Hyperpolarizing Shift In Steady-state Inactivation Research Articles

The Anti-Epileptic Drugs Lamotrigine and Valproic Acid Reduce the Cardiac Sodium Current.

Anti-epileptic drugs (AEDs) are associated with increased risk of sudden cardiac death. To establish whether gabapentin, lamotrigine, levetiracetam, pregabalin, and valproic acid reduce the Nav1.5 current, we conducted whole-cell patch-clamp studies to study the effects of the five AEDs on currents of human cardiac Nav1.5 channels stably expressed in HEK293 cells, and on action potential (AP) properties of freshly isolated rabbit ventricular cardiomyocytes. Lamotrigine and valproic acid exhibited inhibitory effects on the Nav1.5 current in a concentration-dependent manner with an IC50 of 142 ± 36 and 2022 ± 25 µM for lamotrigine and valproic acid, respectively. In addition, these drugs caused a hyperpolarizing shift of steady-state inactivation and a delay in recovery from inactivation. The changes on the Nav1.5 properties were reflected by a reduction in AP upstroke velocity (43.0 ± 6.8% (lamotrigine) and 23.7 ± 10.6% (valproic acid) at 1 Hz) and AP amplitude; in contrast, AP duration was not changed. Gabapentin, levetiracetam, and pregabalin had no effect on the Nav1.5 current. Lamotrigine and valproic acid reduce the Nav1.5 current density and affect its gating properties, resulting in a decrease of the AP upstroke velocity. Gabapentin, levetiracetam, and pregabalin have no effects on the Nav1.5 current.

Conditional knockout of Fgf13 in murine hearts increases arrhythmia susceptibility and reveals novel ion channel modulatory roles

The intracellular fibroblast growth factors (iFGF/FHFs) bind directly to cardiac voltage gated Na+ channels, and modulate their function. Mutations that affect iFGF/FHF-Na+ channel interaction are associated with arrhythmia syndromes. Although suspected to modulate other ionic currents, such as Ca2+ channels based on acute knockdown experiments in isolated cardiomyocytes, the in vivo consequences of iFGF/FHF gene ablation on cardiac electrical activity are still unknown. We generated inducible, cardiomyocyte-restricted Fgf13 knockout mice to determine the resultant effects of Fgf13 gene ablation. Patch clamp recordings from ventricular myocytes isolated from Fgf13 knockout mice showed a ~25% reduction in peak Na+ channel current density and a hyperpolarizing shift in steady-state inactivation. Electrocardiograms on Fgf13 knockout mice showed a prolonged QRS duration. The Na+ channel blocker flecainide further prolonged QRS duration and triggered ventricular tachyarrhythmias only in Fgf13 knockout mice, suggesting that arrhythmia vulnerability resulted, at least in part, from a loss of functioning Na+ channels. Consistent with these effects on Na+ channels, action potentials in Fgf13 knockout mice, compared to Cre control mice, exhibited slower upstrokes and reduced amplitude, but unexpectedly had longer durations. We investigated candidate sources of the prolonged action potential durations in myocytes from Fgf13 knockout mice and found a reduction of the transient outward K+ current (Ito). Fgf13 knockout did not alter whole-cell protein levels of Kv4.2 and Kv4.3, the Ito pore-forming subunits, but did decrease Kv4.2 and Kv4.3 at the sarcolemma. No changes were seen in the sustained outward K+ current or voltage-gated Ca2+ current, other candidate contributors to the increased action potential duration. These results implicate that FGF13 is a critical cardiac Na+ channel modulator and Fgf13 knockout mice have increased arrhythmia susceptibility in the setting of Na+ channel blockade. The unanticipated effect on Ito revealed new FGF13 properties and the unexpected lack of an effect on voltage-gated Ca2+ channels highlight potential compensatory changes in vivo not readily revealed with acute Fgf13 knockdown in cultured cardiomyocytes.

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 (hNaV1.4F1705I) has been linked to cold aggravated myotonia. The functional effects of altered regulation of hNaV1.4F1705I are incompletely understood. The location of the hNaV1.4F1705I 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 NaV1.4 channel regulation by Ca2+ and CaM. hNaV1.4F1705I inactivation gating is Ca2+-sensitive compared to wild type hNaV1.4 which is Ca2+ insensitive and the mutant channel exhibits a depolarizing shift of the V1/2 of inactivation with CaM over expression. In contrast the same mutation in the rNaV1.4 channel background (rNaV1.4F1698I) 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 NaV1.4 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 INa decay in a Ca2+-sensitive fashion. The combination of the altered voltage dependence and kinetics of INa decay contribute to the myotonic phenotype and may involve the Ca2+-sensing apparatus in the CT of NaV1.4.

Ethanol Inhibition of a T‐Type Ca2+ Channel Through Activity of Protein Kinase C

T-type calcium channels (T-channels) are widely distributed in the central and peripheral nervous system, where they mediate calcium entry and regulate the intrinsic excitability of neurons. T-channels are dysregulated in response to alcohol administration and withdrawal. We therefore investigated acute ethanol (EtOH) effects and the underlying mechanism of action in human embryonic kidney (HEK) 293 cell lines, as well as effects on native currents recorded from dorsal root ganglion (DRG) neurons cultured from Long-Evans rats. Whole-cell voltage-clamp recordings were performed at 32 to 34°C in both HEK cell lines and DRG neurons. The recordings were taken after a 10-minute application of EtOH or protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate [PMA]). We recorded T-type Ca²⁺ currents (T-currents) from 3 channel isoforms (CaV3.1, CaV3.2, and CaV3.3) before and during administration of EtOH. We found that only 1 isoform, CaV3.2, was significantly affected by EtOH. EtOH reduced current density as well as producing a hyperpolarizing shift in steady-state inactivation of both CaV3.2 currents from HEK 293 cell lines and in native T-currents from DRG neurons that are known to be enriched in CaV3.2. A myristoylated PKC peptide inhibitor (MPI) blocked the major EtOH effects, in both the cell lines and the DRG neurons. However, PMA effects were more complex. Lower concentration PMA (100 nM) replicated the major effects of EtOH, while higher concentration PMA (1 μM) did not, suggesting that the EtOH effects operate through activation of PKC and were mimicked by lower concentration of PMA. EtOH primarily affects the CaV3.2 isoform of T-type Ca²⁺ channels acting through PKC, highlighting a novel target and mechanism for EtOH effects on excitable membranes.

RyR(R4496C) Mutant Mice Model Reveals a New Paradigm on Local Ca 2+ Control of I CaL

The Ca2+-dependent inactivation (CDI) of the widely distributed L-type Ca2+ channels is a major intrinsic feedback mechanism for Ca2+ homeostasis. CDI manifests by the time-dependent current decay during prolonged depolarization but also the voltage-dependent availability of Ca2+ channel during double-pulse protocols. Whereas the first one has received most of the attention cumulating with the identification of calmodulin (CaM) as an important Ca2+ sensor that mediates CDI, the latter has not received extensive investigation. Here we evidence that Ca2+ sparks control L-type Ca2+ current (ICaL) availability in cardiomyocytes and consequently the steady-state window current. Comparing RyRR4496C catecholaminergic polymorphic ventricular tachycardia (CPVT) mutant mice to wild type (WT) littermates, we show that whereas peak density of ICaL and global [Ca2+]i transient are not modified, RyR2R4496C myocytes demonstrated a significantly lower ICaL density at −30 and −20 mV. This decrease is the consequence of a depolarizing shift in steady-state activation and a hyperpolarizing shift of steady-state inactivation that result in a decreased window current. These differences are eliminated in presence of fast the Ca2+ buffer BAPTA and by depleting the sarcoplasmic reticulum (SR) with thapsigargin, pointing out to a local control by the observed increase in Ca2+ spark occurrence at low voltages in CPVT mice. Somewhat surprisingly, CaM antagonists did not have any effect on CPVT cells but altered the inactivation and activation curves in the WT cells to make these similar to the CPVT. Our data echo the retrograde coupling of RyR and L-type Ca2+ channel observed in skeletal muscle, representing a novel mechanism to counteract excessive SR Ca2+ leak and to preserve CPVT mice from arrhythmic trigger activity such as early afterdepolarization due to enhancement of ICaL window current.

Cardiac Dynamics In-Silico: Pharmacological Targeting of Long QT3 Syndrome

The treatment of cardiac arrhythmia is largely empirical due to incomplete understanding of antiarrhythmic drug-receptor dynamics. However, our laboratory has recently determined that drug receptor interactions of commonly prescribed local anesthetics (LAs) can be described using theoretical models based on a limited number of experimentally determined parameters. Here, we use experimental function data from cardiac Na+ channels harboring disease-linked mutations to build models that simulate measured properties of drug interactions with mutant channels. We developed computational models of 3 closely linked LQT3 mutants: deltaKPQ, D1790G, and Y1795C that account for 1) mutant-specific biophysical channel gating characteristics including mean open time and persistent Na+ current; 2) drug partitioning that underlies situation-dependent blockade; and 3) drug sensitivity to LAs that matches experimental data for steady-state inactivation (SSI), tonic block (TB), concentration and frequency dependence of use-dependent block (UDB), and recovery from UDB. Experimental 30υM flecainide block of D1790G induces a 5.5 ± 1.4mV hyperpolarizing shift in SSI, similar to our simulations (6.25mV). Experimental fits to TB reveal an IC50 of 48υM (simulations: 53.5υM), and for UDB, an IC50 of 1.7υM (simulations: 2υM). Frequency dependence of UDB is in excellent agreement with experimental data over a broad range of pacing frequencies (1 - 10Hz). We have built similarly predictive models of LA interactions for deltaKPQ and Y1795C. The in-silico platform that we have developed to predict drug-receptor interactions can be used to predict how subtle alterations to gating resulting from the mutations, as well as differential sensitivities to LAs, predispose patients to under- or overly-effective therapy with commonly prescribed antiarrhythmic drugs.

Mechanisms of Inhibition of T-Type Calcium Current in the Reticular Thalamic Neurons by 1-Octanol: Implication of the Protein Kinase C Pathway

Recent studies indicate that T-type calcium channels (T-channels) in the thalamus are cellular targets for general anesthetics. Here, we recorded T-currents and underlying low-threshold calcium spikes from neurons of nucleus reticularis thalami (nRT) in brain slices from young rats and investigated the mechanisms of their modulation by an anesthetic alcohol, 1-octanol. We found that 1-octanol inhibited native T-currents at subanesthetic concentrations with an IC(50) of approximately 4 muM. In contrast, 1-octanol was up to 30-fold less potent in inhibiting recombinant Ca(V)3.3 T-channels heterologously expressed in human embryonic kidney cells. Inhibition of both native and recombinant T-currents was accompanied by a hyperpolarizing shift in steady-state inactivation, indicating that 1-octanol stabilized inactive states of the channel. To explore the mechanisms underlying higher 1-octanol potency in inhibiting native nRT T-currents, we tested the effect of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and PKC inhibitors. We found that PMA caused a modest increase of T-current, whereas the inactive PMA analog 4alpha-PMA failed to affect T-current in nRT neurons. In contrast, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Go 6976), an inhibitor of calcium-dependent PKC, decreased baseline T-current amplitude in nRT cells and abolished the effects of subsequently applied 1-octanol. The effects of 1-octanol were also abolished by chelation of intracellular calcium ions with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Taken together, these results suggest that inhibition of calcium-dependent PKC signaling is a possible molecular substrate for modulation of T-channels in nRT neurons by 1-octanol.

Dilated Cardiomyopathy due to Sodium Channel Dysfunction

The cardiac sodium channel mediates the rapid upstroke of the cardiac action potential and thereby constitutes a critical determinant of cardiac excitability and conduction. Mutations in the SCN5A gene encoding the α-subunit of this channel have been linked to a broad clinical spectrum of arrhythmia disorders, including long QT syndrome, Brugada syndrome, sick sinus syndrome, conduction disease, and most recently, atrial fibrillation.1,2 These primary arrhythmia syndromes were originally considered pure electrical entities occurring in the absence of structural heart disease, and the presence of myocardial abnormalities in these disorders was originally actually excluded by definition. Evidence is now accumulating, however, that sodium channelopathy can also be associated with the development of cardiac fibrosis, dilatation, and hypertrophy.3–8 Such structural changes within the myocardium may in turn further predispose to the development of ventricular arrhythmias. These findings have led to reevaluation of the initial view that mutations in a cardiac ion channel would only lead to pure electrical dysfunction and raised the intriguing possibility that the structural alterations could be a direct consequence of sodium channel dysfunction rather than a consequence of long-standing arrhythmia. The mechanism by which a dysfunctional sodium channel leads to structural changes in myocardial tissue, however, remains unclear. Article p 83 In this issue of Circulation: Arrhythmia and Electrophysiology , Ge et al8 report on a novel SCN5A mutation (A1180V) linked to dilated cardiomyopathy (DCM), expanding the repertoire of SCN5A mutations associated with DCM. Previously, the D1275N mutation was discovered independently by McNair et al5 and Olson et al6 in the same large family with the syndrome of DCM, atrial arrhythmias, sinus node dysfunction, and conduction disease. This family had been originally described by Greenlee et al9 in 1986. The D1275N mutation has also been associated with mild ventricular structural alterations in …

T-Type Calcium Channels Are Inhibited by Fluoxetine and Its Metabolite Norfluoxetine

Fluoxetine, a widely used antidepressant that primarily acts as a selective serotonin reuptake inhibitor, also inhibits various neuronal ion channels. Using the whole-cell patch-clamp technique, we have examined the effects of fluoxetine and norfluoxetine, its major active metabolite, on cloned low-voltage-activated T-type calcium channels (T channels) expressed in tsA 201 cells. Fluoxetine inhibited the three T channels Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3 in a concentration-dependent manner (IC(50) = 14, 16, and 30 microM, respectively). Norfluoxetine was a more potent inhibitor than fluoxetine, especially on the Ca(V)3.3 T current (IC(50) = 5 microM). The fluoxetine block of T channels was voltage-dependent because it was significantly enhanced for T channels in the inactivated state. Fluoxetine caused a hyperpolarizing shift in steady-state inactivation, with a slower rate of recovery from the inactivated state. These results indicated a tighter binding of fluoxetine to the inactivated state than to the resting state of T channels, suggesting a more potent inhibition of T channels at physiological resting membrane potential. Indeed, fluoxetine and norfluoxetine at 1 microM strongly inhibited cloned T currents (approximately 50 and approximately 75%, respectively) in action potential clamp experiments performed with firing activities of thalamocortical relay neurons. Altogether, these data demonstrate that clinically relevant concentrations of fluoxetine exert a voltage-dependent block of T channels that may contribute to this antidepressant's pharmacological effects.

Topiramate Inhibits the Initiation of Plateau Potentials in CA1 Neurons by Depressing R‐type Calcium Channels

Cholinergic-dependent plateau potentials (PPs) are intrinsically generated conductances that can elicit ictal-type seizure activity. The aim of this study was to investigate the actions of topiramate (TPM) on the generation of PPs. We used whole-cell patch-clamp recordings from CA1 pyramidal neurons in rat hippocampal slices to examine the effects of TPM on the PPs. In current-clamp mode, action potentials evoked PPs after cholinergic receptor stimulation. Therapeutically relevant concentrations of TPM (50 microM) depressed the PPs evoked by action potentials. Surprisingly, in voltage-clamp mode, we discovered that the cyclic nucleotide-gated (CNG) current that underlies PP generation (denoted as I(tail)) was not depressed. However, significantly longer depolarizing voltage steps were required to elicit I(tail). This suggested that the calcium entry trigger for evoking PPs was depressed by TPM and not I(tail) itself. TPM had no effect on calcium spikes in control conditions; however, TPM did reduce calcium spikes after cholinergic-receptor stimulation. We recently found that R-type calcium spikes are enhanced by cholinergic-receptor stimulation. Therefore we isolated R-type calcium spikes with a cocktail containing tetrodotoxin, omega-conotoxin MVIIC, omega-conotoxin-GVIA, omega-agatoxin IVA, and nifedipine. R-type calcium spikes were significantly depressed by TPM. We also examined the effects of TPM on recombinant Ca(V)2.3 calcium channels expressed in tsA-201 cells. TPM depressed currents mediated by Ca(V)2.3 subunits by a hyperpolarizing shift in steady-state inactivation. We have found that TPM reduces ictal-like activity in CA1 hippocampal neurons through a novel inhibitory action of R-type calcium channels.

Somatic Gene Transfer of Tagged K+ Channel Fragments to Probe Trafficking and Electrical Function in Epithelial Cells and Cardiac Myocytes

To evaluate the roles of the C-termini of K + channels in subcellular targeting and protein-protein interactions, we created fusion constructs of the cell-surface antigen CD8 and the C-termini of Kv4.3, Kv1.4 and KvLQT1. Using a Cre-lox recombination system, we made 3 adenoviruses containing a fusion of the N-terminal-and transmembrane segments of CD8 with the C-termini of each of the 3 K + channels. Expression in polarized Opossum Kidney (OK) epithelial cells led to localization of CD8-Kv4.3 and CD8-Kv1.4 into the apical and basolateral membranes, while CD8-KvLQT1 remained in the endoplasmic reticulum (ER), even when co-expressed with MinK. When expressed in rat cardiac myocytes in culture, all the 3 constructs were diffusely targeted to the surface membrane. The ER retention of CD8-KvLQT1 in OK cells but not in cardiomyocytes thus reveals functional differences in trafficking between these two cell types. To probe functional roles of C-termini, we studied K + currents in cardiac myocytes expressing CD8-Kv4.3. Patch-clamp recordings of transient outward current revealed a hyperpolarizing shift of steady-state inactivation, implying that CD8-Kv4.3 may be disrupting the interaction of Kv4.x channels with one or more as-yet-undefined regulatory subunits. Thus, expression of tagged ion-channel fragments represents a novel, generalizable approach that may help to elucidate assembly, localization and function of these important signaling proteins.

A novel SCN5A mutation associated with idiopathic ventricular fibrillation without typical ECG findings of Brugada syndrome

Mutations in the human cardiac Na + channel α subunit gene ( SCN5A) are responsible for Brugada syndrome, an idiopathic ventricular fibrillation (IVF) subgroup characterized by right bundle branch block and ST elevation on an electrocardiogram (ECG). However, the molecular basis of IVF in subgroups lacking these ECG findings has not been elucidated. We performed genetic screenings of Japanese IVF patients and found a novel SCN5A missense mutation (S1710L) in one symptomatic IVF patient that did not exhibit the typical Brugada ECG. Heterologously expressed S1710L channels showed marked acceleration in the current decay together with a large hyperpolarizing shift of steady-state inactivation and depolarizing shift of activation. These findings suggest that SCN5A is one of the responsible genes for IVF patients who do not show typical ECG manifestations of the Brugada syndrome.

Volatile anesthetic inhibition of neuronal Ca channel currents expressed in Xenopus oocytes

The genes encoding the α 1A, α 1B, α 1C and α 1E subunits of neuronal high voltage-gated Ca channels (HVGCCs) were separately expressed with β 1B and α 2/δ subunits in Xenopus oocytes to determine the effects of volatile anesthetics (VAs) on currents through each specific channel. VA effects were determined on currents carried by Ba 2+ ( I Ba) using the two electrode voltage clamp technique. Although time to peak was unaffected, both halothane (0.59 mM) and isoflurane (0.70 mM) reversibly inhibited peak I Ba by 25–35% and late current (at 830 ms) by 50–60%. A hyperpolarizing shift in steady-state inactivation of α 1E-current was found which could contribute up to one third of observed decrease in the peak current. The rate of inactivation of I Ba seen with α 1A, α 1B and α 1E-type Ca channels was consistently increased by halothane and isoflurane. To more clearly quantify these effects, I Ba inactivation was fit by a single exponential function. The anesthetics depressed both the inactivating and non-inactivating residual components of I Ba and decreased the time constant of inactivation. In the case of I Ba through α 1C-type channels, inactivation was minimal; however, the average current was inhibited by VAs. Similar inhibition of all these HVGCCs by halothane and isoflurane suggests that a common structural component may be involved. Furthermore, the inhibition of such neuronal HVGCCs in situ could alter synaptic neurotransmitter release and contribute to the anesthetic state.

Volatile anesthetics significantly suppress central and peripheral mammalian sodium channels

1. Voltage-dependent sodium channels are important for neuronal signal propagation and integration. 2. Non-mammalian preparations, such as squid giant axon, have sodium channels which have been found to be insensitive to clinical anesthetic concentrations. 3. On the other hand, sodium channels from mammalian neurons are much more sensitive to block by volatile anesthetics. 4. Due to a significant hyperpolarizing shift in steady-state inactivation, IC 50s for sodium channel block at potentials close to the resting membrane potential overlapped with clinical anesthetic concentrations. 5. Hence, sodium channels in mammalian neurons may be sensitive molecular targets of volatile anesthetics.

Effects of temperature and mexiletine on the F1473S Na+ channel mutation causing paramyotonia congenita.

The F1473S mutation of the adult human skeletal muscle Na+ channel causes paramyotonia congenita, a disease characterized by muscle stiffness sometimes followed by weakness in a cold environment. The symptoms are relieved by the local anaesthetic mexiletine. This mutation, which resides in the cytoplasmic S4-S5 loop in domain IV of the alpha-subunit, was studied by heterologous expression in HEK293 cells using standard patch-clamp techniques. Compared to wild-type (WT) channels, those with the F1473S mutation exhibit a twofold slowing of fast inactivation, an increased persistent Na+ current, a +18-mV shift in steady-state inactivation and a fivefold acceleration of recovery from fast inactivation; slow inactivation was similar for both clones. Single-channel recordings for the F1473S mutation revealed a prolonged mean open time and an increased number of channel reopenings that increased further upon cooling. The pharmacological effects of mexiletine on cells expressing either WT, F1473S or G1306E channels were studied. G1306E is a myotonia-causing mutation located within the inactivation gate that displays similar but stronger inactivation defects than F1473S. The hyperpolarizing shift in steady-state inactivation induced by mexiletine was almost identical for all three clones. In contrast, this agent had a reduced effectiveness on the phasic (use-dependent) block of Na+ currents recorded from the mutants: the relative order of block was WT>F1473S>G1306E. We suggest that the relative effectiveness of mexiletine is associated with the degree of abnormal channel inactivation and that the relative binding affinity of mexiletine is not substantially different between the mutations or the WT.

Effect of intracellular and extracellular acidosis on sodium current in ventricular myocytes

Conduction slowing is an essential element in the generation of ischemic ventricular arrhythmias and is determined in part by the inward Na+ current (INa). Because intracellular acidosis is an early consequence of ischemia, we hypothesized that lowering intracellular pH (pHi) would reduce or kinetically modulate INa and thus affect cardiac conduction. To test this hypothesis, the whole cell patch-clamp method was used to measure INa in neonatal rat ventricular myocytes exposed to varying extracellular pH (pHo 6.4-7.4), while perfusing the cells with acidic solutions (pHi 6.2-7.2). With simultaneous acidification of pHo and pHi there was a progressive increase in time to peak current, a 31% decrease in peak INa (298 +/- 18 to 206 +/- 16 pA/pF), and a complex slowing of inactivation kinetics. At the most extreme levels of acidification, there was a 5-mV hyperpolarizing shift in steady-state inactivation and a 6-mV depolarizing shift in activation. Independent changes of pHo and pHi indicate that the reduction of peak INa is a function of pHo. However, steady-state inactivation is modulated by pHi. The time course of activation and inactivation appears to depend on both pHo and pHi. We conclude that both intracellular and extracellular acidosis are significant but distinct modulators of INa amplitude and kinetics in cardiac myocytes.

PROTHROMBIN

Although tricyclic antidepressant (TCA) blockade of cardiac Na⁺ channels is appreciated, actions on neuronal Na⁺ channels are less clear. Therefore, the effects of TCAs (amitriptyline, doxepin and desipramine) as well as trazadone and fluoxetine on voltage-gated Na⁺ current (I_Na) were examined in bovine adrenal chromaffin cells using the whole-cell patch-clamp method. Amitriptyline produced concentration-dependent depression of peak I_Na evoked from a holding potential of −80 mV with <i>K</i>_<i>D</i> value of 20.2 μM and a Hill coefficient of 1.2. Although 20 μM amitriptyline induced no change in the rate or voltage dependence of I_Naactivation, steady-state inactivation demonstrated a 15-mV hyperpolarizing shift. Similar results were observed for doxepin and desipramine. This shift in steady-state inactivation was associated with a slowed rate of recovery from the inactivated state. Contrasting results were observed with the atypical antidepressants: while 20 μM fluoxetine depressed peak I_Na by 61% and caused a 7-mV hyperpolarizing shift in steady-state inactivation, 100 μM trazodone decreased peak I_Na by only 19% and caused only a 3-mV shift. Although the magnitude of fluoxetine effects was similar to those of the TCAs, the onset of fluoxetine effects was substantially slower than for amitriptyline. In voltage-clamp and current-clamp measurements from neonatal rat dorsal root ganglion neurons, 20 μM amitriptyline decreased I_Na by 52% and depressed action potential dynamics consistent with enhanced Na⁺ channel inactivation. The effects of the TCAs on I_Na are similar to local anesthetic behavior and could contribute to certain analgesic actions.