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PubMed2005

Endogenous protease activation of ENaC: effect of serine protease inhibition on ENaC single channel properties.

Endogenous serine proteases have been reported to control the reabsorption of Na(+) by kidney- and lung-derived epithelial cells via stimulation of electrogenic Na(+) transport mediated by the epithelial Na(+) channel (ENaC). In this study we investigated the effects of aprotinin on ENaC single channel properties using transepithelial fluctuation analysis in the amphibian kidney epithelium, A6. Aprotinin caused a time- and concentration-dependent inhibition (84 +/- 10.5%) in the amiloride-sensitive sodium transport (I(Na)) with a time constant of 18 min and half maximal inhibition constant of 1 microM. Analysis of amiloride analogue blocker-induced fluctuations in I(Na) showed linear rate-concentration plots with identical blocker on and off rates in control and aprotinin-inhibited conditions. Verification of open-block kinetics allowed for the use of a pulse protocol method (Helman, S.I., X. Liu, K. Baldwin, B.L. Blazer-Yost, and W.J. Els. 1998. Am. J. Physiol. 274:C947-C957) to study the same cells under different conditions as well as the reversibility of the aprotinin effect on single channel properties. Aprotinin caused reversible changes in all three single channel properties but only the change in the number of open channels was consistent with the inhibition of I(Na). A 50% decrease in I(Na) was accompanied by 50% increases in the single channel current and open probability but an 80% decrease in the number of open channels. Washout of aprotinin led to a time-dependent restoration of I(Na) as well as the single channel properties to the control, pre-aprotinin, values. We conclude that protease regulation of I(Na) is mediated by changes in the number of open channels in the apical membrane. The increase in the single channel current caused by protease inhibition can be explained by a hyperpolarization of the apical membrane potential as active Na(+) channels are retrieved. The paradoxical increase in channel open probability caused by protease inhibition will require further investigation but does suggest a potential compensatory regulatory mechanism to maintain I(Na) at some minimal threshold value.

Amiloride↗
PubMed1995

The effects of calcium and calcium channel blockers on sodium pump.

The effects of 10 mM Ca2+ and Ca2+ channel blockers verapamil, diltiazem and flunarizine on the ouabain-sensitive electrogenic Na+, K+ pump activity of mouse diaphragm muscle fibres enriched with Na+ were compared with the changes in cytosolic [Ca2+]. The electrogenic Na+ pump activity produced by adding K+ to muscles previously bathed for 4 h in a K(+)-free, 2-mM [Ca2+] solution increased the resting membrane potential by about 18 mV. This hyperpolarization was completely inhibited after 10 min incubation in 10 mM Ca2+. Verapamil 10(-5) M, 10(-5) M diltiazem and 10(-7) M flunarizine effectively prevented the effect of elevated [Ca2+]. At these concentrations, these drugs did not affect the K(+)-induced hyperpolarization. In mouse diaphragm, the basal cytosolic [Ca2+] measured by the fluorescent indicator 1-[2-(5-carboxyoxazol-2-yl)-6- aminobenzofuran-5-oxy]2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N '- tetraacetic acid acetoxymethyl ester (fura-2/AM) was 261 +/- 6 nM. After 4 h in a Liley K(+)-free, 2 mM [Ca2+] solution, the cytosolic [Ca2+] increased to 314 +/- 28 nM. Increase in [Ca2+] from 2 to 10 mM caused a twofold increase of cytosolic [Ca2+] to 637 +/- 26 nM. This rise was, like the Ca(2+)-induced inhibition of electrogenic pump, prevented by 10(-5) M verapamil, 10(-5) M diltiazem and 10(-7) M flunarizine. The results suggest that substances which block Ca2+ entry into the cell prevent the Ca(2+)-induced inhibition of the Na+ pump.

Animals↗
PubMed2004

Characterization of the participation of sodium channels on the rise in Na+ induced by 4-aminopyridine (4-AP) in synaptosomes.

The participation of voltage-sensitive Na+ channels (VSSC) on the changes on internal (i) Na+, K+, Ca2+, and on DA, Glu, and GABA release caused by different concentrations of 4-AP was investigated in striatum synaptosomes. TTX, which abolished the increase in Na(i) (as determined with SBFI), induced by 0.1 mM 4-AP only inhibited by 30% the rise in Na(i) induced by 1 mM 4-AP. One millimolar 4-AP markedly decreased the fluorescence of the K+ indicator dye PBFI but 0.1 mM 4-AP did not. Like 1 mM 4-AP, ouabain decreased PBFI fluorescence and increased a considerable fraction of Na(i) in a TTX-insensitive manner. In contrast with the different TTX sensitivity of the rise in Na(i) induced by 0.1 and 1 mM 4-AP, the rise in Ca(i) (as determined with fura-2) induced by the two concentrations of 4-AP was markedly inhibited by TTX, as well as by omega-agatoxin in combination with omega-conotoxin GVIA, indicating that only the TTX-sensitive fraction of the rise in Na(i) induced by 4-AP is linked with the activation of presynaptic Ca2+ channels. It is concluded that the TTX-sensitive fraction of neurotransmitter release evoked by 4-AP is released by exocytosis, and the TTX insensitive fraction involves reversal of the neurotransmitters transporters. This contrasts with the exocytosis evoked by high K+ that is unchanged by TTX and with the neurotransmitter-transporter-mediated release evoked by veratridine, which is highly TTX sensitive and does not require activation of Ca2+ channels.

4-Aminopyridine↗
PubMed2003

Dependence of mu-conotoxin block of sodium channels on ionic strength but not on the permeating [Na+]: implications for the distinctive mechanistic interactions between Na+ and K+ channel pore-blocking toxins and their molecular targets.

Mu-conotoxins (mu-CTXs) are Na+ channel-blocking, 22-amino acid peptides produced by the sea snail Conus geographus. Although K+ channel pore-blocking toxins show specific interactions with permeant ions and strong dependence on the ionic strength (mu), no such dependence has been reported for mu-CTX and Na+ channels. Such properties would offer insight into the binding and blocking mechanism of mu-CTX as well as functional and structural properties of the Na+ channel pore. Here we studied the effects of mu and permeant ion concentration ([Na+]) on mu-CTX block of rat skeletal muscle (mu1, Nav1.4) Na+ channels. Mu-CTX sensitivity of wild-type and E758Q channels increased significantly (by approximately 20-fold) when mu was lowered by substituting external Na+ with equimolar sucrose (from 140 to 35 mm Na+); however, toxin block was unaltered (p > 0.05) when mu was maintained by replacement of [Na+] with N-methyl-d-glucamine (NMG+), suggesting that the enhanced sensitivity at low mu was not due to reduction in [Na+]. Single-channel recordings identified the association rate constant, k(on), as the primary determinant of the changes in affinity (k(on) increased 40- and 333-fold for mu-CTX D2N/R13Q and D12N/R13Q, respectively, when symmetric 200 mm Na+ was reduced to 50 mm). In contrast, dissociation rates changed <2-fold for the same derivatives under the same conditions. Experiments with additional mu-CTX derivatives identified toxin residues Arg-1, Arg-13, and Lys-16 as important contributors to the sensitivity to external mu. Taken together, our findings indicate that mu-CTX block of Na+ channels depends critically on mu but not specifically on [Na+], contrasting with the known behavior of pore-blocking K+ channel toxins. These findings suggest that different degrees of ion interaction, underlying the fundamental conduction mechanisms of Na+ and K+ channels, are mirrored in ion interactions with pore-blocking toxins.

Animals↗
PubMed2000

Intrinsic membrane properties underlying spontaneous tonic firing in neostriatal cholinergic interneurons.

Neostriatal cholinergic interneurons produce spontaneous tonic firing in the absence of synaptic input. Perforated patch recording and whole-cell recording combined with calcium imaging were used in vitro to identify the intrinsic membrane properties underlying endogenous excitability. Spontaneous firing was driven by the combined action of a sodium current and the hyperpolarization-activated cation current (I(h)), which together ensured that there was no zero current point in the subthreshold voltage range. Blockade of sodium channels or I(h) established a stable subthreshold resting membrane potential. A tetrodotoxin-sensitive region of negative slope conductance was observed between approximately -60 mV and threshold (approximately -50 mV) and the h-current was activated at all subthreshold voltages. Calcium imaging experiments revealed that there was minimal calcium influx at subthreshold membrane potentials but that action potentials produced elevations of calcium in both the soma and dendrites. Spike-triggered calcium entry shaped the falling phase of the action potential waveform and activated calcium-dependent potassium channels. Blockade of big-conductance channels caused spike broadening. Application of apamin, which blocks small-conductance channels, abolished the slow spike afterhyperpolarization (AHP) and caused a transition to burst firing. In the absence of synaptic input, a range of tonic firing patterns are observed, suggesting that the characteristic spike sequences described for tonically active cholinergic neurons (TANs) recorded in vivo are intrinsic in origin. The pivotal role of the AHP in regulating spike patterning indicates that burst firing of TANs in vivo could arise from direct or indirect modulation of the AHP without requiring phasic synaptic input.

Action Potentials↗
PubMed1998

Mechanisms of calcium and sodium fluxes in anoxic myelinated central nervous system axons.

Electron probe X-ray microanalysis was used to measure water content and concentrations of elements (i.e. Na, K, Cl and Ca) in selected morphological compartments of rat optic nerve myelinated axons. Transaxolemmal movements of Na+ and Ca2+ were modified experimentally and corresponding effects on axon element and water compositions were determined under control conditions and following in vitro anoxic challenge. Also characterized were effects of modified ion transport on axon responses to postanoxia reoxygenation. Blockade of Na+ entry by tetrodotoxin (1 microM) or zero Na+/Li(+)-substituted perfusion reduced anoxic increases in axonal Na and Ca concentrations. Incubation with zero-Ca2+/EGTA perfusate prevented axoplasmic and mitochondrial Ca accumulation during anoxia but did not affect Na increases or K losses in these compartments. Inhibition of Na(+)-Ca2+ exchange with bepridil (30 microM) selectively prevented increases in intra-axonal Ca, whereas neither nifedipine (5 microM) nor nimodipine (5 microM) influenced the effects of anoxia on axonal Na, K or Ca. X-ray microanalysis also showed that prevention of Na and Ca influx during anoxia obtunded severe elemental deregulation normally associated with reoxygenation. Results of the present study suggest that during anoxia, Na+ enters axons mainly through voltage-gated Na+ channels and that subsequent increases in axoplasmic Na+ are functionally coupled to extra-axonal Ca2+ import. Na+i-dependent, Ca2+o entry is consistent with reverse operation of the axolemmal Na(+)-Ca2+ exchanger and we suggest this route represents a primary mechanism of Ca2+ influx. Our findings also implicate a minor route of Ca2+ entry directly through Na+ channels.

Animals↗
PubMed2006

Translating basic research on sodium channels in human neuropathic pain.

UNLABELLED: A number of experimental studies in animals have suggested that voltage-gated sodium channels may play a crucial role in neuropathic pain. However, it is still difficult to translate the pathophysiological mechanisms identified in animal studies to the clinic and several questions regarding the role of sodium channels in neuropathic pain must therefore be addressed primarily in the clinical setting. Despite providing indirect information, pharmacologic challenge using sodium channel blockers, such as some antiepileptics, local anesthetics and derivatives, is the best way to investigate the role of sodium channels in the various clinical symptoms of neuropathies (eg, spontaneous pain, mechanical or thermal allodynia, and hyperalgesia). Randomized controlled trials have demonstrated the efficacy of these compounds for various neuropathic pain conditions. Recent psychophysical studies in which symptoms and signs are more accurately assessed indicate that these compounds act as antihyperalgesic agents rather than as simple analgesics. They also show that the sensitivity to these drugs is not affected by the aetiology of pain and the peripheral or central location of the nerve lesion. These data emphasize the role of peripheral and central sodium channels in neuropathic pain. Studies using more selective sodium channel blockers are required to gain further insight into the role of the various subtypes of sodium channel in these pain conditions. PERSPECTIVE: Pharmacological challenge using sodium channel blockers is the best way to translate basic research on sodium channels in human neuropathic pain. To date, the role of sodium channels in neuropathic pain symptoms/signs is mostly documented for mechanical static and dynamic allodynia, and either peripheral or central sodium channels may be involved.

Humans↗
PubMed2007

Pharmacological dissection and distribution of NaN/Nav1.9, T-type Ca2+ currents, and mechanically activated cation currents in different populations of DRG neurons.

Low voltage-activated (LVA) T-type Ca(2+) (I(Ca)T) and NaN/Nav1.9 currents regulate DRG neurons by setting the threshold for the action potential. Although alterations in these channels have been implicated in a variety of pathological pain states, their roles in processing sensory information remain poorly understood. Here, we carried out a detailed characterization of LVA currents in DRG neurons by using a method for better separation of NaN/Nav1.9 and I(Ca)T currents. NaN/Nav1.9 was inhibited by inorganic I(Ca) blockers as follows (IC(50), microM): La(3+) (46) > Cd(2+) (233) > Ni(2+) (892) and by mibefradil, a non-dihydropyridine I(Ca)T antagonist. Amiloride, however, a preferential Cav3.2 channel blocker, had no effects on NaN/Nav1.9 current. Using these discriminative tools, we showed that NaN/Nav1.9, Cav3.2, and amiloride- and Ni(2+)-resistant I(Ca)T (AR-I(Ca)T) contribute differentially to LVA currents in distinct sensory cell populations. NaN/Nav1.9 carried LVA currents into type-I (CI) and type-II (CII) small nociceptors and medium-Adelta-like nociceptive cells but not in low-threshold mechanoreceptors, including putative Down-hair (D-hair) and Aalpha/beta cells. Cav3.2 predominated in CII-nociceptors and in putative D-hair cells. AR-I(Ca)T was restricted to CII-nociceptors, putative D-hair cells, and Aalpha/beta-like cells. These cell types distinguished by their current-signature displayed different types of mechanosensitive channels. CI- and CII-nociceptors displayed amiloride-sensitive high-threshold mechanical currents with slow or no adaptation, respectively. Putative D-hair and Aalpha/beta-like cells had low-threshold mechanical currents, which were distinguished by their adapting kinetics and sensitivity to amiloride. Thus, subspecialized DRG cells express specific combinations of LVA and mechanosensitive channels, which are likely to play a key role in shaping responses of DRG neurons transmitting different sensory modalities.

Action Potentials↗
PubMed2000

Block of wild-type and inactivation-deficient cardiac sodium channels IFM/QQQ stably expressed in mammalian cells.

The role of inactivation as a central mechanism in blockade of the cardiac Na(+) channel by antiarrhythmic drugs remains uncertain. We have used whole-cell and single channel recordings to examine the block of wild-type and inactivation-deficient mutant cardiac Na(+) channels, IFM/QQQ, stably expressed in HEK-293 cells. We studied the open-channel blockers disopyramide and flecainide, and the lidocaine derivative RAD-243. All three drugs blocked the wild-type Na(+) channel in a use-dependent manner. There was no use-dependent block of IFM/QQQ mutant channels with trains of 20 40-ms pulses at 150-ms interpulse intervals during disopyramide exposure. Flecainide and RAD-243 retained their use-dependent blocking action and accelerated macroscopic current relaxation. All three drugs reduced the mean open time of single channels and increased the probability of their failure to open. From the abbreviation of the mean open times, we estimated association rates of approximately 10(6)/M/s for the three drugs. Reducing the burst duration contributed to the acceleration of macroscopic current relaxation during exposure to flecainide and RAD-243. The qualitative differences in use-dependent block appear to be the result of differences in drug dissociation rate. The inactivation gate may play a trapping role during exposure to some sodium channel blocking drugs.

Amino Acid Substitution↗
PubMed2000

Effects of blockade of voltage-sensitive Ca(2+)/Na(+) channels by a novel phenylpyrimidine derivative, NS-7, on CREB phosphorylation in focal cerebral ischemia in the rat.

NS-7 is a novel blocker of voltage-sensitive Ca(2+) and Na(+) channels, and it significantly reduces infarct size after occlusion of the middle cerebral artery. Persistent activation of cyclic AMP response element binding protein (CREB), which can be induced by increase in intracellular Ca(2+) concentrations or other second messengers, has recently been found to be closely associated with neuronal survival in cerebral ischemia. The present study was therefore undertaken to evaluate the neuroprotective effects of NS-7 by analyzing changes in CREB phosphorylation in a focal cerebral ischemia model. CREB phosphorylation in the brain of rats was investigated immunohistochemically at 3.5-48-h recirculation after 1. 5-h occlusion of the middle cerebral artery. NS-7 (1 mg/kg; NS-7 group) or saline (saline group) was intravenously injected 5 min after the start of recirculation. The NS-7 group showed significantly milder activation of CREB phosphorylation in various cortical regions after 3.5 h of recirculation than the saline group. The inner border zone of ischemia in the NS-7 group subsequently exhibited a moderate, but persistent, increase in number of phosphorylated CREB-positive neurons with no apparent histological damage. By contrast, the saline group displayed a marked, but only transient, increase in number of immunopositive neurons in this border zone after 3.5 h of recirculation, and this was followed by clear suppression of CREB phosphorylation and subsequent loss of normal neurons. These findings suggest that: (1) the marked enhancement of CREB phosphorylation in the acute post-ischemic phase may be triggered largely by an influx of calcium ions as a result of activation of the voltage-sensitive Ca(2+) and Na(+) channels; and that (2) the neuroprotective effects of NS-7 may be accompanied by persistent activation of CREB phosphorylation in the inner border zone of ischemia.

Animals↗
PubMed2001

Structural and gating changes of the sodium channel induced by mutation of a residue in the upper third of IVS6, creating an external access path for local anesthetics.

Membrane-impermeant quaternary amine local anesthetics QX314 and QX222 can access their binding site on the cytoplasmic side of the selectivity filter from the outside in native cardiac Na(+) channels. Mutation of domain IV S6 Ile-1760 of rat brain IIA Na(+) channel or the equivalent (Ile-1575) in the adult rat skeletal muscle isoform (mu 1) creates an artificial access path for QX. We examined the characteristics of mutation of mu 1-I1575 and the resulting QX path. In addition to allowing external QX222 access, I1575A accelerated decay of Na(+) current and shifted steady-state availability by -27 mV. I1575A had negligible effects on inorganic or organic cation selectivity and block by tetrodotoxin (TTX), saxitoxin (STX), or mu-conotoxin (mu-CTX). It exposed a site within the protein that binds membrane-permeant methanethiosulfonate ethylammonium (MTSEA), but not membrane-impermeant methanethiosulfonate ethyltrimethylammonium (MTSET) and methanethiosulfonate ethylsulfonate (MTSES). MTSEA binding abolished the QX path created by this mutation, without effects on toxin binding. The mu-CTX derivative R13N, which partially occluded the pore, had no effect on QX access. I1575A exposed two Cys residues because a disulfide bond was formed under oxidative conditions, but the exposed Cys residues are not those in domain IV S6, adjacent to Ile-1575. The Cys mutant I1575C was insensitive to external Cd(2+) and MTS compounds (MTSEA, MTSET, MTSES), and substitution of Ile with a negatively charged residue (I1575E) did not affect toxin binding. Ile-1575 seems to be buried in the protein, and its mutation disrupts the protein structure to create the QX path without disturbing the outer vestibule and its selectivity function.

Amino Acid Substitution↗
PubMed2004

Voltage-gated sodium channels and pain pathways.

Acute, inflammatory, and neuropathic pain can all be attenuated or abolished by local treatment with sodium channel blockers such as lidocaine. The peripheral input that drives pain perception thus depends on the presence of functional voltage-gated sodium channels. Remarkably, two voltage-gated sodium channel genes (Nav1.8 and Nav1.9) are expressed selectively in damage-sensing peripheral neurons, while a third channel (Nav1.7) is found predominantly in sensory and sympathetic neurons. An embryonic channel (Nav1.3) is also upregulated in damaged peripheral nerves and associated with increased electrical excitability in neuropathic pain states. A combination of antisense and knock-out studies support a specialized role for these sodium channels in pain pathways, and pharmacological studies with conotoxins suggest that isotype-specific antagonists should be feasible. Taken together, these data suggest that isotype-specific sodium channel blockers could be useful analgesics.

Amino Acid Sequence↗
PubMed1997

Ca2+ and Na+ permeability of high-threshold Ca2+ channels and their voltage-dependent block by Mg2+ ions in chick sensory neurones.

1. The Mg2+ block of Na+ and Ca2+ currents through high-voltage activated (HVA; L- and N-type) Ca2+ channels was studied in chick dorsal root ganglion neurones. 2. In low extracellular [Ca2+] (< 10(-8) M) and with Na+o and Cs+i as the main charge carriers (120 mM), HVA Na+ currents started to activate at -40 mV, reached inward peak values near 0 mV and reversed at about +40 mV. 3. Addition of 30-500 microM Mg2+ to the bath caused a strong depression of inward Na+ currents that was voltage and dose dependent (KD = 39 microM in 120 mM Na+ at -10 mV). The block was maximal at negative potentials (< -70 mV) and decreased with increasing positive potentials, suggesting that Mg2+ cannot escape to the cell interior. 4. Block of Ca2+ currents by Mg2+ was also voltage dependent, but by three orders of magnitude less potent than with Na+ currents (KD = 24 mM in 2 mM Ca2+ at -30 mV). The high concentration of Mg2+ caused a prominent voltage shift of channel gating kinetics induced by surface charge screening effects. To compensate for this, Mg2+ block of inward Ca2+ currents was estimated from the instantaneous I-V relationships on return from very positive potentials (+100 mV). 5. Inward Na+ and Ca2+ tail currents following depolarization to +90 mV were markedly depressed, suggesting that channels cleared of Mg2+ ions during strong depolarization are quickly re-blocked on return to negative potentials. The kinetics of re-block by Mg2+ was too fast (< 100 microseconds) to be resolved by our recording apparatus. This implies a rate of entry for Mg2+ > 1.45 x 10(8) M-1 S-1 when Na+ is the permeating ion and a rate approximately 3 orders of magnitude smaller for Ca2+. 6. Mg2+ unblock of HVA Na+ currents at +100 mV was independent of the size of outward currents, whether Na+, Cs+ or NMG+ were the main internal cations. 7. Consistent with the idea of a high-affinity binding site for Ca2+ inside the channel, micromolar amounts of Ca2+ caused a strong depression of Na+ currents between -40 and 0 mV, which was effectively relieved with more positive as well as with negative potentials (KD = 0.7 microM in 120 mM Na+ at -20 mV). In this case, the kinetics of re-block could be resolved and gave rates of entry and exit for Ca2+ of 1.4 x 10(8) M-1 S-1 and 2.95 x 10(2) s-1, respectively. 8. The strong voltage dependence and weak current dependence of HVA channel block by divalent cations and the markedly different KD values of Na+ and Ca2+ current block by Mg2+ can be well described by a previously proposed model for Ca2+ channel permeation based on interactions between the permeating ion and the negative charges forming the high-affinity binding site for Ca2+ inside the pore (Lux, Carbone & Zucker, 1990).

Animals↗
PubMed1998

The heterotetrameric architecture of the epithelial sodium channel (ENaC).

The epithelial sodium channel (ENaC) is a key element for the maintenance of sodium balance and the regulation of blood pressure. Three homologous ENaC subunits (alpha, beta and gamma) assemble to form a highly Na+-selective channel. However, the subunit stoichiometry of ENaC has not yet been solved. Quantitative analysis of cell surface expression of ENaC alpha, beta and gamma subunits shows that they assemble according to a fixed stoichiometry, with alpha ENaC as the most abundant subunit. Functional assays based on differential sensitivities to channel blockers elicited by mutations tagging each alpha, beta and gamma subunit are consistent with a four subunit stoichiometry composed of two alpha, one beta and one gamma. Expression of concatameric cDNA constructs made of different combinations of ENaC subunits confirmed the four subunit channel stoichiometry and showed that the arrangement of the subunits around the channel pore consists of two alpha subunits separated by beta and gamma subunits.

Amiloride↗
PubMed1997

Sodium channels and therapy of central nervous system diseases.

Voltage-dependent Na+ channels have long been recognized targets for anti-arrhythmic and local anesthetic drugs. Since the mid-1980s, Na+ channels have become widely accepted as the primary target of anticonvulsants with pharmacological profiles similar to phenytoin, carbamazepine, and lamotrigine. Results from animal models and a few preliminary clinical trials suggest that this class of drugs may also offer significant potential for reducing the neuronal damage caused by ischemic stroke, head trauma, and perhaps certain neurodegenerative diseases. Studies using site-directed mutations of Na+ channels with electrophysiology have provided extensive insight into both the physiology and the interaction of drug molecules with ion channels. This review includes an introduction to Na+ channel structure, molecular biology, and physiology as they relate to pharmacology. A review of several in vitro actions of Na+ channel blockers is provided. Neuroprotective actions with a variety of Na+ channel blockers in models of central nervous system disease in animals and in vitro models are reviewed. Although many voltage-dependent Na+ channel blockers have additional pharmacological targets, the hypothesis that anticonvulsant and neuroprotective actions results from the blockade of Na+ channels is explored.

Amino Acid Sequence↗
PubMed2003

Veratridine block of rat skeletal muscle Nav1.4 sodium channels in the inner vestibule.

Veratridine (VTD) is an alkaloid toxin found in Liliaceae plants. VTD causes persistent opening of the voltage-gated Na+ channel and reduces its single-channel conductance by 75 %. The mechanisms for these different VTD actions are unknown. Recent reports indicate that the VTD receptor aligns closely with the local anaesthetic (LA) receptor, which resides at D1S6, D3S6 and D4S6 of the Na+ channel alpha-subunit. To study this alignment, we created a mutant with cysteine substitutions at three S6 residues (rNav1.4-N434C/L1280C/F1579C). Under voltage-clamp conditions, amitriptyline and bupivacaine remained as potent blockers of this mutant channel when expressed in human embryonic kidney cells, whereas VTD completely failed to cause persistent opening. Unexpectedly, VTD at 100 microM progressively blocked mutant currents by 90.4 +/- 1.6 % (n = 5), as assayed at 0.1 Hz for 15 min. This VTD block was reversed little during wash-off: approximately 70 % of mutant currents did not return in 30 min. An increase in channel opening either by repetitive pulses at 1 Hz or by the inhibition of the fast inactivation hastened the VTD block. Co-application of amitriptyline or bupivacaine, which targeted the LA receptor, prevented this VTD block. Our data suggest that (a) the VTD receptor and the LA receptor overlap extensively, (b) receptor-bound VTD lies in the inner vestibule, and (c) VTD blocks this mutant channel as a bona fide Na+ channel blocker. We propose that VTD likewise blocks the wild-type open Na+ channel, albeit partially, to decrease the unitary conductance and to stabilize the open conformation for persistent opening.

Amino Acid Sequence↗
PubMed1995

Myocardial recovery during post-ischemic reperfusion: optimal concentrations of Na+ and Ca2+ in the reperfusate and protective effects of amiloride added to cardioplegic solution.

The effects of Na+ and Ca2+ concentrations in the reperfusate on post-ischemic myocardial recovery were examined. Also, the myocardial protective effects of amiloride, an inhibitor of the Na+/Ca2+ and Na+/H+ exchange systems, added to cardioplegic solutions were assessed, using an isolated working rat heart perfusion system. Global myocardial ischemia was induced by 30-min normothermic cardioplegic arrest, using St. Thomas' solution. The concentration of Na+ in the reperfusate varied, stepwise, from 75 to 145 mM/l, and that of Ca2+, from 0.1 to 2.5 mM/l. In this study post-ischemic functional recovery was best at 110mM/l Na+ and 1.2-1.8 mM/l Ca2+ in the reperfusate. A significantly greater post-ischemic functional recovery and a lower creatine kinase release were observed when amiloride was added to the cardioplegic solution. Ca2+ overload via Na+/Ca2+ and Na+/H+ exchange systems would, thus, appear to be due, at least in part, to post-ischemic reperfusion injury.

Amiloride↗
PubMed1999

Origin of extracellular dopamine increase induced by lactic acid striatal perfusion monitored by microdialysis in the awake rat.

In previous studies we showed that a striatal lactic acid perfusion-induced lactacidosis produces a diphasic increase in extracellular dopamine (DA). In the present study, different pharmacological reagents were used to determine the origin of accumulated DA. Our data show that both DA accumulations were totally suppressed by tetrodotoxin and nicardipine, indicating a relationship with membrane depolarization and a Ca(2+)-dependent effect. The first DA peak was largely reduced by a specific inhibitor of DA uptake such as GBR-12935, and the second was totally suppressed by tyramine and reserpine and lowered and delayed by GBR-12935. These results compared to data in the literature suggest that the first increase in extracellular DA resulted mainly from a release of cytosolic DA by reversal of the DA transporter, while the second was mainly due to a release of vesicular DA by exocytosis. These data indicate that lactic acid perfusion helps clarify the mechanisms involved in this process and could be useful for the study of new treatments against the hyperactive dopaminergic reaction occuring during ischemia.

Adrenergic Uptake Inhibitors↗