Familial hemiplegic migraine type 1 mutations K1336E, W1684R, and V1696I alter Cav2.1 Ca2+ channel gating: evidence for beta-subunit isoform-specific effects

Mutations in the Cav2.1 alpha1-subunit of P/Q-type Ca2+ channels cause human diseases, including familial hemiplegic migraine type-1 (FHM1). FHM1 mutations alter channel gating and enhanced channel activity at negative potentials appears to be a common pathogenetic mechanism. Different beta-subunit isoforms (primarily beta4 and beta3) participate in the formation of Cav2.1 channel complexes in mammalian brain. Here we investigated not only whether FHM1 mutations K1336E (KE), W1684R (WR), and V1696I (VI) can affect Cav2.1 channel function but focused on the important question whether mutation-induced changes on channel gating depend on the beta-subunit isoform. Mutants were co-expressed in Xenopus oocytes together with beta1, beta3, or beta4 and alpha2delta1 subunits, and channel function was analyzed using the two-electrode voltage-clamp technique. WR shifted the voltage dependence for steady-state inactivation of Ba2+ inward currents (IBa) to more negative voltages with all beta-subunits tested. In contrast, a similar shift was observed for KE only when expressed with beta3. All mutations promoted IBa decay during pulse trains only when expressed with beta1 or beta3 but not with beta4. Enhanced decay could be explained by delayed recovery from inactivation. KE accelerated IBa inactivation only when co-expressed with beta3, and VI slowed inactivation only with beta1 or beta3. KE and WR shifted channel activation of IBa to more negative voltages. As the beta-subunit composition of Cav2.1 channels varies in different brain regions, our data predict that the functional FHM1 phenotype also varies between different neurons or even within different neuronal compartments.     

Use-dependent potentiation of the Nav1.6 sodium channel

Nav1.2 and Nav1.6 are two voltage-gated sodium channel isoforms that are abundant in the adult central nervous system. These channels are expressed in different cells and localized in different neuronal regions, which may reflect functional specialization. To examine this possibility, we compared the properties of Nav1.2 and Nav1.6 in response to a rapid series of repetitive depolarizations. Currents through Nav1.6 coexpressed with beta1 demonstrated use-dependent potentiation during a rapid train of depolarizations. This potentiation was in contrast to the use-dependent decrease in current for Nav1.2 with beta1. The voltage dependence of potentiation correlated with the voltage dependence of activation, and it still occurred when fast inactivation was removed by mutation. Rapid stimulation accelerated a slow phase of activation in the Nav1.6 channel that had fast inactivation removed, resulting in faster channel activation. Although the Nav1.2 channel with fast inactivation removed also demonstrated slightly faster activation, that channel showed very pronounced slow inactivation compared to Nav1.6. These results indicate that potentiation of Nav1.6 sodium currents results from faster channel activation, and that this effect is masked by slow inactivation in Nav1.2. The data suggest that Nav1.6 might be more resistant to inactivation, which might be helpful for high-frequency firing at nodes of Ranvier compared to Nav1.2.     

The poison Dart frog's batrachotoxin modulates Nav1.8

Batrachotoxin is a potent modulator of voltage-gated sodium channels, leading to irreversible depolarisation of nerves and muscles, fibrillation, arrhythmias and eventually cardiac failure. Since its discovery, field researchers also reported numbness after their skin came into contact with this toxin. Intrigued by this phenomenon, we determined the effect of batrachotoxin on the voltage-gated sodium channel Nav1.8, which is considered to be a key player in nociception. As a result, we discovered that batrachotoxin profoundly modulates this channel: the inactivation process is severely altered, the voltage-dependence of activation is shifted towards more hyperpolarised potentials resulting in the opening of Nav1.8 at more negative membrane potentials and the ion selectivity is modified.     

Individual variation and hormonal modulation of a sodium channel beta subunit in the electric organ correlate with variation in a social signal

The sodium channel beta1 subunit affects sodium channel gating and surface density, but little is known about the factors that regulate beta1 expression or its participation in the fine control of cellular excitability. In this study we examined whether graded expression of the beta1 subunit contributes to the gradient in sodium current inactivation, which is tightly controlled and directly related to a social behavior, the electric organ discharge (EOD), in a weakly electric fish Sternopygus macrurus. We found the mRNA and protein levels of beta1 in the electric organ both correlate with EOD frequency. We identified a novel mRNA splice form of this gene and found the splicing preference for this novel splice form also correlates with EOD frequency. Androgen implants lowered EOD frequency and decreased the beta1 mRNA level but did not affect splicing. Coexpression of each splice form in Xenopus oocytes with either the human muscle sodium channel gene, hNav1.4, or a Sternopygus ortholog, smNav1.4b, sped the rate of inactivation of the sodium current and shifted the steady-state inactivation toward less negative membrane potentials. The translational product of the novel mRNA splice form lacks a previously identified important tyrosine residue but still functions normally. The properties of the fish alpha and coexpressed beta1 subunits in the oocyte replicate those of the electric organ's endogenous sodium current. These data highlight the role of ion channel beta subunits in regulating cellular excitability.     

Resting potential-dependent regulation of the voltage sensitivity of sodium channel gating in rat skeletal muscle in vivo

Normal muscle has a resting potential of -85 mV, but in a number of situations there is depolarization of the resting potential that alters excitability. To better understand the effect of resting potential on muscle excitability we attempted to accurately simulate excitability at both normal and depolarized resting potentials. To accurately simulate excitability we found that it was necessary to include a resting potential-dependent shift in the voltage dependence of sodium channel activation and fast inactivation. We recorded sodium currents from muscle fibers in vivo and found that prolonged changes in holding potential cause shifts in the voltage dependence of both activation and fast inactivation of sodium currents. We also found that altering the amplitude of the prepulse or test pulse produced differences in the voltage dependence of activation and inactivation respectively. Since only the Nav1.4 sodium channel isoform is present in significant quantity in adult skeletal muscle, this suggests that either there are multiple states of Nav1.4 that differ in their voltage dependence of gating or there is a distribution in the voltage dependence of gating of Nav1.4. Taken together, our data suggest that changes in resting potential toward more positive potentials favor states of Nav1.4 with depolarized voltage dependence of gating and thus shift voltage dependence of the sodium current. We propose that resting potential-induced shifts in the voltage dependence of sodium channel gating are essential to properly regulate muscle excitability in vivo.     

Cloning and functional expression of two families of beta-subunits of the large conductance calcium-activated K+ channel

We report here a characterization of two families of calcium-activated K(+) channel beta-subunits, beta2 and beta3, which are encoded by distinct genes that map to 3q26.2-27. A single beta2 family member and four alternatively spliced variants of beta3 were investigated. These subunits have predicted molecular masses of 27. 1-31.6 kDa, share approximately 30-44% amino acid identity with beta1, and exhibit distinct but overlapping expression patterns. Coexpression of the beta2 or beta3a-c subunits with a BK alpha-subunit altered the functional properties of the current expressed by the alpha-subunit alone. The beta2 subunit rapidly and completely inactivated the current and shifted the voltage dependence for activation to more polarized membrane potentials. In contrast, coexpression of the beta3a-c subunits resulted in only partial inactivation of the current, and the beta3b subunit conferred an apparent inward rectification. Furthermore, unlike the beta1 and beta2 subunits, none of the beta3 subunits increased channel sensitivity to calcium or voltage. The tissue-specific expression of these beta-subunits may allow for the assembly of a large number of distinct BK channels in vivo, contributing to the functional diversity of native BK currents.     

Scorpion toxin BmK I directly activates Nav1.8 in primary sensory neurons to induce neuronal hyperexcitability in rats

Voltage-gated sodium channels (VGSCs) in primary sensory neurons play a key role in transmitting pain signals to the central nervous system. BmK I, a site-3 sodium channel-specific toxin from scorpion Buthus martensi Karsch, induces pain behaviors in rats. However, the subtypes of VGSCs targeted by BmK I were not entirely clear. We therefore investigated the effects of BmK I on the current amplitude, gating and kinetic properties of Nav1.8, which is associated with neuronal hyperexcitability in DRG neurons. It was found that BmK I dose-dependently increased Nav1.8 current in smallsized (<25 μm) acutely dissociated DRG neurons, which correlated with its inhibition on both fast and slow inactivation. Moreover, voltage-dependent activation and steady-state inactivation curves of Nav1.8 were shifted in a hyperpolarized direction. Thus, BmK I reduced the threshold of neuronal excitability and increased action potential firing in DRG neurons. In conclusion, our data clearly demonstrated that BmK I modulated Nav1.8 remarkably, suggesting BmK I as a valuable probe for studying Nav1.8. And Nav1.8 is an important target related to BmK I-evoked pain.     

The sialic acid component of the beta1 subunit modulates voltage-gated sodium channel function

Voltage-gated sodium channels (Nav) are responsible for initiation and propagation of nerve, skeletal muscle, and cardiac action potentials. Nav are composed of a pore-forming alpha subunit and often one to several modulating beta subunits. Previous work showed that terminal sialic acid residues attached to alpha subunits affect channel gating. Here we show that the fully sialylated beta1 subunit induces a uniform, hyperpolarizing shift in steady state and kinetic gating of the cardiac and two neuronal alpha subunit isoforms. Under conditions of reduced sialylation, the beta1-induced gating effect was eliminated. Consistent with this, mutation of beta1 N-glycosylation sites abolished all effects of beta1 on channel gating. Data also suggest an interaction between the cis effect of alpha sialic acids and the trans effect of beta1 sialic acids on channel gating. Thus, beta1 sialic acids had no effect gating on the of the heavily glycosylated skeletal muscle alpha subunit. However, when glycosylation of the skeletal muscle alpha subunit was reduced through chimeragenesis such that alpha sialic acids did not impact gating, beta1 sialic acids caused a significant hyperpolarizing shift in channel gating. Together, the data indicate that beta1 N-linked sialic acids can modulate Nav gating through an apparent saturating electrostatic mechanism. A model is proposed in which a spectrum of differentially sialylated Nav can directly modulate channel gating, thereby impacting cardiac, skeletal muscle, and neuronal excitability.     

Calmodulin and calcium differentially regulate the neuronal Nav1.1 voltage-dependent sodium channel

Mutations in the neuronal Nav1.1 voltage-gated sodium channel are responsible for mild to severe epileptic syndromes. The ubiquitous calcium sensor calmodulin (CaM) bound to rat brain Nav1.1 and to the human Nav1.1 channel expressed by a stably transfected HEK-293 cell line. The C-terminal region of the channel, as a fusion protein or in the yeast two-hybrid system, interacted with CaM via a consensus C-terminal motif, the IQ domain. Patch clamp experiments on HEK1.1 cells showed that CaM overexpression increased peak current in a calcium-dependent way. CaM had no effect on the voltage-dependence of fast inactivation, and accelerated the inactivation kinetics. Elevating Ca⁺⁺ depolarized the voltage-dependence of fast inactivation and slowed down the fast inactivation kinetics, and for high concentrations this effect competed with the acceleration induced by CaM alone. Similarly, the depolarizing action of calcium antagonized the hyperpolarizing shift of the voltage-dependence of activation due to CaM overexpression. Fluorescence spectroscopy measurements suggested that Ca⁺⁺ could bind the Nav1.1 C-terminal region with micromolar affinity.     

The beta1 subunit enhances oxidative regulation of large-conductance calcium-activated K+ channels

Oxidative stress may alter the functions of many proteins including the Slo1 large conductance calcium-activated potassium channel (BKCa). Previous results demonstrated that in the virtual absence of Ca2+, the oxidant chloramine-T (Ch-T), without the involvement of cysteine oxidation, increases the open probability and slows the deactivation of BKCa channels formed by human Slo1 (hSlo1) alpha subunits alone. Because native BKCa channel complexes may include the auxiliary subunit beta1, we investigated whether beta1 influences the oxidative regulation of hSlo1. Oxidation by Ch-T with beta1 present shifted the half-activation voltage much further in the hyperpolarizing direction (-75 mV) as compared with that with alpha alone (-30 mV). This shift was eliminated in the presence of high [Ca2+]i, but the increase in open probability in the virtual absence of Ca2+ remained significant at physiologically relevant voltages. Furthermore, the slowing of channel deactivation after oxidation was even more dramatic in the presence of beta1. Oxidation of cysteine and methionine residues within beta1 was not involved in these potentiated effects because expression of mutant beta1 subunits lacking cysteine or methionine residues produced results similar to those with wild-type beta1. Unlike the results with alpha alone, oxidation by Ch-T caused a significant acceleration of channel activation only when beta1 was present. The beta1 M177 mutation disrupted normal channel activation and prevented the Ch-T-induced acceleration of activation. Overall, the functional effects of oxidation of the hSlo1 pore-forming alpha subunit are greatly amplified by the presence of beta1, which leads to the additional increase in channel open probability and the slowing of deactivation. Furthermore, M177 within beta1 is a critical structural determinant of channel activation and oxidative sensitivity. Together, the oxidized BKCa channel complex with beta1 has a considerable chance of being open within the physiological voltage range even at low [Ca2+]i.     

The human heart and rat brain IIA Na+ channels interact with different molecular regions of the beta1 subunit

The alpha subunit of voltage-gated Na(+) channels of brain, skeletal muscle, and cardiomyocytes is functionally modulated by the accessory beta(1), but not the beta(2) subunit. In the present study, we used beta(1)/beta(2) chimeras to identify molecular regions within the beta(1) subunit that are responsible for both the increase of the current density and the acceleration of recovery from inactivation of the human heart Na(+) channel (hH1). The channels were expressed in Xenopus oocytes. As a control, we coexpressed the beta(1)/beta(2) chimeras with rat brain IIA channels. In agreement with previous studies, the beta(1) extracellular domain sufficed to modulate IIA channel function. In contrast to this, the extracellular domain of the beta(1) subunit alone was ineffective to modulate hH1. Instead, the putative membrane anchor plus either the intracellular or the extracellular domain of the beta(1) subunit was required. An exchange of the beta(1) membrane anchor by the corresponding beta(2) subunit region almost completely abolished the effects of the beta(1) subunit on hH1, suggesting that the beta(1) membrane anchor plays a crucial role for the modulation of the cardiac Na(+) channel isoform. It is concluded that the beta(1) subunit modulates the cardiac and the neuronal channel isoforms by different molecular interactions: hH1 channels via the membrane anchor plus additional intracellular or extracellular regions, and IIA channels via the extracellular region only.     

Human ether-à-go-go-related gene K+ channel gating probed with extracellular ca2+. Evidence for two distinct voltage sensors

Human ether-à-go-go-related gene (HERG) encoded K+ channels were expressed in Chinese hamster ovary (CHO-K1) cells and studied by whole-cell voltage clamp in the presence of varied extracellular Ca2+ concentrations and physiological external K+. Elevation of external Ca2+ from 1.8 to 10 mM resulted in a reduction of whole-cell K+ current amplitude, slowed activation kinetics, and an increased rate of deactivation. The midpoint of the voltage dependence of activation was also shifted +22.3 +/- 2.5 mV to more depolarized potentials. In contrast, the kinetics and voltage dependence of channel inactivation were hardly affected by increased extracellular Ca2+. Neither Ca2+ screening of diffuse membrane surface charges nor open channel block could explain these changes. However, selective changes in the voltage-dependent activation, but not inactivation gating, account for the effects of Ca2+ on Human ether-à-go-go-related gene current amplitude and kinetics. The differential effects of extracellular Ca2+ on the activation and inactivation gating indicate that these processes have distinct voltage-sensing mechanisms. Thus, Ca2+ appears to directly interact with externally accessible channel residues to alter the membrane potential detected by the activation voltage sensor, yet Ca2+ binding to this site is ineffective in modifying the inactivation gating machinery.     

Comparison of heterologously expressed human cardiac and skeletal muscle sodium channels.

In this study we have expressed and characterized recombinant cardiac and skeletal muscle sodium channel alpha subunits in tsA-201 cells under identical experimental conditions. Unlike the Xenopus oocyte expression system, in tsA-201 cells (transformed human embryonic kidney) both channels seem to gate rapidly, as in native tissue. In general, hSkM1 gating seemed faster than hH1 both in terms of rate of inactivation and rate of recovery from inactivation as well as time to peak current. The midpoint of the steady-state inactivation curve was approximately 25 mV more negative for hH1 compared with hSkM1. In both isoforms, the steady-state channel availability relationships ("inactivation curves") shifted toward more negative membrane potentials with time. The cardiac isoform showed a minimal shift in the activation curve as a function of time after whole-cell dialysis, whereas hSkM1 showed a continued and marked negative shift in the activation voltage dependence of channel gating. This observation suggests that the mechanism underlying the shift in inactivation voltage dependence may be similar to the one that is causing the shift in the activation voltage dependence in hSkM1 but that this is uncoupled in the cardiac isoform. These results demonstrate the utility and limitations of measuring cardiac and skeletal muscle recombinant Na+ channels in tsA-201 cells. This baseline characterization will be useful for future investigations on channel mutants and pharmacology.     

Voltage-gated rearrangements associated with differential beta-subunit modulation of the L-type Ca(2+) channel inactivation

Auxiliary beta-subunits bound to the cytoplasmic alpha(1)-interaction domain of the pore-forming alpha(1C)-subunit are important modulators of voltage-gated Ca(2+) channels. The underlying mechanisms are not yet well understood. We investigated correlations between differential modulation of inactivation by beta(1a)- and beta(2)- subunits and structural responses of the channel to transition into distinct functional states. The NH(2)-termini of the alpha(1C)- and beta-subunits were fused with cyan or yellow fluorescent proteins, and functionally coexpressed in COS1 cells. Fluorescence resonance energy transfer (FRET) between them or with membrane-trapped probes was measured in live cells under voltage clamp. It was found that in the resting state, the tagged NH(2)-termini of the alpha(1C)- and beta-subunit fluorophores are separated. Voltage-dependent inactivation generates strong FRET between alpha(1C) and beta(1a) suggesting mutual reorientation of the NH(2)-termini, but their distance vis-à-vis the plasma membrane is not appreciably changed. These voltage-gated rearrangements were substantially reduced when the beta(1a)-subunit was replaced by beta(2). Differential beta-subunit modulation of inactivation and of FRET between alpha(1C) and beta were eliminated by inhibition of the slow inactivation. Thus, differential beta-subunit modulation of inactivation correlates with the voltage-gated motion between the NH(2)-termini of alpha(1C)- and beta-subunits and targets the mechanism of slow voltage-dependent inactivation.     

Inherited Pain: SODIUM CHANNEL NAV1.7 A1632T MUTATION CAUSES ERYTHROMELALGIA DUE TO A SHIFT OF FAST INACTIVATION*

Inherited erythromelalgia (IEM) causes debilitating episodic neuropathic pain characterized by burning in the extremities. Inherited “paroxysmal extreme pain disorder” (PEPD) differs in its clinical picture and affects proximal body areas like the rectal, ocular, or jaw regions. Both pain syndromes have been linked to mutations in the voltage-gated sodium channel Nav1.7. Electrophysiological characterization shows that IEM-causing mutations generally enhance activation, whereas mutations leading to PEPD alter fast inactivation. Previously, an A1632E mutation of a patient with overlapping symptoms of IEM and PEPD was reported (Estacion, M., Dib-Hajj, S. D., Benke, P. J., Te Morsche, R. H., Eastman, E. M., Macala, L. J., Drenth, J. P., and Waxman, S. G. (2008) NaV1.7 Gain-of-function mutations as a continuum. A1632E displays physiological changes associated with erythromelalgia and paroxysmal extreme pain disorder mutations and produces symptoms of both disorders. J. Neurosci. 28, 11079–11088), displaying a shift of both activation and fast inactivation. Here, we characterize a new mutation of Nav1.7, A1632T, found in a patient suffering from IEM. Although transfection of A1632T in sensory neurons resulted in hyperexcitability and spontaneous firing of dorsal root ganglia (DRG) neurons, whole-cell patch clamp of transfected HEK cells revealed that Nav1.7 activation was unaltered by the A1632T mutation but that steady-state fast inactivation was shifted to more depolarized potentials. This is a characteristic normally attributed to PEPD-causing mutations. In contrast to the IEM/PEPD crossover mutation A1632E, A1632T failed to slow current decay (i.e. open-state inactivation) and did not increase resurgent currents, which have been suggested to contribute to high-frequency firing in physiological and pathological conditions. Reduced fast inactivation without increased resurgent currents induces symptoms of IEM, not PEPD, in the new Nav1.7 mutation, A1632T. Therefore, persistent and resurgent currents are likely to determine whether a mutation in Nav1.7 leads to IEM or PEPD.     

Functional roles of the extracellular segments of the sodium channel alpha subunit in voltage-dependent gating and modulation by beta1 subunits.

Voltage-gated sodium channels consist of a pore-forming alpha subunit associated with beta1 subunits and, for brain sodium channels, beta2 subunits. Although much is known about the structure and function of the alpha subunit, there is little information on the functional role of the 16 extracellular loops. To search for potential functional activities of these extracellular segments, chimeras were studied in which an individual extracellular loop of the rat heart (rH1) alpha subunit was substituted for the corresponding segment of the rat brain type IIA (rIIA) alpha subunit. In comparison with rH1, wild-type rIIA alpha subunits are characterized by more positive voltage-dependent activation and inactivation, a more prominent slow gating mode, and a more substantial shift to the fast gating mode upon coexpression of beta1 subunits in Xenopus oocytes. When alpha subunits were expressed alone, chimeras with substitutions from rH1 in five extracellular loops (IIS5-SS1, IISS2-S6, IIIS1-S2, IIISS2-S6, and IVS3-S4) had negatively shifted activation, and chimeras with substitutions in three of these (IISS2-S6, IIIS1-S2, and IVS3-S4) also had negatively shifted steady-state inactivation. rIIA alpha subunit chimeras with substitutions from rH1 in five extracellular loops (IS5-SS1, ISS2-S6, IISS2-S6, IIIS1-S2, and IVS3-S4) favored the fast gating mode. Like wild-type rIIA alpha subunits, all of the chimeric rIIA alpha subunits except chimera IVSS2-S6 were shifted almost entirely to the fast gating mode when coexpressed with beta1 subunits. In contrast, substitution of extracellular loop IVSS2-S6 substantially reduced the effectiveness of beta1 subunits in shifting rIIA alpha subunits to the fast gating mode. Our results show that multiple extracellular loops influence voltage-dependent activation and inactivation and gating mode of sodium channels, whereas segment IVSS2-S6 plays a dominant role in modulation of gating by beta1 subunits. Evidently, several extracellular loops are important determinants of sodium channel gating and modulation.     

Sodium channel beta1 subunit-mediated modulation of Nav1.2 currents and cell surface density is dependent on interactions with contactin and ankyrin

Voltage-gated sodium channels are composed of a pore-forming alpha subunit and at least one auxiliary beta subunit. Both beta1 and beta2 are cell adhesion molecules that interact homophilically, resulting in ankyrin recruitment. In contrast, beta1, but not beta2, interacts heterophilically with contactin, resulting in increased levels of cell surface sodium channels. We took advantage of these results to investigate the molecular basis of beta1-mediated enhancement of sodium channel cell surface density, including elucidating structure-function relationships for beta1 association with contactin, ankyrin, and Nav1.2. beta1/beta2 subunit chimeras were used to assign putative sites of contactin interaction to two regions of the beta1 Ig loop. Recent studies have shown that glutathione S-transferase fusion proteins containing portions of Nav1.2 intracellular domains interact directly with ankyrinG. We show that native Nav1.2 associates with ankyrinG in cells in the absence of beta subunits and that this interaction is enhanced in the presence of beta1 but not beta1Y181E, a mutant that does not interact with ankyrinG. beta1Y181E does not modulate Nav1.2 channel function despite efficient association with Nav1.2 and contactin. beta1Y181E increases Nav1.2 cell surface expression, but not as efficiently as wild type beta1. beta1/beta2 chimeras exchanging various regions of the beta1 Ig loop were all ineffective in increasing Nav1.2 cell surface density. Our results demonstrate that full-length beta1 is required for channel modulation and enhancement of sodium channel cell surface expression.     

Modulation of human Nav1.7 channel gating by synthetic α-scorpion toxin OD1 and its analogs

Nine different voltage-gated sodium channel isoforms are responsible for inducing and propagating action potentials in the mammalian nervous system. The Na_v1.7 channel isoform plays an important role in conducting nociceptive signals. Specific mutations of this isoform may impair gating behavior of the channel resulting in several pain syndromes. In addition to channel mutations, similar or opposite changes in gating may be produced by spider and scorpion toxins binding to different parts of the voltage-gated sodium channel. In the present study, we analyzed the effects of the α-scorpion toxin OD1 and 2 synthetic toxin analogs on the gating properties of the Na_v1.7 sodium channel. All toxins potently inhibited channel inactivation, however, both toxin analogs showed substantially increased potency by more than one order of magnitude when compared with that of wild-type OD1. The decay phase of the whole-cell Na⁺ current was substantially slower in the presence of toxins than in their absence. Single-channel recordings in the presence of the toxins revealed that Na⁺ current inactivation slowed due to prolonged flickering of the channel between open and closed states. Our findings support the voltage-sensor trapping model of α-scorpion toxin action, in which the toxin prevents a conformational change in the domain IV voltage sensor that normally leads to fast channel inactivation.     

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 (I_Na). 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 I_Na with IC₅₀ values of 10.3 and 21.5 μM (holding potential=-120 or -100 mV, respectively). The potency of I_Na 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 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 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.     

Understanding LTP in pain pathways

Background

Small 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 Na_v1.8.

Results

In 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 V_1/2 values of -73 and -37 mV. These values are similar to the V_1/2 values for steady-state fast inactivation of tetrodotoxin-sensitive (TTX-s) sodium channels, and the tetrodotoxin-resistant (TTX-r) Na_v1.8 sodium channel, respectively. Addition of TTX eliminates the more hyperpolarized V_1/2 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 Na_v1.8(-/-) mice, the voltage-dependent decrease in AP amplitude is characterized by a single Boltzmann equation with a V_1/2 value of -55 mV, suggesting a shift in the steady-state fast inactivation properties of TTX-s sodium channels. Transfection of Na_v1.8(-/-) DRG neurons with DNA encoding Na_v1.8 results in a membrane potential-dependent decrease in AP amplitude that recapitulates WT properties.

Conclusion

We conclude that the presence of Na_v1.8 allows AP amplitude to be maintained in DRG neurons and their centrally projecting axons even when depolarized within the dorsal horn.