Steady-state Fast Inactivation Research Articles

Enhancement of Slow Inactivation of Voltage-Gated Na + Channels by Ranolazine

Ranolazine is an anti-anginal drug that blocks cardiac (Nav1.5) late Na+ current (INa) at therapeutic concentrations (2-10 μM). Recent electrophysiological studies have shown that ranolazine also blocks skeletal muscle (Nav1.4) and neuronal (Nav1.1, Nav1.7, Nav1.8) INa. We investigated the effects of ranolazine on peak INa and on both sustained repetitive firing (SRF) and 0 mM Mg2+- induced (0 [Mg2+]O) continuous high frequency firing of action potentials in cultured rat hippocampal neurons. Ranolazine caused a voltage (−60 mV) and frequency (10 Hz)-dependent inhibition of INa with IC50 values of 0.48 and 61.8 μM (n=4-6), respectively. Ranolazine (10 μM, n=4) did not shift the voltage-dependence of steady-state fast inactivation; however, it caused a 15-mV hyperpolarizing shift (p<0.05, compared to control, n=4) in the slow inactivation. Consistently, ranolazine (10 μM) reduced SRF only during a 4-sec burst but not with a 1-sec depolarization step (Fig.1). Furthermore, ranolazine (10 μM) reduced 0 [Mg2+]O -induced high frequency firing of spontaneous action potentials from 0.78 to 0.45 Hz (p<0.05, n=5). Taken together, these data suggest that ranolazine suppresses the propagation and conduction of action potentials by preferentially interacting with Na+ channels in the slow inactivated state.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Ranolazine selectively blocks persistent current evoked by epilepsy‐associated NaV1.1 mutations

Mutations of SCN1A, the gene encoding the pore-forming subunit of the voltage-gated sodium channel Na(V) 1.1, have been associated with a spectrum of genetic epilepsies and a familial form of migraine. Several mutant Na(V) 1.1 channels exhibit increased persistent current due to incomplete inactivation and this biophysical defect may contribute to altered neuronal excitability in these disorders. Here, we investigated the ability of ranolazine to preferentially inhibit increased persistent current evoked by mutant Na(V) 1.1 channels. Human wild-type (WT) and mutant Na(V) 1.1 channels were expressed heterologously in human tsA201 cells and whole-cell patch clamp recording was used to assess tonic and use-dependent ranolazine block. Ranolazine (30 µM) did not affect WT Na(V) 1.1 channel current density, activation or steady-state fast inactivation but did produce mild slowing of recovery from inactivation. Ranolazine blocked persistent current with 16-fold selectivity over tonic block of peak current and 3.6-fold selectivity over use-dependent block of peak current. Similar selectivity was observed for ranolazine block of increased persistent current exhibited by Na(V) 1.1 channel mutations representing three distinct clinical syndromes, generalized epilepsy with febrile seizures plus (R1648H, T875M), severe myoclonic epilepsy of infancy (R1648C, F1661S) and familial hemiplegic migraine type 3 (L263V, Q1489K). In vitro application of achievable brain concentrations (1, 3 µM) to cells expressing R1648H channels was sufficient to suppress channel activation during slow voltage ramps, consistent with inhibition of persistent current. Our findings support the feasibility of using selective suppression of increased persistent current as a potential new therapeutic strategy for familial neurological disorders associated with certain sodium channel mutations.

Can robots patch-clamp as well as humans? Characterization of a novel sodium channel mutation

Ion channel missense mutations cause disorders of excitability by changing channel biophysical properties. As an increasing number of new naturally occurring mutations have been identified, and the number of other mutations produced by molecular approaches such as in situ mutagenesis has increased, the need for functional analysis by patch-clamp has become rate limiting. Here we compare a patch-clamp robot using planar-chip technology with human patch-clamp in a functional assessment of a previously undescribed Nav1.7 sodium channel mutation, S211P, which causes erythromelalgia. This robotic patch-clamp device can increase throughput (the number of cells analysed per day) by 3- to 10-fold. Both modes of analysis show that the mutation hyperpolarizes activation voltage dependence (8 mV by manual profiling, 11 mV by robotic profiling), alters steady-state fast inactivation so that it requires an additional Boltzmann function for a second fraction of total current (approximately 20% manual, approximately 40% robotic), and enhances slow inactivation (hyperpolarizing shift--15 mV by human,--13 mV robotic). Manual patch-clamping demonstrated slower deactivation and enhanced (approximately 2-fold) ramp response for the mutant channel while robotic recording did not, possibly due to increased temperature and reduced signal-to-noise ratio on the robotic platform. If robotic profiling is used to screen ion channel mutations, we recommend that each measurement or protocol be validated by initial comparison to manual recording. With this caveat, we suggest that, if results are interpreted cautiously, robotic patch-clamp can be used with supervision and subsequent confirmation from human physiologists to facilitate the initial profiling of a variety of electrophysiological parameters of ion channel mutations.

PH Modulation of the Cardiac Voltage Gated Sodium Channel, Nav1.5

Alterations in the function of the cardiac voltage-gated sodium channel (NaV1.5) are a known cause of cardiac disease and arrhythmia. Elevated concentrations of protons decrease conductance and depolarize the voltage dependence of activation and steady-state fast inactivation (SSFI) of NaV1.5 channels (Zhang & Siegelbaum, 1991, Khan et al., 2006). A complete analysis of the effects of low pH on NaV1.5 channel kinetics has not previously been reported. We sought to characterize the effects of low pH on NaV1.5 kinetics. NaV1.5 was co-expressed in Xenopus laevis oocytes with the β1 subunit, and currents were recorded at 20 °C using the cut-open voltage clamp technique with the extracellular solution titrated to either pH 7.4 (control) or pH 6.0. Application of solution at pH 6.0 significantly depolarized the voltage dependence of activation and SSFI; −34.4 ± 0.3mV to −25.2 ± 0.2mV and −76.4 ± 0.1mV to −72.7 ± 0.2mV, respectively. The apparent valences of activation and SSFI were significantly decreased; from 3.4 ± 0.12e to 2.5 ± 0.04e, and from −4.6 ± 0.07e to −4.1 ± 0.09e, respectively. At pH 6.0, the fast time constant of use-dependent inactivation was significantly increased and the use dependent current reduction was decreased from 40.6 ± 0.12% to 34.8 ± 0.05%. The rates of open-state fast inactivation onset were significantly decreased at potentials between −30mV and +30mV, and the rates of recovery at −90mV and −80mV were significantly increased. There was also a visible increase in window current. All effects were reversible upon reperfusion of solution at pH 7.4. Taken together, these data suggest that lowering extracellular pH from 7.4 to 6.0 destabilizes the fast-inactivated state of NaV1.5 channels, an effect that could act as an arrhythmogenic trigger during ischemic events.

From Plastic-Bottle-Toxin to Sodium Channel Blocker: A New Role for Bisphenol A

Bisphenol A has reached public attention due to its presence in food and beverages following leaching from plastic containers.. It is a monomer that is polymerized to manufacture polycarbonate plastic food wrappings and it is detectable in the blood of human populations in developed countries (Palanza et al. 2008, Environmental Research). In animal studies and in vitro bisphenol A was shown to have estrogenic effects. Data link bisphenol A exposure to a variety of diseases including miscarriage, menstrual pain and cardiovascular syndromes.The human heart voltage-gated sodium channel hNav1.5 was expressed in HEK293t cells to determine the effect of bisphenol A on sodium channel function.With whole-cell patch clamp analysis, we show that bisphenol A reduces the peak sodium current through hNav1.5 from a holding potential of −120 mV with an EC50 of 54 ± 8 µM This concentration is considerably higher than has been found in beverages from plastic bottles. However, compared to other known sodium channel blockers, such as lamotrigine or lidocaine, its efficacy is approximately ten-fold higher.Bisphenol A shifts steady-state fast inactivation to more hyperpolarized potentials, whereas voltage-dependence of activation is unaffected. As with local anesthetics, bisphenol A binds preferentially to the inactivated state. The association time constant, as determined by a single exponential fit of peak current decline induced by 30 µM bisphenol A is approximately 14 times faster than that induced by 300 µM lidocaine.In conclusion, we have determined that bisphenol A has blocking effects on hNav1.5 sodium currents that are more pronounced than those of known blockers, such as lidocaine or lamotrigine.Funding: AOR and BAW: BBSRC. AL: Robert-Bosch-Foundation.

Differential pH-Dependent Regulation of NaV Channels

Brain and skeletal muscle NaV channels play a crucial role in neuronal and muscle excitability. Using whole-cell recordings we studied effects of low extracellular pH on the biophysical properties of rNaV1.2 and hNaV1.4, stably expressed in CHO cells. Activation in both channel isoforms was unaffected at low pH. In hNaV1.4, low pH slightly increased the apparent valence of steady-state fast inactivation and accelerated recovery from the fast-inactivated state, although voltage dependence of fast inactivation was not shifted. Time course of cumulative inactivation in hNaV1.4 was unchanged at pH 6.0. In contrast, both fast and slow inactivation in rNaV1.2 were susceptible to acidification. Consistent with our previous studies, the fast-inactivated state in rNaV1.2 was destabilized at pH 6.0, as suggested first-order two-state Eyring model. Slow inactivation at pH 6.0 was more complete than at pH 7.0 and cumulative inactivation was enhanced at low pH. Thus, our data suggest that pH differentially regulates brain and skeletal muscle NaV channels. This differential regulation might reflect unique physiological roles of these isoforms and tissue-specific distributions of NaV1.2 and NaV1.4 channels.

Cold‐induced disruption of Na+ channel slow inactivation underlies paralysis in highly thermosensitive paramyotonia

The Q270K mutation of the skeletal muscle Na(+) channel alpha subunit (Nav1.4) causes atypical paramyotonia with a striking sensitivity to cold. Attacks of paralysis and a drop in the compound muscle action potential (CMAP) are exclusively observed at cold. To understand the pathogenic process, we studied the consequences of this mutation on channel gating at different temperatures. WT or Q270K recombinant Nav1.4 channels fused at their C-terminal end to the enhanced green fluorescent protein (EGFP) were expressed in HEK-293 cells. Whole-cell Na(+) currents were recorded using the patch clamp technique to examine channel gating at 30 degrees C and after cooling the bathing solution to 20 degrees C. Mutant channel fast inactivation was impaired at both temperatures. Cooling slowed the kinetics and enhanced steady-state fast inactivation of both mutant and WT channels. Mutant channel slow inactivation was fairly comparable to that of the WT at 30 degrees C, but became clearly abnormal at 20 degrees C. Cooling enhanced slow inactivation in the WT by shifting the voltage dependence toward hyperpolarization, but induced the opposite effect in the mutant. Destabilization of mutant channel slow inactivation in combination with defective fast inactivation is expected to increase the susceptibility to prolonged membrane depolarization, and can ultimately lead to membrane inexcitability and paralysis at cold. Thus, abnormal temperature sensitivity of slow inactivation can be a determinant pathogenic factor, and should therefore be more widely considered in thermosensitive Na(+) channelopathies.

Block of Tetrodotoxin-sensitive, Nav1.7, and Tetrodotoxin-resistant, Nav1.8, Na+ Channels by Ranolazine

Evidence supports a role for the tetrodotoxin-sensitive Na(V)1.7 and the tetrodotoxin-resistant Na(V)1.8 in the pathogenesis of pain. Ranolazine, an anti-ischemic drug, has been shown to block cardiac (Na(V)1.5) late sodium current (I(Na)). In this study, whole-cell patch-clamp techniques were used to determine the effects of ranolazine on human Na(V)1.7 (hNa(V)1.7 + beta(1) subunits) and rat Na(V)1.8 (rNa(V)1.8) channels expressed in HEK293 and ND7-23 cells, respectively. Ranolazine reduced hNa(V)1.7 and rNa(V)1.8 I(Na) with IC50 values of 10.3 and 21.5 microM (holding potential = -120 or -100 mV, respectively). The potency of I(Na) block by ranolazine increased to 3.2 and 4.3 microM when 5-sec depolarizing prepulses to -70 (hNa(V)1.7) and -40 (rNa(V)1.8) mV were applied. Ranolazine caused a preferential hyperpolarizing shift of the steady-state fast, intermediate and slow inactivation of hNa(V)1.7 and intermediate and slow inactivation of rNa(V)1.8, suggesting preferential interaction of the drug with the inactivated states of both channels. Ranolazine (30 microM) caused a use-dependent block (10-msec pulses at 1, 2 and 5 Hz) of hNa(V)1.7 and rNa(V)1.8 I(Na) and significantly accelerated the onset of, and slowed the recovery from inactivation, of both channels. An increase of depolarizing pulse duration from 3 to 200 msec did not affect the use-dependent block of I(Na) by 100 microM ranolazine. Taken together, the data suggest that ranolazine blocks the open state and may interact with the inactivated states of Na(V)1.7 and Na(V)1.8 channels. The state-and use-dependent modulation of hNa(V)1.7 and rNa(V)1.8 Na+ channels by ranolazine could lead to an increased effect of the drug at high firing frequencies, as in injured neurons.

PH-Dependent Regulation of rNaV1.2 Channel Inactivation

Brain sodium channels play a crucial role in neuronal excitability. Ischemia leads to decreased sodium influx that, as suggested by previous studies, may be caused by channel protonation. Using whole-cell recordings we studied effects of low extracellular pH on the biophysical properties of rNaV1.2 stably expressed in CHO cells. We confirmed that acidification causes a decrease of peak Na currents with no measurable effects on the voltage dependence of activation. By contrast, low pH has a significant effect on rNaV1.2 inactivation properties. At pH 6.0 the effective charge of the steady state fast inactivation curve is significantly reduced, whereas the midpoint voltage is unchanged. The kinetics of fast inactivation are accelerated and shifted to less negative potentials, indicating a destabilization of the fast inactivated state. This was confirmed by first-order two-state Eyring model fit to τ(V) dependence. Slow inactivation is enhanced at low pH, as demonstrated by experiments using cumulative inactivation of rNaV1.2 with 45Hz stimulation. Thus, our data suggest Na+ channel fast and slow inactivation, but not activation, might be the target for protonation at low pH and might play role in rNaV1.2 down-regulation in low extracellular pH during ischemic events.

Block of Tetrodotoxin-sensitive, Nav1.7, and Tetrodotoxin-resistant, Nav1.8, Na+ channels by Ranolazine

Evidence supports a role for the tetrodotoxin-sensitive Nav1.7 and the tetrodotoxin-resistant Nav1.8 in the pathogenesis of pain. Ranolazine, an anti-ischemic drug, has been shown to block cardiac (Nav1.5) late sodium current (INa). In this study, whole-cell patch-clamp techniques were used to determine the effects of ranolazine on human Nav1.7 (hNav1.7+β1 subunits) and rat Nav1.8 (rNav1.8) channels expressed in HEK293 and ND7-23 cells, respectively. Ranolazine reduced hNav1.7 and rNav1.8 INa with IC50 values of 10.3 and 21.5 μM (holding potential=-120 or -100 mV, respectively). The potency of INa block by ranolazine increased to 3.2 and 4.3 μM when 5-sec depolarizing prepulses to -70 (hNav1.7) and -40 (rNav1.8) mV were applied. Ranolazine caused a preferential hyperpolarizing shift of the steady-state fast, intermediate and slow inactivation of hNav1.7 and and intermediate and slow inactivation of rNav1.8, suggesting preferential interaction of the drug with the inactivated states of both channels. Ranolazine (30 μM) caused a use-dependent block (10-msec pulses at 1, 2 and 5 Hz) of hNav1.7 and rNav1.8 INa and significantly accelerated the onset of, and slowed the recovery from inactivation, of both channels. An increase of depolarizing pulse duration from 3 to 200 msec did not affect the use-dependent block of INa by 100 μM ranolazine. Taken together, the data suggest that ranolazine blocks the open state and may interact with the inactivated states of Nav1.7 and Nav1.8 channels. The state-and use-dependent modulation of hNav1.7 and rNav1.8 Na+ channels by ranolazine could lead to an increased effect of the drug at high firing frequencies, as in injured neurons.

A Pore-blocking Hydrophobic Motif at the Cytoplasmic Aperture of the Closed-state Nav1.7 Channel Is Disrupted by the Erythromelalgia-associated F1449V Mutation

Sodium channel Na(v)1.7 has recently elicited considerable interest as a key contributor to human pain. Gain-of-function mutations of Na(v)1.7 produce painful disorders, whereas loss-of-function Na(v)1.7 mutations produce insensitivity to pain. The inherited erythromelalgia Na(v)1.7/F1449V mutation, within the C terminus of domain III/transmembrane helix S6, shifts channel activation by -7.2 mV and accelerates time to peak, leading to nociceptor hyperexcitability. We constructed a homology model of Na(v)1.7, based on the KcsA potassium channel crystal structure, which identifies four phylogenetically conserved aromatic residues that correspond to DIII/F1449 at the C-terminal end of each of the four S6 helices. The model predicted that changes in side-chain size of residue 1449 alter the pore's cytoplasmic aperture diameter and reshape inter-domain contact surfaces that contribute to closed state stabilization. To test this hypothesis, we compared activation of wild-type and mutant Na(v)1.7 channels F1449V/L/Y/W by whole cell patch clamp analysis. All but the F1449V mutation conserve the voltage dependence of activation. Compared with wild type, time to peak was shorter in F1449V, similar in F1449L, but longer for F1449Y and F1449W, suggesting that a bulky, hydrophobic residue is necessary for normal activation. We also substituted the corresponding aromatic residue of S6 in each domain individually with valine, to mimic the naturally occurring Na(v)1.7 mutation. We show that DII/F960V and DIII/F1449V, but not DI/Y405V or DIV/F1752V, regulate Na(v)1.7 activation, consistent with well established conformational changes in DII and DIII. We propose that the four aromatic residues contribute to the gate at the cytoplasmic pore aperture, and that their ring side chains form a hydrophobic plug which stabilizes the closed state of Na(v)1.7.

Differential Block of Sensory Neuronal Voltage-Gated Sodium Channels by Lacosamide [(2R)-2-(Acetylamino)-N-benzyl-3-methoxypropanamide], Lidocaine, and Carbamazepine

Voltage-gated sodium channels play a critical role in excitability of nociceptors (pain-sensing neurons). Several different sodium channels are thought to be potential targets for pain therapeutics, including Na(v)1.7, which is highly expressed in nociceptors and plays crucial roles in human pain and hereditary painful neuropathies, Na(v)1.3, which is up-regulated in sensory neurons following chronic inflammation and nerve injury, and Na(v)1.8, which has been implicated in inflammatory and neuropathic pain mechanisms. We compared the effects of lacosamide [(2R)-2-(acetylamino)-N-benzyl-3-methoxypropanamide], a new pain therapeutic, with those of lidocaine and carbamazepine on recombinant Na(v)1.7 and Na(v)1.3 currents and neuronal tetrodotoxin-resistant (Na(v)1.8-type) sodium currents using whole-cell patch-clamp electrophysiology. Lacosamide is able to substantially reduce all three current types. However, in contrast to lidocaine and carbamazepine, 1 mM lacosamide did not alter steady-state fast inactivation. Inhibition by lacosamide exhibited substantially slower kinetics, consistent with the proposal that lacosamide interacts with slow-inactivated sodium channels. The estimated IC(50) values for inhibition by lacosamide of Na(v)1.7-, Na(v)1.3-, and Na(v)1.8-type channels following prolonged inactivation were 182, 415, and 16 microM, respectively. Na(v)1.7-, Na(v)1.3-, and Na(v)1.8-type channels in the resting state were 221-, 123-, and 257-fold less sensitive, respectively, to lacosamide than inactivated channels. Interestingly, the ratios of resting to inactivated IC(50)s for carbamazepine and lidocaine were much smaller (ranging from 3 to 16). This suggests that lacosamide should be more effective than carbamazepine and lidocaine at selectively blocking the electrical activity of neurons that are chronically depolarized compared with those at more normal resting potentials.

The Investigational Anticonvulsant Lacosamide Selectively Enhances Slow Inactivation of Voltage-Gated Sodium Channels

We hypothesized that lacosamide modulates voltage-gated sodium channels (VGSCs) at clinical concentrations (32-100 muM). Lacosamide reduced spiking evoked in cultured rat cortical neurons by 30-s depolarizing ramps but not by 1-s ramps. Carbamazepine and phenytoin reduced spike-firing induced by both ramps. Lacosamide inhibited sustained repetitive firing during a 10-s burst but not within the first second. Tetrodotoxin-sensitive VGSC currents in N1E-115 cells were reduced by 100 muM lacosamide, carbamazepine, lamotrigine, and phenytoin from V(h) of -60 mV. Hyperpolarization (500 ms) to -100 mV removed the block by carbamazepine, lamotrigine, and phenytoin but not by lacosamide. The voltage-dependence of activation was not changed by lacosamide. The inactive S-stereoisomer did not inhibit VGSCs. Steady-state fast inactivation curves were shifted in the hyperpolarizing direction by carbamazepine, lamotrigine, and phenytoin but not at all by lacosamide. Lacosamide did not retard recovery from fast inactivation in contrast to carbamazepine. Carbamazepine, lamotrigine, and phenytoin but not lacosamide all produced frequency-dependent facilitation of block of a 3-s, 10-Hz pulse train. Lacosamide shifted the slow inactivation voltage curve in the hyperpolarizing direction and significantly promoted the entry of channels into the slow inactivated state (carbamazepine weakly impaired entry into the slow inactivated state) without altering the rate of recovery. Lacosamide is the only analgesic/anticonvulsant drug that reduces VGSC availability by selective enhancement of slow inactivation but without apparent interaction with fast inactivation gating. The implications of this unique profile are being explored in phase III clinical trials for epilepsy and neuropathic pain.

Isoform-selective Effects of Isoflurane on Voltage-gated Na+ Channels

Voltage-gated Na channels modulate membrane excitability in excitable tissues. Inhibition of Na channels has been implicated in the effects of volatile anesthetics on both nervous and peripheral excitable tissues. The authors investigated isoform-selective effects of isoflurane on the major Na channel isoforms expressed in excitable tissues. Rat Nav1.2, Nav1.4, or Nav1.5 alpha subunits heterologously expressed in Chinese hamster ovary cells were analyzed by whole cell voltage clamp recording. The effects of isoflurane on Na current activation, inactivation, and recovery from inactivation were analyzed. The cardiac isoform Nav1.5 activated at more negative potentials (peak INa at -30 mV) than the neuronal Nav1.2 (0 mV) or skeletal muscle Nav1.4 (-10 mV) isoforms. Isoflurane reversibly inhibited all three isoforms in a concentration- and voltage-dependent manner at clinical concentrations (IC50 = 0.70, 0.61, and 0.45 mm, respectively, for Nav1.2, Nav1.4, and Nav1.5 from a physiologic holding potential of -70 mV). Inhibition was greater from a holding potential of -70 mV than from -100 mV, especially for Nav1.4 and Nav1.5. Isoflurane enhanced inactivation of all three isoforms due to a hyperpolarizing shift in the voltage dependence of steady state fast inactivation. Inhibition of Nav1.4 and Nav1.5 by isoflurane was attributed primarily to enhanced inactivation, whereas inhibition of Nav1.2, which had a more positive V1/2 of inactivation, was due primarily to tonic block. Two principal mechanisms contribute to Na channel inhibition by isoflurane: enhanced inactivation due to a hyperpolarizing shift in the voltage dependence of steady state fast inactivation (Nav1.5 approximately Nav1.4 > Nav1.2) and tonic block (Nav1.2 > Nav1.4 approximately Nav1.5). These novel mechanistic differences observed between isoforms suggest a potential pharmacologic basis for discrimination between Na channel isoforms to enhance anesthetic specificity.

NA+‐ and K+‐channels as molecular targets of the alkaloid ajmaline in skeletal muscle fibres

Ajmaline is a widely used antiarrhythmic drug. Its action on voltage-gated ion channels in skeletal muscle is not well documented and we have here elucidated its effects on Na(+) and K(+) channels. Sodium (I(Na)) and potassium (I(K)) currents in amphibian skeletal muscle fibres were recorded using 'loose-patch' and two-microelectrode voltage clamp techniques (2-MVC). Action potentials were generated using current clamp. Under 'loose patch' clamp conditions, the IC(50) for I(Na) was 23.2 microM with Hill-coefficient h=1.21. For I(K), IC(50) was 9.2 microM, h=0.87. Clinically relevant ajmaline concentrations (1-3 microM) reduced peak I(Na) by approximately 5% but outward I(K) values were reduced by approximately 20%. Na(+) channel steady-state activation and fast inactivation were concentration-dependently shifted towards hyperpolarized potentials ( approximately 10 mV at 25 microM). Inactivation curves were markedly flattened by ajmaline. Peak-I(K) under maintained depolarisation was reduced to approximately 30% of control values by 100 microM ajmaline. I(K) activation time constants were increased at least two-fold. Lower concentrations (10 or 25 microM) reduced steady-state-I(K) slightly but peak-I(K) significantly. Action potential generation threshold was increased by 10 microM ajmaline and repolarisation prolonged. Ajmaline acts differentially on Na(+) and K(+) channels in skeletal muscle. This suggests at least multiple sites of action including the S4 subunit. Our data may provide a first insight into specific mechanisms of ajmaline-ion channel interaction in tissues other than cardiac muscle and could suggest possible side-effects that need to be further evaluated.

(725): Lacosamide has a dual mode of action: Selective enhancement of sodium channel slow inactivation

Lacosamide is a functionalized amino acid which showed analgesic and anticonvulsant effects in a variety of animal models. This analgesic activity is currently being evaluated in Phase III clinical trials. Lacosamide did not bind with significant affinity to any of more than 100 receptors, enzymes, transporters, or ion-channels tested. Lacosamide bound to collapsin response mediator protein 2 (CRMP-2, see corresponding poster) and displayed low binding affinity to binding site 2 of the sodium ion channel. The aim of the present experiments was to assess the effect of lacosamide on Na-currents in detailed electrophysiological studies. In neuroblastoma cells held at a potential of -60 mV lacosamide inhibited Na-current by about 30%. This inhibition was not affected by a prepulse to -100 mV to remove Na-channel fast inactivation, whereas inhibition by carbamazepine (CBZ), phenytoin (PHT) and lamotrigine (LTG) was almost completely abolished by this hyperpolarizing prepulse. Lacosamide did not influence the steady state fast inactivation curve. In contrast, CBZ, PHT and LTG shifted this curve to more hyperpolarized potentials. Lacosamide shifted the availability curve of steady state slow inactivation to more negative potentials and increased the number of slow inactivated Na-channels. In neuroblastoma cells it enhanced entry of Na-channels into slow inactivation without influencing recovery from slow inactivation or Na-channel activation. Enhanced slow inactivation by lacosamide was also detected in TTX-resistant Na-channels of rat dorsal root ganglia in vitro. When compared to the fast inactivation Na-channel modifier lamotrigine in animal models of neuropathic pain, lacosamide showed clear antinociceptive effects under conditions where lamotrigine was without effect. Since slow inactivation of Na-channels is an endogenous mechanism by which neurons reduce stimulated or ectopic hyperactivity, this modulation represents one molecular mechanism involved in the pharmacological activity of lacosamide. In summary, these experiments (see also corresponding abstract) demonstrate a dual mode of action for lacosamide.

Inactivation Properties of Sodium Channel Nav1.8 Maintain Action Potential Amplitude in Small DRG Neurons in the Context of Depolarization

BackgroundSmall neurons of the dorsal root ganglion (DRG) express five of the nine known voltage-gated sodium channels. Each channel has unique biophysical characteristics which determine how it contributes to the generation of action potentials (AP). To better understand how AP amplitude is maintained in nociceptive DRG neurons and their centrally projecting axons, which are subjected to depolarization within the dorsal horn, we investigated the dependence of AP amplitude on membrane potential, and how that dependence is altered by the presence or absence of sodium channel Nav1.8.ResultsIn small neurons cultured from wild type (WT) adult mouse DRG, AP amplitude decreases as the membrane potential is depolarized from -90 mV to -30 mV. The decrease in amplitude is best fit by two Boltzmann equations, having V1/2 values of -73 and -37 mV. These values are similar to the V1/2 values for steady-state fast inactivation of tetrodotoxin-sensitive (TTX-s) sodium channels, and the tetrodotoxin-resistant (TTX-r) Nav1.8 sodium channel, respectively. Addition of TTX eliminates the more hyperpolarized V1/2 component and leads to increasing AP amplitude for holding potentials of -90 to -60 mV. This increase is substantially reduced by the addition of potassium channel blockers. In neurons from Nav1.8(-/-) mice, the voltage-dependent decrease in AP amplitude is characterized by a single Boltzmann equation with a V1/2 value of -55 mV, suggesting a shift in the steady-state fast inactivation properties of TTX-s sodium channels. Transfection of Nav1.8(-/-) DRG neurons with DNA encoding Nav1.8 results in a membrane potential-dependent decrease in AP amplitude that recapitulates WT properties.ConclusionWe conclude that the presence of Nav1.8 allows AP amplitude to be maintained in DRG neurons and their centrally projecting axons even when depolarized within the dorsal horn.

Nav1.7 Mutant A863P in Erythromelalgia: Effects of Altered Activation and Steady-State Inactivation on Excitability of Nociceptive Dorsal Root Ganglion Neurons

Inherited erythromelalgia/erythermalgia (IEM) is a neuropathy characterized by pain and redness of the extremities that is triggered by warmth. IEM has been associated with missense mutations of the voltage-gated sodium channel Na(V)1.7, which is preferentially expressed in most nociceptive dorsal root ganglia (DRGs) and sympathetic ganglion neurons. Several mutations occur in cytoplasmic linkers of Na(V)1.7, with only two mutations in segment 4 (S4) and S6 of domain I. We report here a simplex case with an alanine 863 substitution by proline (A863P) in S5 of domain II of Na(V)1.7. The functional effect of A863P was investigated by voltage-clamp analysis in human embryonic kidney 293 cells and by current-clamp analysis to determine the effects of A863P on firing properties of small DRG neurons. Activation of mutant channels was shifted by -8 mV, whereas steady-state fast inactivation was shifted by +10 mV, compared with wild-type (WT) channels. There was a marked decrease in the rate of deactivation of mutant channels, and currents elicited by slow ramp depolarizations were 12 times larger than for WT. These results suggested that A863P could render DRG neurons hyperexcitable. We tested this hypothesis by studying properties of rat DRG neurons transfected with either A863P or WT channels. A863P depolarized resting potential of DRG neurons by +6 mV compared with WT channels, reduced the threshold for triggering single action potentials to 63% of that for WT channels, and increased firing frequency of neurons when stimulated with suprathreshold stimuli. Thus, A863P mutant channels produce hyperexcitability in DRG neurons, which contributes to the pathophysiology of IEM.

How Batrachotoxin Modifies the Sodium Channel Permeation Pathway: Computer Modeling and Site-Directed Mutagenesis

A structural model of the rNav1.4 Na+ channel with batrachotoxin (BTX) bound within the inner cavity suggested that the BTX pyrrole moiety is located between a lysine residue at the DEKA selectivity filter (Lys1237) and an adjacent phenylalanine residue (Phe1236). We tested this pyrrole-binding model by site-directed mutagenesis of Phe1236 at D3/P-loop with 11 amino acids. Mutants F1236D and F1236E expressed poorly, whereas nine other mutants either expressed robust Na+ currents, like the wild-type (F1236Y/Q/K), or somewhat reduced current (F1236G/A/C/N/W/R). Gating properties were altered modestly in most mutant channels, with F1236G displaying the greatest shift in activation and steady-state fast inactivation (-10.1 and -7.5 mV, respectively). Mutants F1236K and F1236R were severely resistant to BTX after 1000 repetitive pulses (+50 mV/20 ms at 2 Hz), whereas seven other mutants were sensitive but with reduced magnitudes compared with the wild type. It is noteworthy that rNav1.4-F1236K mutant Na+ channels remained highly sensitive to block by the local anesthetic bupivacaine, unlike several other BTX-resistant mutant channels. Our data thus support a model in which BTX, when bound within the inner cavity, interacts with the D3/P-loop directly. Such a direct interaction provides clues on how BTX alters the Na+ channel selectivity and conductance.

The pH-dependent effect of mexiletine on IVS4 sodium channel mutants R1448H/C

The effects of extracellular pH (6.2, 7.4 and 8.2) and 0.1mM mexiletine, a channel blocker of the lidocaine type, are studied on two sodium channel mutations of the fourth voltage sensor, R1448H/C. The fast inactivated channel state to which mexiletine preferentially binds is destabilized by the mutations. In contrast to the expected low response of R1448H/C carriers, mexiletine is particularly effective in preventing exercise-induced stiffness and paralysis from which these patients suffer. Our measurements performed in the whole-cell mode on stably transfected HEK cells show for the first time that the mutations strikingly accelerate closed-state inactivation and, as steady-state fast inactivation is shifted to more negative potentials, stabilize the fast inactivated channel state in the potential range around the resting potential. At pH 7.4 and 8.2, the phasic mexiletine block is larger for R1448C (55%) and R1448H (47%) than for wild type (WT) channels (31%) due to slowed recovery from block (520 ms for R1448C versus 270 ms for WT at pH 7.4) although the recovery from inactivation is slightly faster for the mutants (1.9 ms for R1448C versus 3.8 ms for WT at pH 7.4). At pH 6.2, the effect of mexiletine is smaller and shows a relatively fast recovery from block. We conclude that enhanced closed-state inactivation expands the concept of a mutation-induced uncoupling of channel inactivation from activation to a new potential range and that the higher mexiletine efficacy in R1448H/C carriers than other myotonic patients, offering a pharmacogenetic strategy for mutation-specific treatment.