open-channel block of Shaker (Kv1) potassium channels by s-nitrosodithiothreitol (SNDTT).

Large quaternary ammonium (QA) ions block voltage-gated K(+) (Kv) channels by binding with a 1:1 stoichiometry in an aqueous cavity that is exposed to the cytoplasm only when channels are open. S-nitrosodithiothreitol (SNDTT; ONSCH(2)CH(OH)CH(OH)CH(2)SNO) produces qualitatively similar "open-channel block" in Kv channels despite a radically different structure. SNDTT is small, electrically neutral, and not very hydrophobic. In whole-cell voltage-clamped squid giant fiber lobe neurons, bath-applied SNDTT causes reversible time-dependent block of Kv channels, but not Na(+) or Ca(2)+ channels. Inactivation-removed ShakerB (ShBDelta) Kv1 channels expressed in HEK 293 cells are similarly blocked and were used to study further the action of SNDTT. Dose-response data are consistent with a scheme in which two SNDTT molecules bind sequentially to a single channel, with binding of the first being sufficient to produce block. The dissociation constant for the binding of the second SNDTT molecule (K(d2) = 0.14 mM) is lower than that of the first molecule (K(d1) = 0.67 mM), indicating cooperativity. The half-blocking concentration (K(1/2)) is approximately 0.2 mM. Steady-state block by this electrically neutral compound has a voltage dependence (about -0.3 e(0)) similar in magnitude but opposite in directionality to that reported for QA ions. Both nitrosyl groups on SNDTT (one on each sulfur atom) are required for block, but transfer of these reactive groups to channel cysteine residues is not involved. SNDTT undergoes a slow intramolecular reaction (tau approximately 770 s) in which these NO groups are liberated, leading to spontaneous reversal of the SNDTT effect. Competition with internal tetraethylammonium indicates that bath-applied SNDTT crosses the cell membrane to act at an internal site, most likely within the channel cavity. Finally, SNDTT is remarkably selective for Kv1 channels. When individually expressed in HEK 293 cells, rat Kv1.1-1.6 display profound time-dependent block by SNDTT, an effect not seen for Kv2.1, 3.1b, or 4.2.     

Structure,function and expression of voltage-dependent sodium channels

Voltage-dependent sodium channels control the transient inward current responsible for the action potential in most excitable cells. Members of this multigene family have been cloned, sequenced, and functionally expressed from various tissues and species, and common features of their structure have clearly emerged. Site-directed mutagenesis coupled with in vitro expression has provided additional insight into the relationship between structure and function. Subtle differences between sodium channel isoforms are also important, and aspects of the regulation of sodium channel gene expression and the modulation of channel function are becoming topics of increasing importance. Finally, sodium channel mutations have been directly linked to human disease, yielding insight into both disease pathophysiology and normal channel function. After a brief discussion of previous work, this review will focus on recent advances in each of these areas.     

Gating of the bacterial sodium channel, NaChBac: voltage-dependent charge movement and gating currents

The bacterial sodium channel, NaChBac, from Bacillus halodurans provides an excellent model to study structure-function relationships of voltage-gated ion channels. It can be expressed in mammalian cells for functional studies as well as in bacterial cultures as starting material for protein purification for fine biochemical and biophysical studies. Macroscopic functional properties of NaChBac have been described previously (Ren, D., B. Navarro, H. Xu, L. Yue, Q. Shi, and D.E. Clapham. 2001. Science. 294:2372-2375). In this study, we report gating current properties of NaChBac expressed in COS-1 cells. Upon depolarization of the membrane, gating currents appeared as upward inflections preceding the ionic currents. Gating currents were detectable at -90 mV while holding at -150 mV. Charge-voltage (Q-V) curves showed sigmoidal dependence on voltage with gating charge saturating at -10 mV. Charge movement was shifted by -22 mV relative to the conductance-voltage curve, indicating the presence of more than one closed state. Consistent with this was the Cole-Moore shift of 533 micros observed for a change in preconditioning voltage from -160 to -80 mV. The total gating charge was estimated to be 16 elementary charges per channel. Charge immobilization caused by prolonged depolarization was also observed; Q-V curves were shifted by approximately -60 mV to hyperpolarized potentials when cells were held at 0 mV. The kinetic properties of NaChBac were simulated by simultaneous fit of sodium currents at various voltages to a sequential kinetic model. Gating current kinetics predicted from ionic current experiments resembled the experimental data, indicating that gating currents are coupled to activation of NaChBac and confirming the assertion that this channel undergoes several transitions between closed states before channel opening. The results indicate that NaChBac has several closed states with voltage-dependent transitions between them realized by translocation of gating charge that causes activation of the channel.     

Investigating the size and dynamics of voltage-gated sodium channel fenestrations: A molecular dynamics study

Eukaryotic voltage-gated sodium channels (VGSCs) are essential for the initiation and propagation of action potentials in electrically excitable cells, and are important pharmaceutical targets for the treatment of neurological disorders such as epilepsy, cardiac arrhythmias, and chronic pain. Evidence suggests that small, hydrophobic, VGSC-blocking drugs can gain access to binding residues within the central cavity of these channels by passing through lateral, lipid-filled “fenestrations” which run between the exterior of the protein and its central pore. Here, we use molecular dynamics simulations to investigate how the size and shape of fenestrations change over time in several bacterial VGSC models and a homology model of Nav1.4. We show that over the course of the simulations, the size of the fenestrations is primarily influenced by rapid protein motions, such as amino acid side-chain rotation, and highlight that differences between fenestration bottleneck-contributing residues are the primary cause of variations in fenestration size between the 6 bacterial models. In the eukaryotic channel model, 2 fenestrations are wide, but 2 are narrow due to differences in the amino acid sequence in the 4 domains. Lipid molecules are found to influence the size of the fenestrations by protruding acyl chains into the fenestrations and displacing amino acid side-chains. Together, the results suggest that fenestrations provide viable pathways for small, flexible, hydrophobic drugs.     

Potassium, sodium, calcium and glutamate-gated channels: pore architecture and ligand action

In the last decade, the idea of common organization of certain ion channel families exhibiting diverse physiological and pharmacological properties has received strong experimental support. Transmembrane topologies and patterns of the pore-facing residues are conserved in P-loop channels that include high-selective cation channels and certain ligand-gated channels. X-ray structures of bacterial K+ channels, KcsA, MthK and KvAP, help to understand structure-function relationships of other P-loop channels. Data on binding sites and mechanisms of action of ligands of K+, Na+, Ca2+ and glutamate gated ion channels are considered in view of their possible structural similarity to the bacterial K+ channels. Emphasized are structural determinants of ligand-receptor interactions within the channels and mechanisms of state-dependent action of the ligands.     

烟碱对脑钾、钠和钙通道表达的调节作用

目的:利用基因表达芯片检测反复摄取烟碱对大鼠脑内钾、钠和钙通道基因表达的调节作用.方法:大鼠每天两次皮下注射烟碱(1.2 mg/kg),连续用药两周后取全脑,提取RNA,逆转录合成cDNA,转录合成生物素化RNA,并将其片断化后与芯片杂交,对荧光信号扫描分析.结合RT-PCR方法对芯片分析结果进行验证.结果:反复摄入烟碱,大鼠脑内钾、钠和钙通道的基因表达均发生变化:电压依赖性K 通道中外向整流K 通道和Ca2 激活的K 通道表达下调,而Kv2.3r等电压依赖性K 通道表达上调;电压依赖性Na 通道中β2亚基表达增加,而α和β1亚基基因表达减少;电压依赖性Ca2 通道的β3亚基基因表达增加.结论:反复摄取烟碱诱导脑N受体失敏时,可引起相关钾、钠和钙通道基因表达发生改变.     

Investigating the size and dynamics of voltage-gated sodium channel fenestrations

Eukaryotic voltage-gated sodium channels (VGSCs) are essential for the initiation and propagation of action potentials in electrically excitable cells, and are important pharmaceutical targets for the treatment of neurological disorders such as epilepsy, cardiac arrhythmias, and chronic pain. Evidence suggests that small, hydrophobic, VGSC-blocking drugs can gain access to binding residues within the central cavity of these channels by passing through lateral, lipid-filled “fenestrations” which run between the exterior of the protein and its central pore. Here, we use molecular dynamics simulations to investigate how the size and shape of fenestrations change over time in several bacterial VGSC models and a homology model of Nav1.4. We show that over the course of the simulations, the size of the fenestrations is primarily influenced by rapid protein motions, such as amino acid side-chain rotation, and highlight that differences between fenestration bottleneck-contributing residues are the primary cause of variations in fenestration size between the 6 bacterial models. In the eukaryotic channel model, 2 fenestrations are wide, but 2 are narrow due to differences in the amino acid sequence in the 4 domains. Lipid molecules are found to influence the size of the fenestrations by protruding acyl chains into the fenestrations and displacing amino acid side-chains. Together, the results suggest that fenestrations provide viable pathways for small, flexible, hydrophobic drugs.     

Permeation of large tetra-alkylammonium cations through mutant and wild-type voltage-gated sodium channels as revealed by relief of block at high voltage

Many large organic cations are potent blockers of K(+) channels and other cation-selective channels belonging to the P-region superfamily. However, the mechanism by which large hydrophobic cations enter and exit the narrow pores of these proteins is obscure. Previous work has shown that a conserved Lys residue in the DEKA locus of voltage-gated Na(+) channels is an important determinant of Na(+)/K(+) discrimination, exclusion of Ca(2+), and molecular sieving of organic cations. In this study, we sought to determine whether the Lys(III) residue of the DEKA locus interacts with internal tetra-alkylammonium cations (TAA(+)) that block Na(+) channels in a voltage-dependent fashion. We investigated block by a series of TAA(+) cations of the wild-type rat muscle Na(+) channel (DEKA) and two different mutants of the DEKA locus, DEAA and DERA, using whole-cell recording. TEA(+) and larger TAA(+) cations block both wild-type and DEAA channels. However, DEAA exhibits dramatic relief of block by large TAA(+) cations as revealed by a positive inflection in the macroscopic I-V curve at voltages greater than +140 mV. Paradoxically, relief of block at high positive voltage is observed for large (e.g., tetrapentylammonium) but not small (e.g., TEA(+)) symmetrical TAA(+) cations. The DEKA wild-type channel and the DERA mutant exhibit a similar relief-of-block phenomenon superimposed on background current rectification. The results indicate: (a) hydrophobic TAA(+) cations with a molecular diameter as large as 15 A can permeate Na(+) channels from inside to outside when driven by high positive voltage, and (b) the Lys(III) residue of the DEKA locus is an important determinant of inward rectification and internal block in Na(+) channels. From these observations, we suggest that hydrophobic interfaces between subunits, pseudosubunits, or packed helices of P-region channel proteins may function in facilitating blocker access to the pore, and may thus play an important role in the blocking and permeation behavior of large TAA(+) cations and potentially other kinds of local anesthetic molecules.     

Activation of Shaker Potassium Channels : III. An Activation Gating Model for Wild-Type and V2 Mutant Channels

A functional kinetic model is developed to describe the activation gating process of the Shaker potassium channel. The modeling in this paper is constrained by measurements described in the preceding two papers, including macroscopic ionic and gating currents and single channel ionic currents. These data were obtained from the normally activating wild-type channel as well as a mutant channel V2, in which the leucine at position 382 has been mutated to a valine. Different classes of models that incorporate Shaker''s symmetrical tetrameric structure are systematically examined. Many simple gating models are clearly inadequate, but a model that can account for all of the qualitative features of the data has the channel open after its four subunits undergo three transitions in sequence, and two final transitions that reflect the concerted action of the four subunits. In this model, which we call Scheme 3+2′, the channel can also close to several states that are not part of the activation path. Channel opening involves a large total charge movement (10.8 e₀), which is distributed among a large number of small steps each with rather small charge movements (between 0.6 and 1.05 e₀). The final two transitions are different from earlier steps by having slow backward rates. These steps confer a cooperative mechanism of channel opening at Shaker''s activation voltages. In the context of Scheme 3+2′, significant effects of the V2 mutation are limited to the backward rates of the final two transitions, implying that L382 plays an important role in the conformational stability of the final two states.     

Blockade of potassium channels in the plasmalemma ofChara corallina by tetraethylammonium,Ba2+, Na+ and Cs+

Summary The outer membranes of plant cells contain channels which are highly selective for K⁺. In the giant-celled green algaChara corallina, K⁺ currents in the plasmalemma were measured during the action potential and when the cell was depolarized to the K⁺ equilibrium potential in high external K⁺ concentrations. Currents in both conditions were reduced by externally added tetraethylammonium (TEA⁺), Ba²⁺, Na⁺ and Cs⁺. In contrast to inhibition by TEA⁺, the latter three ions inhibited inward K⁺ current in a voltage-dependent manner, and reduced inward current more than outward. Ba²⁺ and Na⁺ also appeared to inhibit outward current in a strongly voltage-dependent manner. The blockade by Cs⁺ is studied in more detail in the following paper. TEA⁺ inhibited both inward and outward currents in a largely voltage-independent manner, with an apparentK _D of about 0.7 to 1.1mm, increasing with increasing external K⁺. All inhibitors reduced current towards a similar linear leak, suggesting an insensitivity of the background leak inChara to these various K⁺ channel inhibitors. The selectivity of the channel to various monovalent cations varied depending on the method of measurement, suggesting that ion movement through the K⁺-selective channel may not be independent.     

Common molecular determinants of flecainide and lidocaine block of heart Na+ channels: evidence from experiments with neutral and quaternary flecainide analogues

Flecainide (pKa 9.3, 99% charged at pH 7.4) and lidocaine (pKa 7.6-8.0, approximately 50% neutral at pH 7.4) have similar structures but markedly different effects on Na(+) channel activity. Both drugs cause well-characterized use-dependent block (UDB) of Na(+) channels due to stabilization of the inactivated state, but flecainide requires that channels first open before block develops, whereas lidocaine is believed to bind directly to the inactivated state. To test whether the charge on flecainide might determine its state specificity of Na(+) channel blockade, we developed two flecainide analogues, NU-FL (pKa 6.4), that is 90% neutral at pH 7.4, and a quaternary flecainide analogue, QX-FL, that is fully charged at physiological pH. We examined the effects of flecainide, NU-FL, QX-FL, and lidocaine on human cardiac Na(+) channels expressed in human embryonic kidney (HEK) 293 cells. At physiological pH, NU-FL, like lidocaine but not flecainide, interacts preferentially with inactivated channels without prerequisite channel opening, and causes minimal UDB. We find that UDB develops predominantly by the charged form of flecainide as evidenced by investigation of QX-FL at physiological pH and NU-FL investigated over a more acidic pH range where its charged fraction is increased. QX-FL is a potent blocker of channels when applied from inside the cell, but acts very weakly with external application. UDB by QX-FL, like flecainide, develops only after channels open. Once blocked, channels recover very slowly from QX-FL block, apparently without requisite channel opening. Our data strongly suggest that it is the difference in degree of ionization (pKa) between lidocaine and flecainide, rather than gross structural features, that determines distinction in block of cardiac Na(+) channels. The data also suggest that the two drugs share a common receptor but, consistent with the modulated receptor hypothesis, reach this receptor by distinct routes dictated by the degree of ionization of the drug molecules.     

Current view on regulation of voltage‐gated sodium channels by calcium and auxiliary proteins

In cardiac and skeletal myocytes, and in most neurons, the opening of voltage‐gated Na⁺ channels (Na_V channels) triggers action potentials, a process that is regulated via the interactions of the channels’ intercellular C‐termini with auxiliary proteins and/or Ca²⁺. The molecular and structural details for how Ca²⁺ and/or auxiliary proteins modulate Na_V channel function, however, have eluded a concise mechanistic explanation and details have been shrouded for the last decade behind controversy about whether Ca²⁺ acts directly upon the Na_V channel or through interacting proteins, such as the Ca²⁺ binding protein calmodulin (CaM). Here, we review recent advances in defining the structure of Na_V intracellular C‐termini and associated proteins such as CaM or fibroblast growth factor homologous factors (FHFs) to reveal new insights into how Ca²⁺ affects Na_V function, and how altered Ca²⁺‐dependent or FHF‐mediated regulation of Na_V channels is perturbed in various disease states through mutations that disrupt CaM or FHF interaction.     

Elimination and restoration of voltage dependence in the mitochondrial channel,VDAC, by graded modification with succinic anhydride

Summary The major permeability pathways of the outer mitochondrial membrane are the voltage-gated channels called VDAC. It is known that the conductance of these channels decreases as the transmembrane voltage is increased in the positive or negative direction. These channels are known to display a preference for anions over cations of similar size and valence. It was proposed (Doring & Colombini, 1985b) that a set of positive charges lining the channel may be responsible for both voltage dependence and selectivity. A prediction of this proposal is that progressive replacement of the positive charges with negative charges should at first diminish, and then restore, voltage dependence. At the same time, the channel's preference for anions over cations should diminish then reverse. Succinic anhydride was used to perform these experiments as it replaces positively charged amino groups with negatively charged carboxyl groups. When channels, which had been inserted into phospholipid membranes, were treated with moderate amounts of the anhydride, they lost their voltage dependence and preference for anions. With further succinylation, voltage dependence was regenerated while the channels became cation selective. The voltage needed to close one-half of the channels increased in those treatments in which voltage dependence was diminished. As voltage dependence was restored, the voltage needed to close half of the channels decreased. The energy difference between the open and closed state in the absence of an applied field changed little with succinylation, indicating that the procedure did not cause large changes in VDAC's structure but specifically altered those charges responsible for voltage gating and selectivity.     

Probing the structure of the mitochondrial channel,VDAC, by site-directed mutagenesis: A progress report

The voltage-dependent anion-selective channel (VDAC) of the mitochondrial outer membrane is formed by a small ( 30 kDa) polypeptide, but shares with more complex channels the properties of voltage-dependent gating and ion selectivity. Thus, it is a useful model for studying these properties. The molecular biology techniques available in yeast allow us to construct mutant versions of the cloned yeast VDAC genein vitro, using oligonucleotide-directed mutagenesis, and to express the mutant genes in yeast cells in the absence of wild-type VDAC. We find that one substitution mutation (lys 61 to glu) alters the selectivity of VDAC.