Cardiac Sodium Channel Mutations: Why so Many Phenotypes?

The cardiac Na(+) channel (Nav1.5) conducts a depolarizing inward Na(+) current that is responsible for the generation of the upstroke Phase 0 of the action potential. In heart tissue, changes in Na(+) currents can affect conduction velocity and impulse propagation. The cardiac Nav1.5 is also involved in determination of the action potential duration, since some channels may reopen during the plateau phase, generating a persistent or late inward current. Mutations of cardiac Nav1.5 can induce gain or loss of channel function because of an increased late current or a decrease of peak current, respectively. Gain-of-function mutations cause Long QT syndrome type 3 and possibly atrial fibrillation, while loss-of-function channel mutations are associated with a wider variety of phenotypes, such as Brugada syndrome, cardiac conduction disease, dilated cardiomyopathy, and sick sinus node syndrome. The penetrance and phenotypes resulting from Nav1.5 mutations also vary with age, gender, body temperature, circadian rhythm, and between regions of the heart. This phenotypic variability makes it difficult to correlate genotype-phenotype. We propose that mutations are only one contributor to the phenotype and additional modifications on Nav1.5 lead to the phenotypic variability. Possible modifiers include other genetic variations and alterations in the life cycle of Nav1.5 such as gene transcription, RNA processing, translation, posttranslational modifications, trafficking, complex assembly, and degradation. In this chapter, we summarize potential modifiers of cardiac Nav1.5 that could help explain the clinically observed phenotypic variability. Consideration of these modifiers could help improve genotype-phenotype correlations and lead to new therapeutic strategies.

Gene and cell therapies for the failing heart to prevent sudden arrhythmic death.

Current therapies for treatment and prevention of sudden cardiac death have certain limitations, and a search for new therapeutic approaches is desirable to reduce the burden of sudden arrhythmic death. Gene therapy and stem cell therapy have been investigated as new, valuable tools in treating cardiac diseases such as arrhythmias. In this review, the basics of each modality, important related experimental and clinical studies, and potential advantages and limitations of these treatments will be discussed. The future success of gene and cell therapy to become practical clinical tools greatly depends on our understanding of the mechanisms of ventricular arrhythmia and the mechanisms of action of gene and cell therapy.

The selectivity filter of the voltage-gated sodium channel is involved in channel activation.

Amino acids located in the outer vestibule of the voltage-gated Na+ channel determine the permeation properties of the channel. Recently, residues lining the outer pore have also been implicated in channel gating. The domain (D) IV P-loop residue alanine 1529 forms a part of the putative selectivity filter of the adult rat skeletal muscle (mu1) Na+ channel. Here we report that replacement of alanine 1529 by aspartic acid enhances entry to an ultra-slow inactivated state. Ultra-slow inactivation is characterized by recovery time constants on the order of approximately 100 s from prolonged depolarizations and by the fact that entry to this state can be reduced by binding to the pore of a mutant mu-conotoxin GIIIA, suggesting that ultra-slow inactivation may reflect a structural rearrangement of the outer vestibule. The voltage dependence of ultra-slow inactivation in DIV-A1529D is U-shaped, with a local maximum near -60 mV, whereas activation is maximal only above -20 mV. Furthermore, a train of brief depolarizations produces more ultra-slow inactivation than a single maintained depolarization of the same duration. These data suggest that ultra-slow inactivation emanates from "partially activated" closed states and that the P-loop in DIV may undergo a conformational change during channel activation, which is accentuated by DIV-A1529D.

Specific neosaxitoxin interactions with the Na+ channel outer vestibule determined by mutant cycle analysis.

The voltage-gated Na+ channel alpha-subunit consists of four homologous domains arranged circumferentially to form the pore. Several neurotoxins, including saxitoxin (STX), block the pore by binding to the outer vestibule of this permeation pathway, which is composed of four pore-forming loops (P-loops), one from each domain. Neosaxitoxin (neoSTX) is a variant of STX that differs only by having an additional hydroxyl group at the N1 position of the 1,2,3 guanidinium (N1-OH). We used this structural variant in mutant cycle experiments to determine interactions of the N1-OH and its guanidinium with the outer vestibule. NeoSTX had a higher affinity for the adult rat skeletal muscle Na+ channel (muI or Scn4a) than for STX (DeltaG approximately = 1.3 kcal/mol). Mutant cycle analysis identified groups that potentially interacted with each other. The N1 toxin site interacted most strongly with muI Asp-400 and Tyr-401. The interaction between the N1-OH of neoSTX and Tyr-401 was attractive (DeltaDeltaG = -1.3 +/- 0.1 kcal/mol), probably with formation of a hydrogen bond. A second possible attractive interaction to Asp-1532 was identified. There was repulsion between Asp-400 and the N1-OH (DeltaDeltaG = 1.4 +/- 0.1 kcal/mol), and kinetic analysis further suggested that the N1-OH was interacting negatively with Asp-400 at the transition state. Changes in pH altered the affinity of neoSTX, as would be expected if the N1-OH site were partially deprotonated. These interactions offer an explanation for most of the difference in blocking efficacy between neoSTX and STX and for the sensitivity of neoSTX to pH. Kinetic analysis suggested significant differences in coupling energies between the transition and the equilibrium, bound states. This is the first report to identify points of interaction between a channel and a non-peptide toxin. This interaction pattern was consistent with previous proposals describing the interactions of STX with the outer vestibule (Lipkind, G. M., and H. A. Fozzard. 1994. Biophys. J. 66:1-13; Penzotti, J. L., G. Lipkind, H. A. Fozzard, and S. C. Dudley, Jr. 1998. Biophys. J. 75:2647-2657).

Clockwise domain arrangement of the sodium channel revealed by (mu)-conotoxin (GIIIA) docking orientation.

mu-Conotoxins (mu-CTXs) specifically inhibit Na(+) flux by occluding the pore of voltage-gated Na(+) channels. Although the three-dimensional structures of mu-CTXs are well defined, the molecular configuration of the channel receptor is much less certain; even the fundamental question of whether the four homologous Na(+) channel domains are arranged in a clockwise or counter-clockwise configuration remains unanswered. Residues Asp(762) and Glu(765) from domain II and Asp(1241) from domain III of rat skeletal muscle Na(+) channels are known to be critical for mu-CTX binding. We probed toxin-channel interactions by determining the potency of block of wild-type, D762K, E765K, and D1241C channels by wild-type and point-mutated mu-CTXs (R1A, Q14D, K11A, K16A, and R19A). Individual interaction energies for different toxin-channel pairs were quantified from the half-blocking concentrations using mutant cycle analysis. We find that Asp(762) and Glu(765) interact strongly with Gln(14) and Arg(19) but not Arg(1) and that Asp(1241) is tightly coupled to Lys(16) but not Arg(1) or Lys(11). These newly identified toxin-channel interactions within adjacent domains, interpreted in light of the known asymmetric toxin structure, fix the orientation of the toxin with respect to the channel and reveal that the four internal domains of Na(+) channels are arranged in a clockwise configuration as viewed from the extracellular surface.

mu-conotoxin GIIIA interactions with the voltage-gated Na(+) channel predict a clockwise arrangement of the domains.

Voltage-gated Na(+) channels underlie the electrical activity of most excitable cells, and these channels are the targets of many antiarrhythmic, anticonvulsant, and local anesthetic drugs. The channel pore is formed by a single polypeptide chain, containing four different, but homologous domains that are thought to arrange themselves circumferentially to form the ion permeation pathway. Although several structural models have been proposed, there has been no agreement concerning whether the four domains are arranged in a clockwise or a counterclockwise pattern around the pore, which is a fundamental question about the tertiary structure of the channel. We have probed the local architecture of the rat adult skeletal muscle Na(+) channel (mu1) outer vestibule and selectivity filter using mu-conotoxin GIIIA (mu-CTX), a neurotoxin of known structure that binds in this region. Interactions between the pore-forming loops from three different domains and four toxin residues were distinguished by mutant cycle analysis. Three of these residues, Gln-14, Hydroxyproline-17 (Hyp-17), and Lys-16 are arranged approximately at right angles to each other in a plane above the critical Arg-13 that binds directly in the ion permeation pathway. Interaction points were identified between Hyp-17 and channel residue Met-1240 of domain III and between Lys-16 and Glu-403 of domain I and Asp-1532 of domain IV. These interactions were estimated to contribute -1.0+/-0.1, -0.9+/-0.3, and -1.4+/-0.1 kcal/mol of coupling energy to the native toxin-channel complex, respectively. mu-CTX residues Gln-14 and Arg-1, both on the same side of the toxin molecule, interacted with Thr-759 of domain II. Three analytical approaches to the pattern of interactions predict that the channel domains most probably are arranged in a clockwise configuration around the pore as viewed from the extracellular surface.

Ultra-slow inactivation in mu1 Na+ channels is produced by a structural rearrangement of the outer vestibule.

While studying the adult rat skeletal muscle Na+ channel outer vestibule, we found that certain mutations of the lysine residue in the domain III P region at amino acid position 1237 of the alpha subunit, which is essential for the Na+ selectivity of the channel, produced substantial changes in the inactivation process. When skeletal muscle alpha subunits (micro1) with K1237 mutated to either serine (K1237S) or glutamic acid (K1237E) were expressed in Xenopus oocytes and depolarized for several minutes, the channels entered a state of inactivation from which recovery was very slow, i.e., the time constants of entry into and exit from this state were in the order of approximately 100 s. We refer to this process as "ultra-slow inactivation". By contrast, wild-type channels and channels with the charge-preserving mutation K1237R largely recovered within approximately 60 s, with only 20-30% of the current showing ultra-slow recovery. Coexpression of the rat brain beta1 subunit along with the K1237E alpha subunit tended to accelerate the faster components of recovery from inactivation, as has been reported previously of native channels, but had no effect on the mutation-induced ultra-slow inactivation. This implied that ultra-slow inactivation was a distinct process different from normal inactivation. Binding to the pore of a partially blocking peptide reduced the number of channels entering the ultra-slow inactivation state, possibly by interference with a structural rearrangement of the outer vestibule. Thus, ultra-slow inactivation, favored by charge-altering mutations at site 1237 in micro1 Na+ channels, may be analogous to C-type inactivation in Shaker K+ channels.

Differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the Na+ channel outer vestibule.

The marine guanidinium toxins, saxitoxin (STX) and tetrodotoxin (TTX), have played crucial roles in the study of voltage-gated Na+ channels. Because they have similar actions, sizes, and functional groups, they have been thought to associate with the channel in the same manner, and early mutational studies supported this idea. Recent experiments by. Biophys. J. 67:2305-2315) have suggested that the toxins bind differently to the isoform-specific domain I Phe/Tyr/Cys location. In the adult skeletal muscle Na+ channel isoform (microliter), we compared the effects on both TTX and STX affinities of mutations in eight positions known to influence toxin binding. The results permitted the assignment of energies contributed by each amino acid to the binding reaction. For neutralizing mutations of Asp400, Glu755, and Lys1237, all thought to be part of the selectivity filter of the channel, the loss of binding energy was identical for the two toxins. However, the loss of binding energy was quite different for vestibule residues considered to be more superficial. Specifically, STX affinity was reduced much more by neutralizations of Glu758 and Asp1532. On the other hand, mutation of Tyr401 to Cys reduced TTX binding energy twice as much as it reduced STX binding energy. Kinetic analysis suggested that all outer vestibule residues tested interacted with both toxins early in the binding reaction (consistent with larger changes in the binding than unbinding rates) before the transition state and formation of the final bound complex. We propose a revised model of TTX and STX binding in the Na+ channel outer vestibule in which the toxins have similar interactions at the selectivity filter, TTX has a stronger interaction with Tyr401, and STX interacts more strongly with the more extracellular residues.

Sodium channel selectivity filter regulates antiarrhythmic drug binding.

Local anesthetic antiarrhythmic drugs block Na+ channels and have important clinical uses. However, the molecular mechanism by which these drugs block the channel has not been established. The family of drugs is characterized by having an ionizable amino group and a hydrophobic tail. We hypothesized that the charged amino group of the drug may interact with charged residues in the channel's selectivity filter. Mutation of the putative domain III selectivity filter residue of the adult rat skeletal muscle Na+ channel (micro1) K1237E increased resting lidocaine block, but no change was observed in block by neutral analogs of lidocaine. An intermediate effect on the lidocaine block resulted from K1237S and there was no effect from K1237R, implying an electrostatic effect of Lys. Mutation of the other selectivity residues, D400A (domain I), E755A (domain II), and A1529D (domain IV) allowed block by externally applied quaternary membrane-impermeant derivatives of lidocaine (QX314 and QX222) and accelerated recovery from block by internal QX314. Neo-saxitoxin and tetrodotoxin, which occlude the channel pore, reduced the amount of QX314 bound in D400A and A1529D, respectively. Block by outside QX314 in E755A was inhibited by mutation of residues in transmembrane segment S6 of domain IV that are thought to be part of an internal binding site. The results demonstrate that the Na+ channel selectivity filter is involved in interactions with the hydrophilic part of the drugs, and it normally limits extracellular access to and escape from their binding site just within the selectivity filter. Participation of the selectivity ring in antiarrhythmic drug binding and access locates this structure adjacent to the S6 segment.

Unitary conductance of Na+ channel isoforms in cardiac and NB2a neuroblastoma cells.

Unitary conductances of native Na+ channel isoforms (gamma Na) have been determined under a variety of conditions, making comparisons of gamma Na difficult. To allow direct comparison, we measured gamma Na in cell-attached patches on NB2a neuroblastoma cells and rabbit ventricular myocytes under identical conditions [pipette solution (in mM): 280 Na+ and 2 Ca2+, pH 7.4; 10 degrees C]. gamma Na of NB2a channels, 22.9 +/- 0.9 pS, was 21% greater than that of cardiac channels, 18.9 +/- 0.9 pS. In contrast, respective extrapolated reversal potentials, +62.4 +/- 4.6 and +57.9 +/- 5.1 mV, were not significantly different. Several kinetic differences between the channel types were also noted. Negative to -20 mV, mean open time (MOT) of the NB2a isoform was significantly less than that of cardiac channels, and, near threshold, latency to channel opening decayed more rapidly in NB2a. On the basis of analysis of MOT between -60 and 0 mV, the rate constants at 0 mV for the open-to-closed (O-->C) and open-to-inactivated (O-->I) transitions were 0.42 +/- 0.11 and 0.47 +/- 0.11 ms-1 in NB2a and 0.10 +/- 0.06 and 1.19 +/- 0.07 ms-1 in myocytes. The slope factors were -38.9 +/- 8.7 and +10.7 +/- 6.1 mV in NB2a and -27.3 +/- 7.1 and +23.7 +/- 4.9 mV in myocytes. Transition rate constants were significantly different in NB2a and cardiac cells, but voltage dependence was not.

A mu-conotoxin-insensitive Na+ channel mutant: possible localization of a binding site at the outer vestibule.

We describe a mutation in the outer vestibule region of the adult rat skeletal muscle voltage-gated Na+ channel (microliter) that dramatically alters binding of mu-conotoxin GIIIA (mu-CTX). Mutating the glutamate at position 758 to glutamine (E758Q) decreased mu-CTX binding affinity by 48-fold. Because the mutant channel showed both low tetrodotoxin (TTX) and mu-CTX affinities, these results suggested that mu-CTX bound to the outer vestibule and implied that the TTX- and mu-CTX-binding sites partially overlapped in this region. The mutation decreased the association rate of the toxin with little effect on the dissociation rate, suggesting that Glu-758 could be involved in electrostatic guidance of mu-CTX to its binding site. We propose a mechanism for mu-CTX block of the Na+ channel based on the analogy with saxitoxin (STX) and TTX, on the requirement of mu-CTX to have an arginine in position 13 to occlude the channel, and on this experimental result suggesting that mu-CTX binds in the outer vestibule. In this model, the guanidinium group of Arg-13 of the toxin interacts with two carboxyls known to be important for selectivity (Asp-400 and Glu-755), with the association rate of the toxin increased by interaction with Glu-758 of the channel.

Bursting of cardiac sodium channels after acute exposure to 3,5,3'-triiodo-L-thyronine.

Physiological concentrations of 3,5,3'-triiodo-L-thyronine (T3) acutely increased burst-mode gating of Na+ channels in rabbit ventricular myocytes. Bursting was measured as the ratio of long events to the total number of events multiplied by 100 (%LE); a long event was defined as a set of openings or a single opening with a total duration greater than or equal to five times the control mean open time (MOT) for cell-attached patches. In the cell-attached configuration, adding either 5 or 50 nM T3 to the pipette increased the %LE. %LE had a biphasic voltage dependence and peaked at -50 mV, although the largest percentage change from control occurred between -30 and -40 mV. Neither unitary conductance nor the overall MOT was altered by T3-induced bursting. However, the MOT of openings within bursts increased, implying a kinetically distinct mode of channel gating during bursts. Long events sometimes were grouped into runs, but the more usual pattern suggested that modal shifts occurred in approximately 1 second. Similar behavior was observed with triiodothyroacetic acid, a T3 analogue that does not elicit protein synthesis. To investigate involvement of soluble second messengers, cell-attached recordings were made with and without T3 in the bath. Placed outside the pipette, 50 and 100 nM T3 failed to alter MOT, unitary current, or %LE. Na+ channel gating also was unaffected by patch excision and by exposing the cytoplasmic face of inside-out patches to 50 nM T3. Nevertheless, excision to the inside-out configuration with 5 nM T3 in the pipette dramatically increased the %LE and lengthened MOT. These results suggest that T3 induced Na+ channel bursting by an extranuclear mechanism that requires proximity of T3 to the extracellular face of the Na+ channel. Furthermore, T3 was not membrane permeant on the time scale of these experiments. Na+ channel bursting may contribute to the propensity for arrhythmias in hyperthyroidism and to the positive inotropic effect of acute T3 administration in the stunned and ischemic myocardium.

Modification of cardiac sodium channels by carboxyl reagents. Trimethyloxonium and water-soluble carbodiimide.

In TTX-sensitive nerve and skeletal muscle Na+ channels, selective modification of external carboxyl groups with trimethyloxonium (TMO) or water-soluble carbodiimide (WSC) prevents voltage-dependent Ca2+ block, reduces unitary conductance, and decreases guanidinium toxin affinity. In the case of TMO, it has been suggested that all three effects result from modification of a single carboxyl group, which causes a positive shift in the channel's surface potential. We studied the effect of these reagents on Ca2+ block of adult rabbit ventricular Na+ channels in cell-attached patches. In unmodified channels, unitary conductance (gamma Na) was 18.6 +/- 0.9 pS with 280 mM Na+ and 2 mM Ca2+ in the pipette and was reduced to 5.2 +/- 0.8 pS by 10 mM Ca2+. In contrast to TTX-sensitive Na+ channels, Ca2+ block of cardiac Na+ channels was not prevented by TMO; after TMO pretreatment, gamma Na was 6.1 +/- 1.0 pS in 10 mM Ca2+. Nevertheless, TMO altered cardiac Na+ channel properties. In 2 mM Ca2+, TMO-treated patches exhibited up to three discrete gamma Na levels: 15.3 +/- 1.7, 11.3 +/- 1.5, and 9.8 +/- 1.8 pS. Patch-to-patch variation in which levels were present and the absence of transitions between levels suggests that at least two sites were modified by TMO. An abbreviation of mean open time (MOT) accompanied each decrease in gamma Na. The effects on channel gating of elevating external Ca2+ differed from those of TMO pretreatment. Increasing pipette Ca2+ from 2 to 10 mM prolonged the MOT at potentials positive to approximately -35 mV by decreasing the open to inactivated (O-->I) transition rate constant. On the other hand, even in 10 mM Ca2+ TMO accelerated the O-->I transition rate constant without a change in its voltage dependence. Ensemble averages after TMO showed a shortening of the time to peak current and an acceleration of the rate of current decay. Channel modification with WSC resulted in analogous effects to those of TMO in failing to show relief from block by 10 mM Ca2+. Further, WSC caused a decrease in gamma Na and an abbreviation of MOT at all potentials tested. We conclude that a change in surface potential caused by a single carboxyl modification is inadequate to explain the effects of TMO and WSC in heart. Failure of TMO and WSC to prevent Ca2+ block of the cardiac Na+ channel is a new distinction among isoforms in the Na+ channel multigene family.

Reversal of low voltage and infarction pattern on the surface electrocardiogram after renal hemodialysis for pulmonary edema.

A case is presented in which markedly low surface electrocardiographic (ECG) voltage and an infarction pattern are rapidly reversed with renal hemodialysis for pulmonary edema. A patient presenting with QRS voltages below 0.1 mV in all the limb and augmented limb leads and a waveform pattern suggestive of an anterior and inferior myocardial infarction experienced a dramatic increase in voltage and a reversal of the infarction pattern after dialysis. A hypothesis is proposed in which alterations in chest wall impedance and in electrolytes are involved in the ECG changes resulting from dialysis. This case illustrates one source of diagnostic error, and that dialysis may result in large, rapid changes in the ECG. Diagnostic errors may be avoided by obtaining serial ECGs in patients undergoing dialysis.