Important Role of Asparagines in Coupling the Pore and Votage-Sensor Domain in Voltage-Gated Sodium Channels.

Voltage-gated sodium (NaV) channels contain an α-subunit incorporating the channel's pore and gating machinery composed of four homologous domains (DI-DIV), with a pore domain formed by the S5 and S6 segments and a voltage-sensor domain formed by the S1-S4 segments. During a membrane depolarization movement, the S4s in the voltage-sensor domains exert downstream effects on the S6 segments to control ionic conductance through the pore domain. We used lidocaine, a local anesthetic and antiarrhythmic drug, to probe the role of conserved Asn residues in the S6s of DIII and DIV in NaV1.5 and NaV1.4. Previous studies have shown that lidocaine binding to the pore domain causes a decrease in the maximum gating (Qmax) charge of ∼38%, and three-fourths of this decrease results from the complete stabilization of DIII-S4 (contributing a 30% reduction in Qmax) and one-fourth is due to partial stabilization of DIV-S4 (a reduction of 8-10%). Even though substitutions for the Asn in DIV-S6 in NaV1.5, N1764A and N1764C, produce little ionic current in transfected mammalian cells, they both express robust gating currents. Anthopleurin-A toxin, which inhibits movement of DIV-S4, still reduced Qmax by nearly 30%, a value similar to that observed in wild-type channels, in both N1764A and N1764C. By applying lidocaine and measuring the gating currents, we demonstrated that Asn residues in the S6s of DIII and DIV are important for coupling their pore domains to their voltage-sensor domains, and that Ala and Cys substitutions for Asn in both S6s result in uncoupling of the pore domains from their voltage-sensor domains. Similar observations were made for NaV1.4, although substitutions for Asn in DIII-S6 showed somewhat less uncoupling.

The sodium channel as a target for local anesthetic drugs.

Na channels are the source of excitatory currents for the nervous system and muscle. They are the target for a class of drugs called local anesthetics (LA), which have been used for local and regional anesthesia and for excitatory problems such as epilepsy and cardiac arrhythmia. These drugs are prototypes for new analgesic drugs. The drug-binding site has been localized to the inner pore of the channel, where drugs interact mainly with a phenylalanine in domain IV S6. Drug affinity is both voltage- and use-dependent. Voltage-dependency is the result of changes in the conformation of the inner pore during channel activation and opening, allowing high energy interaction of drugs with the phenylalanine. LA drugs also reduce the gating current of Na channels, which represents the movement of charged residues in the voltage sensors. Specifically, drug binding to phenylalanine locks the domain III S4 in its outward (activated) position, and slows recovery of the domain IV S4. Although strongly affecting gating, LA drugs almost certainly also block by steric occlusion of the pore. Molecular definition of the binding and blocking interactions may help in new drug development.

A molecular model of the inner pore of the Ca channel in its open state.

Structure of the Ca channel open pore is unlikely to be the same as that of the K channel because Ca channels do not contain the hinge residues Gly or Pro. The Ca channel does not have a wide entry into the inner pore, as is found in K channels. First we sought to simulate the open state of the Ca channel by modeling forced opening of the KcsA channel using a procedure of restrained minimization with distance constraints at the level of the α-helical bundle, corresponding to segments Thr-107-Val-115. This produced an intermediate open state, which was populated by amino acid residues of Ca channels and then successively optimized until the opening of the pore reached a diameter of about 10 Å, large enough to allow verapamil to enter and block the Ca channel from inside. Although this approach produced a sterically plausible structure, it was in significant disagreement with the MTSET accessibility data for single cysteine mutations of S6 segments of the P/Q channel(1) that do not fit with an α-helical pattern. Last we explored the idea that the four S6 segments of Ca channels may contain intra-molecular deformations that lead to reorientation of its side chains. After introduction of π-bulges, the model agreed with the MTSET accessibility data. MTSET modification of a cysteine at the C-end of only one S6 could produce physical occlusion and block of the inner pore of the open Ca channel, as observed experimentally, and as expected if the pore opening is narrower than that of K channels.

A molecular switch between the outer and the inner vestibules of the voltage-gated Na+ channel.

Voltage-gated ion channels are transmembrane proteins that undergo complex conformational changes during their gating transitions. Both functional and structural data from K(+) channels suggest that extracellular and intracellular parts of the pore communicate with each other via a trajectory of interacting amino acids. No crystal structures are available for voltage-gated Na(+) channels, but functional data suggest a similar intramolecular communication involving the inner and outer vestibules. However, the mechanism of such communication is unknown. Here, we report that amino acid Ile-1575 in the middle of transmembrane segment 6 of domain IV (DIV-S6) in the adult rat skeletal muscle isoform of the voltage-gated sodium channel (rNa(V)1.4) may act as molecular switch allowing for interaction between outer and inner vestibules. Cysteine scanning mutagenesis of the internal part of DIV-S6 revealed that only mutations at site 1575 rescued the channel from a unique kinetic state ("ultra-slow inactivation," I(US)) produced by the mutation K1237E in the selectivity filter. A similar effect was seen with I1575A. Previously, we reported that conformational changes of both the internal and the external vestibule are involved in the generation of I(US). The fact that mutations at site 1575 modulate I(US) produced by K1237E strongly suggests an interaction between these sites. Our data confirm a previously published molecular model in which Ile-1575 of DIV-S6 is in close proximity to Lys-1237 of the selectivity filter. Furthermore, these functional data define the position of the selectivity filter relative to the adjacent DIV-S6 segment within the ionic permeation pathway.

Sodium channel molecular conformations and antiarrhythmic drug affinity.

Class I cardiac antiarrhythmic drugs, for example, lidocaine, mexiletine, flecainide, quinidine, and procainamide, continue to play an important role in the therapy for cardiac arrhythmias because of the presence of use-dependent block. Lidocaine, as well as related drugs such as mepivacaine, bupivacaine, and cocaine, also belong to the class of medications referred to as local anesthetics. In this review, we will consider lidocaine as the prototypical antiarrhythmic drug because it continues to be widely used both as an antiarrhythmic drug (first used as an antiarrhythmic drug in 1950) as well as a local anesthetic agent. Both of these clinical uses depend upon block of sodium current (I(Na)), but it is the presence of use-dependent I(Na) block, that is, an increasing amount of block at faster heart rates, which enables a local anesthetic agent to be a useful antiarrhythmic drug. Although many early studies investigated the action of antiarrhythmic drugs on Na currents, the availability of site-directed mutant Na channels has enabled for major advances in understanding their mechanisms of action based upon molecular conformations of the Na channel.

Molecular model of anticonvulsant drug binding to the voltage-gated sodium channel inner pore.

The tricyclic anticonvulsant drugs phenytoin, carbamazepine, and lamotrigine block neuronal voltage-gated Na(+) channels, and their binding sites to domain IV-S6 in the channel's inner pore overlap with those of local anesthetic drugs. These anticonvulsants are neutral, in contrast to the mostly positively charged local anesthetics, but their open/inactivated-state blocking affinities are similar. Using a model of the open pore of the Na(+) channel that we developed by homology with the crystal structures of potassium channels, we have docked these three anticonvulsants with residues identified by mutagenesis as important for their binding energy. The three drugs show a common pharmacophore, including an aromatic ring that has an aromatic-aromatic interaction with Tyr-1771 of Na(V)1.2 and a polar amide or imide that interacts with the aromatic ring of Phe-1764 by a low-energy amino-aromatic hydrogen bond. The second aromatic ring is nearly at a right angle to the pharmacophore and fills the pore lumen, probably interacting with the other S6 segments and physically occluding the inner pore to block Na(+) permeation. Hydrophobic interactions with this second aromatic ring may contribute an important component to binding for anticonvulsants, which compensates energetically for the absence of positive charge in their structures. Voltage dependence of block, their important therapeutic property, results from their interaction with Phe-1764, which connects them to the voltage sensors. Their use dependence is modest and this results from being neutral, with a fast drug off-rate after repolarization, allowing a normal action potential rate in the presence of the drugs.

The tetrodotoxin binding site is within the outer vestibule of the sodium channel.

Tetrodotoxin and saxitoxin are small, compact asymmetrical marine toxins that block voltage-gated Na channels with high affinity and specificity. They enter the channel pore's outer vestibule and bind to multiple residues that control permeation. Radiolabeled toxins were key contributors to channel protein purification and subsequent cloning. They also helped identify critical structural elements called P loops. Spacial organization of their mutation-identified interaction sites in molecular models has generated a molecular image of the TTX binding site in the outer vestibule and the critical permeation and selectivity features of this region. One site in the channel's domain I P loop determines affinity differences in mammalian isoforms.

Using lidocaine and benzocaine to link sodium channel molecular conformations to state-dependent antiarrhythmic drug affinity.

RATIONALE: Lidocaine and other antiarrhythmic drugs bind in the inner pore of voltage-gated Na channels and affect gating use-dependently. A phenylalanine in domain IV, S6 (Phe1759 in Na(V)1.5), modeled to face the inner pore just below the selectivity filter, is critical in use-dependent drug block. OBJECTIVE: Measurement of gating currents and concentration-dependent availability curves to determine the role of Phe1759 in coupling of drug binding to the gating changes. METHODS AND RESULTS: The measurements showed that replacement of Phe1759 with a nonaromatic residue permits clear separation of action of lidocaine and benzocaine into 2 components that can be related to channel conformations. One component represents the drug acting as a voltage-independent, low-affinity blocker of closed channels (designated as lipophilic block), and the second represents high-affinity, voltage-dependent block of open/inactivated channels linked to stabilization of the S4s in domains III and IV (designated as voltage-sensor inhibition) by Phe1759. A homology model for how lidocaine and benzocaine bind in the closed and open/inactivated channel conformation is proposed. CONCLUSIONS: These 2 components, lipophilic block and voltage-sensor inhibition, can explain the differences in estimates between tonic and open-state/inactivated-state affinities, and they identify how differences in affinity for the 2 binding conformations can control use-dependence, the hallmark of successful antiarrhythmic drugs.

Toxin-resistant sodium channels: parallel adaptive evolution across a complete gene family.

Approximately 75% of vertebrate proteins belong to protein families encoded by multiple evolutionarily related genes, a pattern that emerged as a result of gene and genome duplications over the course of vertebrate evolution. In families of genes with similar or related functions, adaptation to a strong selective agent should involve multiple adaptive changes across the entire gene family. However, we know of no evolutionary studies that have explicitly addressed this point. Here, we show how 4 taxonomically diverse species of pufferfishes (Tetraodontidae) each evolved resistance to the guanidinium toxins tetrodotoxin (TTX) and saxitoxin (STX) via parallel amino acid replacements across all 8 sodium channels present in teleost fish genomes. This resulted in diverse suites of coexisting sodium channel types that all confer varying degrees of toxin resistance, yet show remarkable convergence among genes and phylogenetically diverse species. Using site-directed mutagenesis and expression of a vertebrate sodium channel, we also demonstrate that resistance to TTX/STX is enhanced up to 15-fold by single, frequently observed replacements at 2 sites that have not previously been implicated in toxin binding but show similar or identical replacements in pufferfishes and in distantly related vertebrate and nonvertebrate animals. This study presents an example of natural selection acting upon a complete gene family, repeatedly arriving at a diverse but limited number of adaptive changes within the same genome. To be maximally informative, we suggest that future studies of molecular adaptation should consider all functionally similar paralogs of the affected gene family.

Speeding the recovery from ultraslow inactivation of voltage-gated Na+ channels by metal ion binding to the selectivity filter: a foot-on-the-door?

Slow inactivated states in voltage-gated ion channels can be modulated by binding molecules both to the outside and to the inside of the pore. Thus, external K(+) inhibits C-type inactivation in Shaker K(+) channels by a "foot-in-the-door" mechanism. Here, we explore the modulation of a very long-lived inactivated state, ultraslow inactivation (I(US)), by ligand binding to the outer vestibule in voltage-gated Na(+) channels. Blocking the outer vestibule by a mutant mu-conotoxin GIIIA substantially accelerated recovery from I(US). A similar effect was observed if Cd(2+) was bound to a cysteine engineered to the selectivity filter (K1237C). In K1237C channels, exposed to 30 microM Cd(2+), the time constant of recovery from I(US) was decreased from 145.0 +/- 10.2 s to 32.5 +/- 3.3 s (P < 0.001). Recovery from I(US) was only accelerated if Cd(2+) was added to the bath solution during recovery (V = -120 mV) from I(US), but not when the channels were selectively exposed to Cd(2+) during the development of I(US) (-20 mV). These data could be explained by a kinetic model in which Cd(2+) binds with high affinity to a slow inactivated state (I(S)), which is transiently occupied during recovery from I(US). A total of 50 microM Cd(2+) produced an approximately 8 mV hyperpolarizing shift of the steady-state inactivation curve of I(S), supporting this kinetic model. Binding of lidocaine to the internal vestibule significantly reduced the number of channels entering I(US), suggesting that I(US) is associated with a conformational change of the internal vestibule of the channel. We propose a molecular model in which slow inactivation (I(S)) occurs by a closure of the outer vestibule, whereas I(US) arises from a constriction of the internal vestibule produced by a widening of the selectivity filter region. Binding of Cd(2+) to C1237 promotes the closure of the selectivity filter region, thereby hastening recovery from I(US). Thus, Cd(2+) ions may act like a foot-on-the-door, kicking the I(S) gate to close.

Charge at the lidocaine binding site residue Phe-1759 affects permeation in human cardiac voltage-gated sodium channels.

Our homology molecular model of the open/inactivated state of the Na(+) channel pore predicts, based on extensive mutagenesis data, that the local anaesthetic lidocaine docks eccentrically below the selectivity filter, such that physical occlusion is incomplete. Electrostatic field calculations suggest that the drug's positively charged amine produces an electrostatic barrier to permeation. To test the effect of charge at this pore level on permeation in hNa(V)1.5 we replaced Phe-1759 of domain IVS6, the putative binding site for lidocaine's alkylamino end, with positively and negatively charged residues as well as the neutral cysteine and alanine. These mutations eliminated use-dependent lidocaine block with no effect on tonic/rested state block. Mutant whole cell currents were kinetically similar to wild type (WT). Single channel conductance (gamma) was reduced from WT in both F1759K (by 38%) and F1759R (by 18%). The negatively charged mutant F1759E increased gamma by 14%, as expected if the charge effect were electrostatic, although F1759D was like WT. None of the charged mutations affected Na(+)/K(+) selectivity. Calculation of difference electrostatic fields in the pore model predicted that lidocaine produced the largest positive electrostatic barrier, followed by lysine and arginine, respectively. Negatively charged glutamate and aspartate both lowered the barrier, with glutamate being more effective. Experimental data were in rank order agreement with the predicted changes in the energy profile. These results demonstrate that permeation rate is sensitive to the inner pore electrostatic field, and they are consistent with creation of an electrostatic barrier to ion permeation by lidocaine's charge.

Selectivity filter residues contribute unequally to pore stabilization in voltage-gated sodium channels.

Mutations in the putative selectivity filter region of the voltage-gated Na+ channel, the so-called DEKA-motif, not only affect selectivity but also alter the channel's gating properties, suggesting functional coupling between permeation and gating. We have previously reported that charge-altering mutations at position 1237 in the P-loop of domain III (position K of the DEKA-motif in the adult rat skeletal muscle Na+ channel, rNa(v)1.4) dramatically enhanced entry to an inactivated state from which the channels recovered with a very slow time constant on the order of approximately 100 s (Todt, H., Dudley, S. C. J., Kyle, J. W., French, R. J., and Fozzard, H. A. (1999) Biophys. J. 76, 1335-1345). This state, termed "ultra-slow inactivation", may reflect a complex molecular rearrangement of the channel's pore region that involves both the extracellular and the cytoplasmic pore. Here, we tested whether charged DEKA-motif residues other than K1237 were also important determinants of a channel's gating properties. Therefore, we constructed the charge-neutralizing mutations D400A, E755A, and K1237A and studied the effects of these mutations on I(US). We found that, compared to wild-type rNa(v)1.4 channels, mutant D400A and K1237A but not E755A channels exhibited enhanced entry into ultra-slow inactivation. Selectivity for Na+ over K+, as judged from shifts in reversal potentials, was preserved in D400A, reduced in E755A, and completely lost in K1237A. These data suggest that an electrostatic interaction between the positively charged residue K1237 and the negatively charged residue D400 stabilizes the structure of the pore and thereby prevents I(US). Moreover, the interaction between K1237 and E755 appears to provide the basis for selective permeation of Na+ over K+.

Molecular modeling of local anesthetic drug binding by voltage-gated sodium channels.

Voltage-gated sodium (Na+) channels are targets for local anesthetic (LA) drugs that bind in the inner pore of the channel with affinities related to the channel gating states. Our core model of the sodium channel (P loops and S5 and S6 segments from each of the four domains) was closed because it was developed using coordinates from the KcsA channel crystallographic structure. We developed a model of the activated, open channel based on the structure of the open MthK channel, which was characterized by bends at the S6 glycine or serine residues. This created a conformation that allowed energetically appropriate docking of the LA drugs. The alkylamino head of ionizable LA molecules was docked closer to the selectivity filter and in association with Phe-1579 of IVS6 and Leu-1280 of IIIS6 (Nav1.4), and the aromatic ring interacted with Tyr-1586 of IVS6 and Asn-434 of IS6. Comparison of multiple LA drugs showed relative binding affinities in the model consistent with experimental studies. The ionizable LA alkylamino heads interact primarily by van der Waals forces that position the charge so as to create a positive electrostatic barrier for cation permeation. Permanently uncharged benzocaine could be docked in the closed conformation as well, stabilizing the closed conformation. The structurally different anticonvulsant lamotrigine and one of its derivatives have a binding site that fully overlaps with that of the LA drugs. The open, activated channel creates the high-affinity binding site for these sodium channel blocker drugs, and block may be mainly electrostatic.

Accessibility of mid-segment domain IV S6 residues of the voltage-gated Na+ channel to methanethiosulfonate reagents.

The inner pore of the voltage-gated Na+ channel is predicted by the structure of bacterial potassium channels to be lined with the four S6 alpha-helical segments. Our previously published model of the closed pore based on the KcsA structure, and our new model of the open pore based on the MthK structure predict which residues in the mid-portion of S6 face the pore. We produced cysteine mutants of the mid-portion of domain IV-S6 (Ile-1575-Leu-1591) in NaV 1.4 and tested their accessibility to intracellularly and extracellularly placed positively charged methanethiosulfonate (MTS) reagents. We found that only two mutants, F1579C and V1583C, were accessible to both outside and inside 2-(aminoethyl)-methanethiosulfonate hydrobromide (MTSEA) Further study of those mutants showed that efficient closure of the fast inactivation gate prevented block by inside [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) at slow stimulation rates. When fast inactivation was inhibited by exposure to anthropleurin B (ApB), increasing channel open time, both mutants were blocked by inside MTSET at a rate that depended on the amount of time the channel was open. Consistent with the fast inactivation gate limiting access to the pore, in the absence of ApB, inside MTSET produced block when the cells were stimulated at 5 or 20 Hz. We therefore suggest that the middle of IV-S6 is an alpha-helix, and we propose a model of the open channel, based on MthK, in which Phe-1579 and Val-1583 face the pore.

Lidocaine: a foot in the door of the inner vestibule prevents ultra-slow inactivation of a voltage-gated sodium channel.

After opening, Na(+) channels may enter several kinetically distinct inactivated states. Whereas fast inactivation occurs by occlusion of the inner channel pore by the fast inactivation gate, the mechanistic basis of slower inactivated states is much less clear. We have recently suggested that the inner pore of the voltage-gated Na(+) channel may be involved in the process of ultra-slow inactivation (I(US)). The local anesthetic drug lidocaine is known to bind to the inner vestibule of the channel and to interact with slow inactivated states. We therefore sought to explore the effect of lidocaine binding on I(US). rNa(V) 1.4 channels carrying the mutation K1237E in the selectivity filter were driven into I(US) by long depolarizing pulses (-20 mV, 300 s). After repolarization to -120 mV, 53 +/- 5% of the channels recovered with a very slow time constant (tau(rec) = 171 +/- 19 s), typical for recovery from I(US). After exposure to 300 microM lidocaine, the fraction of channels recovering from I(US) was reduced to 13 +/- 4% (P < 0.01, n = 6). An additional mutation in the binding site of lidocaine (K1237E + F1579A) substantially reduced the effect of lidocaine on I(US), indicating that lidocaine has to bind to the inner vestibule of the channel to modulate I(US). We propose that I(US) involves a closure of the inner vestibule of the channel. Lidocaine may interfere with this pore motion by acting as a "foot in the door" in the inner vestibule.

Molecular modeling of interactions of dihydropyridines and phenylalkylamines with the inner pore of the L-type Ca2+ channel.

Domains IIIS5, IIIS6, and IVS6 transmembrane segments of L-type Ca(2+) channels participate in dihydropyridine (DHP) and phenylalkylamine (PAA) binding. The inner pore structure of the Ca(v)1.2 channel was reconstructed from coordinates of the transmembrane alpha-helices of the KcsA channel. S6s were aligned with M2 by comparative analysis of the pore-facing M2 side chains and those required for drug binding. Two neighboring tilted S6 helices of domains III and IV below the selectivity filter formed an interdomain crevice. Docking of DHPs inside this crevice located the DHP ring between Phe-1159 of IIIS6 and Ala-1467 of IVS6, parallel to the pore axis, whereas the 4-aryl ring participated in aromatic and polar interactions with the side chains of Tyr-1152 and Tyr-1463. Nonpolar interactions of the port side ester group with hydrophobic side chains of Ile-1156, Ile-1163, and Ile-1471 on the bottom of the binding cavity, formed by the crossover of IIIS6 and IVS6, could stabilize the channel's closed/inactivated state. Similar arrangements were found for DHP agonist drugs, except for the absence of hydrophobic interactions with the helical crossing. In this arrangement, DHPs do not physically block the pore. Locating the central amine group of desmethoxyverapamil near the selectivity filter domain III glutamic acid allows one aromatic ring through its CH(2)CH(2) linker to interact with the side chain of Tyr-1463 inside the DHP binding site, whereas the opposite aromatic ring is in contact with the side chain of Ile-1470 of IVS6, blocking the pore.

Interaction between fast and ultra-slow inactivation in the voltage-gated sodium channel. Does the inactivation gate stabilize the channel structure?

Recently, we reported that mutation A1529D in the domain (D) IV P-loop of the rat skeletal muscle Na(+) channel mu(1) (DIV-A1529D) enhanced entry to an inactivated state from which the channels recovered with an abnormally slow time constant on the order of approximately 100 s. Transition to this "ultra-slow" inactivated state (USI) was substantially reduced by binding to the outer pore of a mutant mu-conotoxin GIIIA. This indicated that USI reflected a structural rearrangement of the outer channel vestibule and that binding to the pore of a peptide could stabilize the pore structure (Hilber, K., Sandtner, W., Kudlacek, O., Glaaser, I. W., Weisz, E., Kyle, J. W., French, R. J., Fozzard, H. A., Dudley, S. C., and Todt, H. (2001) J. Biol. Chem. 276, 27831-27839). Here, we tested the hypothesis that occlusion of the inner vestibule of the Na(+) channel by the fast inactivation gate inhibits ultra-slow inactivation. Stabilization of the fast inactivated state (FI) by coexpression of the rat brain beta(1) subunit in Xenopus oocytes significantly prolonged the time course of entry to the USI. A reduction in USI was also observed when the FI was stabilized in the absence of the beta(1) subunit, suggesting a causal relation between the occurrence of the FI and inhibition of USI. This finding was further confirmed in experiments where the FI was destabilized by introducing the mutations I1303Q/F1304Q/M1305Q. In DIV-A1529D + I1303Q/F1304Q/M1305Q channels, occurrence of USI was enhanced at strongly depolarized potentials and could not be prevented by coexpression of the beta(1) subunit. These results strongly suggest that FI inhibits USI in DIV-A1529D channels. Binding to the inner pore of the fast inactivation gate may stabilize the channel structure and thereby prevent USI. Some of the data have been published previously in abstract form (Hilber, K., Sandtner, W., Kudlacek, O., Singer, E., and Todt, H. (2002) Soc. Neurosci. Abstr. 27, program number 46.12).