Two TTX-resistant Na+ currents in mouse colonic dorsal root ganglia neurons and their role in colitis-induced hyperexcitability

The composition of Na+ currents in dorsal root ganglia (DRG) neurons depends on their neuronal phenotype and innervation target. Two TTX-resistant (TTX-R) Na+ currents [voltage-gated Na channels (Nav)] have been described in small DRG neurons; one with slow inactivation kinetics (Nav1.8) and the other with persistent kinetics (Nav1.9), and their modulation has been implicated in inflammatory pain. This has not been studied in neurons projecting to the colon. This study examined the relative importance of these currents in inflammation-induced changes in a mouse model of inflammatory bowel disease. Colonic sensory neurons were retrogradely labeled, and colitis was induced by instillation of trinitrobenzenesulfonic acid (TNBS) into the lumen of the distal colon. Seven to ten days later, immunohistochemical properties were characterized in controls, and whole cell recordings were obtained from small (<40 pF) labeled DRG neurons from control and TNBS animals. Most neurons exhibited both fast TTX-sensitive (TTX-S)- and slow TTX-R-inactivating Na+ currents, but persistent TTX-R currents were uncommon (<15%). Most labeled neurons were CGRP (79%), tyrosine kinase A (trkA) (84%) immunoreactive, but only a small minority bind IB4 (14%). TNBS-colitis caused ulceration, thickening of the colon and significantly increased neuronal excitability. The slow TTX-R-inactivating Na current density (Nav1.8) was significantly increased, but other Na currents were unaffected. Most small mouse colonic sensory neurons are CGRP, trkA immunoreactive, but not isolectin B4 reactive and exhibit fast TTX-S, slow TTX-R, but not persistent TTX-R Na+ currents. Colitis-induced hyperexcitability is associated with increased slow TTX-R (Nav1.8) Na+ current. Together, these findings suggest that colitis alters trkA-positive neurons to preferentially increase slow TTX-R Na+ (Nav1.8) currents.     

Na+ channel Scn1b gene regulates dorsal root ganglion nociceptor excitability in vivo

Nociceptive dorsal root ganglion (DRG) neurons express tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na(+) current (I(Na)) mediated by voltage-gated Na(+) channels (VGSCs). In nociceptive DRG neurons, VGSC β2 subunits, encoded by Scn2b, selectively regulate TTX-S α subunit mRNA and protein expression, ultimately resulting in changes in pain sensitivity. We hypothesized that VGSCs in nociceptive DRG neurons may also be regulated by β1 subunits, encoded by Scn1b. Scn1b null mice are models of Dravet Syndrome, a severe pediatric encephalopathy. Many physiological effects of Scn1b deletion on CNS neurons have been described. In contrast, little is known about the role of Scn1b in peripheral neurons in vivo. Here we demonstrate that Scn1b null DRG neurons exhibit a depolarizing shift in the voltage dependence of TTX-S I(Na) inactivation, reduced persistent TTX-R I(Na), a prolonged rate of recovery of TTX-R I(Na) from inactivation, and reduced cell surface expression of Na(v)1.9 compared with their WT littermates. Investigation of action potential firing shows that Scn1b null DRG neurons are hyperexcitable compared with WT. Consistent with this, transient outward K(+) current (I(to)) is significantly reduced in null DRG neurons. We conclude that Scn1b regulates the electrical excitability of nociceptive DRG neurons in vivo by modulating both I(Na) and I(K).     

Early painful diabetic neuropathy is associated with differential changes in tetrodotoxin-sensitive and -resistant sodium channels in dorsal root ganglion neurons in the rat

Diabetic neuropathy is a common form of peripheral neuropathy, yet the mechanisms responsible for pain in this disease are poorly understood. Alterations in the expression and function of voltage-gated tetrodotoxin-resistant (TTX-R) sodium channels have been implicated in animal models of neuropathic pain, including models of diabetic neuropathy. We investigated the expression and function of TTX-sensitive (TTX-S) and TTX-R sodium channels in dorsal root ganglion (DRG) neurons and the responses to thermal hyperalgesia and mechanical allodynia in streptozotocin-treated rats between 4-8 weeks after onset of diabetes. Diabetic rats demonstrated a significant reduction in the threshold for escape from innocuous mechanical pressure (allodynia) and a reduction in the latency to withdrawal from a noxious thermal stimulus (hyperalgesia). Both TTX-S and TTX-R sodium currents increased significantly in small DRG neurons isolated from diabetic rats. The voltage-dependent activation and steady-state inactivation curves for these currents were shifted negatively. TTX-S currents induced by fast or slow voltage ramps increased markedly in neurons from diabetic rats. Immunoblots and immunofluorescence staining demonstrated significant increases in the expression of Na(v)1.3 (TTX-S) and Na(v) 1.7 (TTX-S) and decreases in the expression of Na(v) 1.6 (TTX-S) and Na(v)1.8 (TTX-R) in diabetic rats. The level of serine/threonine phosphorylation of Na(v) 1.6 and In Na(v)1.8 increased in response to diabetes. addition, increased tyrosine phosphorylation of Na(v)1.6 and Na(v)1.7 was observed in DRGs from diabetic rats. These results suggest that both TTX-S and TTX-R sodium channels play important roles and that differential phosphorylation of sodium channels involving both serine/threonine and tyrosine sites contributes to painful diabetic neuropathy.     

The study of sodium channels involved in pain responses using specific modulators

Voltage-gated sodium channels(VGSCs) are transmembrane proteins responsible for generation and conduction of action potentials in excitable cells.Physiological and pharmacological studies have demonstrated that VGSCs play a critical role in chronic pain associated with tissue or nerve injury.Many long-chain peptide toxins(60-76 amino acid residues) purified from the venom of Asian scorpion Buthus martensii Karsch(BmK) are investigated to be sodium channel-specific modulators.The α-like neurotoxins that can ...     

Altered expression and function of sodium channels in large DRG neurons and myelinated A-fibers in early diabetic neuropathy in the rat

Differential alterations of sodium channels in small nociceptive C-fiber DRG neurons have been implicated in diabetic neuropathy. In this study, we investigated sodium currents and the expression of sodium channels in large A-fiber DRG neurons in diabetic rats. Compared with controls, large neurons from diabetic rats showed significant increases in both total and TTX-S sodium currents and approximately -15mV shifts in their voltage-dependent activation kinetics. TTX-R Na(v)1.9 sodium current was also significantly increased, whereas no alteration of TTX-R Na(v)1.8 current was observed in neurons from diabetic rats. Sodium current induced by fast- or slow-voltage ramps increased markedly in the diabetic neurons as well. Immunofluorescence studies showed significant increases in the levels and number of large DRG neurons from diabetic rats expressing Na(v)1.2, Na(v)1.3, Na(v)1.7, and Na(v)1.9 whereas Na(v)1.8 decreased. We also observed a decrease in the number of nodes of Ranvier expressing Na(v)1.8 and in staining intensity of Na(v)1.6 and Na(v)1.8 at nodes. Our results suggest that alterations of sodium channels occur in large DRG neurons and A-fibers, and may play an important role in diabetic sensory neuropathy.     

The cross channel activities of spider neurotoxin huwentoxin-I on rat dorsal root ganglion neurons

In this paper, we investigated the action of huwentoxin-I (HWTX-I) purified from the venom of the Chinese bird spider Ornithoctonus huwena on Ca(2+), Na(+) channels of adult rat dorsal root ganglion (DRG) neurons. The results showed that huwentoxin-I could reduce the peak currents of N-type Ca(2+) channels (IC(50) approximately 100 nM) and TTX-S Na(+) channels (IC(50) approximately 55 nM), whereas no effect was detected on TTX-R Na(+) channels. The comparative studies indicated that the selectivity of HWTX-I on Ca(2+) channels was higher that of MVIIA and approximately the same as that of GVIA. HWTX-I is the first discovered toxin with the cross channel activities from the spider O. huwena venom similar to micro O-conotoxins MrVIA and MrVIB.     

Identification of a spontaneously active, Na+-permeable channel in guinea pig gallbladder smooth muscle

The action potential in gallbladder smooth muscle (GBSM) is caused by Ca2+ entry through voltage-dependent Ca2+ channels (VDCC), which contributes to the GBSM contractions. Action potential generation in GBSM is critically dependent on the resting membrane potential (about -50 mV), which is approximately 35 mV more positive of the K+ equilibrium potential. We hypothesized that a tonic, depolarizing conductance is present in GBSM and contributes to the regulation of the resting membrane potential and action potential frequency. GBSM cells were isolated from guinea pig gallbladders, and the whole cell patch-camp technique was used to record membrane currents. After eliminating the contribution of VDCC and K+ channels, we identified a novel spontaneously active cation conductance (I(cat)) in GBSM. This I(cat) was mediated predominantly by influx of Na+. Na+ substitution with N-methyl-D-glucamine (NMDG), a large relatively impermeant cation, caused a negative shift in the reversal potential of the ramp current and reduced the amplitude of the inward current at -50 mV by 65%. Membrane potential recordings with intracellular microelectrodes or in current-clamp mode of the patch-clamp technique indicated that the inhibition of I(cat) conductance by NMDG is associated with membrane hyperpolarization and inhibition of action potentials. Extracellular Ca2+, Mg2+, and Gd3+ attenuated the I(cat) in GBSM. Muscarinic stimulation did not activate the I(cat). Our results indicate that, in GBSM, an Na+-permeable channel contributes to the maintenance of the resting membrane potential and action potential generation and therefore plays a critical role in the regulation of GBSM excitability and contractility.     

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.     

大鼠海马神经元膜离子通道随培养时间变化的特点

目的和方法:采用膜片钳全细胞记录技术观察新生大鼠海马神经元体外分散培养过程中,基本离子通道和膜参数随培养天数延长而变化的规律.结果:在7 d,14 d和21 d时电压依赖性钠电流(Voltage-dependent Na cur-rent,ⅠNa)和延迟整流性钾电流(Delayed rectifier K current,Ⅰk)的幅度无显著性差异.电压依赖性钙电流(Voltage-dependent Ca2 current,ⅠCa)和ⅠCa密度则持续增大,进一步研究表明,L型钙通道(L-type voltage-dependent Ca2 channel,L-VDCC)的增加是其主要原因.NMDA诱发电流随培养时间延长而明显增加.结论:钙通道和NMDA受体所介导的Ca2 内流是神经元易感于衰老和死亡的重要机制之一.     

Direct activation of Transient Receptor Potential Vanilloid 1(TRPV1) by Diacylglycerol (DAG)

Voltage-gated sodium channels play important roles in modulating dorsal root ganglion (DRG) neuron hyperexcitability and hyperalgesia after peripheral nerve injury or inflammation. We report that chronic compression of DRG (CCD) produces profound effect on tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) sodium currents, which are different from that by chronic constriction injury (CCI) of the sciatic nerve in small DRG neurons. Whole cell patch-clamp recordings were obtained in vitro from L₄ and/or L₅ dissociated, small DRG neurons following in vivo DRG compression or nerve injury. The small DRG neurons were classified into slow and fast subtype neurons based on expression of the slow-inactivating TTX-R and fast-inactivating TTX-S Na⁺ currents. CCD treatment significantly reduced TTX-R and TTX-S current densities in the slow and fast neurons, but CCI selectively reduced the TTX-R and TTX-S current densities in the slow neurons. Changes in half-maximal potential (V_1/2) and curve slope (k) of steady-state inactivation of Na⁺ currents were different in the slow and fast neurons after CCD and CCI treatment. The window current of TTX-R and TTX-S currents in fast neurons were enlarged by CCD and CCI, while only that of TTX-S currents in slow neurons was increased by CCI. The decay rate of TTX-S and both TTX-R and TTX-S currents in fast neurons were reduced by CCD and CCI, respectively. These findings provide a possible sodium channel mechanism underlying CCD-induced DRG neuron hyperexcitability and hyperalgesia and demonstrate a differential effect in the Na⁺ currents of small DRG neurons after somata compression and peripheral nerve injury. This study also points to a complexity of hyperexcitability mechanisms contributing to CCD and CCI hyperexcitability in small DRG neurons.     

Effect of Na(+) flow on Cd(2+) block of tetrodotoxin-resistant Na(+) channels

Tetrodotoxin-resistant (TTX-R) Na(+) channels are 1,000-fold less sensitive to TTX than TTX-sensitive (TTX-S) Na(+) channels. On the other hand, TTX-R channels are much more susceptible to external Cd(2+) block than TTX-S channels. A cysteine (or serine) residue situated just next to the aspartate residue of the presumable selectivity filter "DEKA" ring of the TTX-R channel has been identified as the key ligand determining the binding affinity of both TTX and Cd(2+). In this study we demonstrate that the binding affinity of Cd(2+) to the TTX-R channels in neurons from dorsal root ganglia has little intrinsic voltage dependence, but is significantly influenced by the direction of Na(+) current flow. In the presence of inward Na(+) current, the apparent dissociation constant of Cd(2+) ( approximately 200 microM) is approximately 9 times smaller than that in the presence of outward Na(+) current. The Na(+) flow-dependent binding affinity change of Cd(2+) block is true no matter whether the direction of Na(+) current is secured by asymmetrical chemical gradient (e.g., 150 mM Na(+) vs. 150 mM Cs(+) on different sides of the membrane, 0 mV) or by asymmetrical electrical gradient (e.g., 150 mM Na(+) on both sides of the membrane, -20 mV vs. 20 mV). These findings suggest that Cd(2+) is a pore blocker of TTX-R channels with its binding site located in a multiion, single-file region near the external pore mouth. Quantitative analysis of the flow dependence with the flux-coupling equation reveals that at least two Na(+) ions coexist with the blocking Cd(2+) ion in this pore region in the presence of 150 mM ambient Na(+). Thus, the selectivity filter of the TTX-R Na(+) channels in dorsal root ganglion neurons might be located in or close to a multiion single-file pore segment connected externally to a wide vestibule, a molecular feature probably shared by other voltage-gated cationic channels, such as some Ca(2+) and K(+) channels.     

Antisense-mediated knockdown of Na(V)1.8, but not Na(V)1.9, generates inhibitory effects on complete Freund's adjuvant-induced inflammatory pain in rat

Tetrodotoxin-resistant (TTX-R) sodium channels Na(V)1.8 and Na(V)1.9 in sensory neurons were known as key pain modulators. Comparing with the widely reported Na(V)1.8, roles of Na(V)1.9 on inflammatory pain are poorly studied by antisense-induced specific gene knockdown. Here, we used molecular, electrophysiological and behavioral methods to examine the effects of antisense oligodeoxynucleotide (AS ODN) targeting Na(V)1.8 and Na(V)1.9 on inflammatory pain. Following complete Freund's adjuvant (CFA) inflammation treatment, Na(V)1.8 and Na(V)1.9 in rat dorsal root ganglion (DRG) up-regulated mRNA and protein expressions and increased sodium current densities. Immunohistochemical data demonstrated that Na(V)1.8 mainly localized in medium and small-sized DRG neurons, whereas Na(V)1.9 only expressed in small-sized DRG neurons. Intrathecal (i.t.) delivery of AS ODN was used to down-regulate Na(V)1.8 or Na(V)1.9 expressions confirmed by immunohistochemistry and western blot. Unexpectedly, behavioral tests showed that only Na(V)1.8 AS ODN, but not Na(V)1.9 AS ODN could reverse CFA-induced heat and mechanical hypersensitivity. Our data indicated that TTX-R sodium channels Na(V)1.8 and Na(V)1.9 in primary sensory neurons played distinct roles in CFA-induced inflammatory pain and suggested that antisense oligodeoxynucleotide-mediated blocking of key pain modulator might point toward a potential treatment strategy against certain types of inflammatory pain.     

Persistent tetrodotoxin-resistant Na+ currents are activated by prostaglandin E2 via cyclic AMP-dependent pathway in C-type nodose neurons of adult rats

It has been documented that nodose neurons express TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na(+) channels. However, wheteher nodose neurons functionally express persistent TTX-R Na(+) currents has not been reported. The present study first demonstrated persistent TTX-R Na(+) channel activities in 7/19 C-type nodose neurons in the presence of PGE(2) using whole-cell patch. Voltage-dependent property showed that persistent TTX-R Na(+) currents were activated at near -60mV and channels were maintained open. The average peak was approximately 300-500pA. The mid-point of activation exhibited a greater shift to a more hyperpolarized potential in the neurons co-expressing TTX-R and persistent TTX-R Na(+) currents than those expressing TTX-R only. This effect of PGE(2) was also mimicked by Forskolin. The fact that persistent TTX-R Na(+) currents were only activated by PGE(2) suggested that the modulatory effects of PGE(2) on persistent TTX-R Na(+) currents are crucial in PGE(2)-mediated neuronal excitability, and may have a great impact on specifically physiological significance.     

Ca2+-activated Na+ fluxes in human red cells. Amiloride sensitivity

The effect of Ca2+ on the ouabain- and bumetanide-resistant Na+ fluxes in intact red cells was studied at relatively constant internal Ca2+, membrane potential, and cell volume. The red cell calcium concentration was modified using the ionophore A23187. In fresh red cells, the Na+ influx and efflux (1.2 +/- 0.13 and 0.26 +/- 0.07 mmol/liter cells x h, respectively) were not affected by amiloride (1 mM). When external Ca2+ was raised from 0 to 150 microM, in the presence of A23187, both the Na+ influx and efflux were stimulated (about 3.5-fold). The Ca2+-activated Na+ efflux and influx had an apparent Km for activation by Ca2+o of about 25 microM. The Ca2+-dependent Na+ transport was inhibited 30-60% by amiloride (ID50 = 17.3 +/- 8 microM). Amiloride, however, had no effect on the Ca2+-dependent K+ influx. The amiloride-sensitive (AS) transport pathway was a linear function of the Na+o concentration in the range from 0 to 75 mM. The Ca2+i activation seems to depend on the metabolic integrity of red cells. 1) It does not take place in ATP-depleted red cells; 2) ATP-repletion of ATP-depleted red cells fully restored AS Na influx; and 3) ATP-enrichment (ATP-red cells) enhanced the AS Na influx by about 100%. The Ca2+-activated AS Na+ influx was not affected by either DIDS or trifluoperazine. The present results indicate that in human erythrocytes an increase in internal Ca2+ activates on otherwise silent AS Na+-transport system, which is dependent on the metabolic integrity of the red cells.     

Reductions in external divalent cations evoke novel voltage-gated currents in sensory neurons

It has long been recognized that divalent cations modulate cell excitability. Sensory nerve excitability is of critical importance to peripheral diseases associated with pain, sensory dysfunction and evoked reflexes. Thus we have studied the role these cations play on dissociated sensory nerve activity. Withdrawal of both Mg(2+) and Ca(2+) from external solutions activates over 90% of dissociated mouse sensory neurons. Imaging studies demonstrate a Na(+) influx that then causes depolarization-mediated activation of voltage-gated Ca(2+) channels (Ca(V)), which allows Ca(2+) influx upon divalent re-introduction. Inhibition of Ca(V) (ω-conotoxin, nifedipine) or Na(V) (tetrodotoxin, lidocaine) fails to reduce the Na(+) influx. The Ca(2+) influx is inhibited by Ca(V) inhibitors but not by TRPM7 inhibition (spermine) or store-operated channel inhibition (SKF96365). Withdrawal of either Mg(2+) or Ca(2+) alone fails to evoke cation influxes in vagal sensory neurons. In electrophysiological studies of dissociated mouse vagal sensory neurons, withdrawal of both Mg(2+) and Ca(2+) from external solutions evokes a large slowly-inactivating voltage-gated current (I(DF)) that cannot be accounted for by an increased negative surface potential. Withdrawal of Ca(2+) alone fails to evoke I(DF). Evidence suggests I(DF) is a non-selective cation current. The I(DF) is not reduced by inhibition of Na(V) (lidocaine, riluzole), Ca(V) (cilnidipine, nifedipine), K(V) (tetraethylammonium, 4-aminopyridine) or TRPM7 channels (spermine). In summary, sensory neurons express a novel voltage-gated cation channel that is inhibited by external Ca(2+) (IC(50)～0.5 μM) or Mg(2+) (IC(50)～3 μM). Activation of this putative channel evokes substantial cation fluxes in sensory neurons.     

Purification, subunit structure and pharmacological effects on cardiac and smooth muscle cells of a polypeptide toxin isolated from the marine snail Conus tessulatus

The most active component in smooth muscle contraction, isolated from the whole venom of the marine snail Conus tessulatus, has a molecular mass of about 55 kDa. The toxin protein, tessulatus toxin, appeared to be constituted by two distinct polypeptide bands of 26 kDa and 29 kDa. The pure toxin caused a marked contraction of both guinea-pig ileum and rabbit aorta at nanomolar concentrations. Tessulatus-toxin-induced contraction was indirectly prevented by classical inhibitors of the voltage-dependent Ca2+ channel. Tessulatus toxin caused a large increase in the initial rate of 45Ca2+ uptake by cardiac cells. This uptake was insensitive to Ca2+ channel blockers at concentrations 100-1000 fold higher than those known to block voltage-dependent Ca2+ channels in these cells. Voltage clamp experiments have confirmed that tessulatus toxin was not directly active on the Ca2+ current. Tessulatus-toxin-stimulated 45Ca2+ influx was inhibited by dichlorobenzamil and suppressed when Na+ was substituted by Li+, indicating that the toxin acted via activation of the Na+/Ca2+ exchange system in cardiac cells. Activation by tessulatus toxin of the Na+/Ca2+ exchange system occurred via a toxin-stimulated Na+ entry into cardiac cells and was observed in the same range of toxin concentration which produced 45Ca2+ entry. The Na+ entry system that was activated by tessulatus toxin was insensitive to classic inhibitors of known Na+ entry systems in cardiac cells. Possible mechanisms by which tessulatus toxin induced Na+ entry into cardiac cells and contractions in smooth muscles are discussed. Tessulatus toxin is cytotoxic when used at high concentrations.     

GM1 ganglioside contributes to retain the neuronal conduction and neuronal excitability in visceral and baroreceptor afferents

GM1 ganglioside has a great impact on the function of nodes of Ranvier on myelinated fiber, suggesting its potential role to maintain the electrical and neuronal excitability of neurons. Here we first demonstrate that visceral afferent conduction velocity of myelinated and unmyelinated fibers are reduced significantly by tetrodotoxin (TTX) or cholera toxin-B subunits (CTX-B), and only the effects mediated by CTX-B are prevented by GM1 pre-treatment. At soma of myelinated A and unmyelinated C-type nodose ganglion neurons (NGNs), the action potential spike frequency reduced by CTX-B is also prevented by GM1. Additionally, the current density of both TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na⁺ channels were significantly decreased by CTX-B without changing the voltage-dependent property. These data confirm that endogenous GM1 may play a dominant role in maintaining the electrical and neuronal excitability via modulation of sodium (Na⁺) channel around nodes and soma as well, especially TTX-S Na⁺ channel, which is also confirmed by the reduction of spike amplitude and depolarization. Similar data are also extended to fluorescently identified and electrophysiologically characterized aortic baroreceptor neurons. These findings suggest that GM1 plays an important role in the neural modulation of electric and neuronal excitability in visceral afferent system.     

Characterization of Na(+) currents in isolated dorsal unpaired median neurons of Locusta migratoria and effect of the alpha-like scorpion toxin BmK M1

A primary cell culture was developed for efferent dorsal unpaired median (DUM) neurons of the locust. The isolated somata were able to generate Tetrodotoxin (TTX)-sensitive action potentials in vitro. The alpha-like scorpion toxin BmK M1, from the Asian scorpion Buthus martensi Karsch, prolonged the duration of the action potential up to 50 times. To investigate the mechanism of action of BmK M1, the TTX-sensitive voltage gated Na(+) currents were studied in detail using the whole cell patch clamp technique. BmK M1 slowed down and partially inhibited the inactivation of the TTX-sensitive Na(+) current in a dose dependent manner (EC50=326.8+/-34.5 nM). Voltage and time dependence of the Na(+) current were described in terms of the Hodgkin-Huxley model and compared in control conditions and in the presence of 500 nM BmK M1. The BmK M1 shifted steady state inactivation by 10.8 mV to less negative potentials. The steady state activation was shifted by 5.5 mV to more negative potentials, making the activation window larger. Moreover, BmK M1 increased the fast time constant of inactivation, leaving the activation time constant unchanged. In summary, BmK M1 primarily affected the inactivation parameters of the voltage gated Na(+) current in isolated locust DUM neurons.     

The effect of alteration of extracellular Na+ or Ca2+ and inhibition of Ca2+ entry, Na(+)-H+ exchange, and Na(+)-Ca2+ exchange by diltiazem, amiloride, and dichlorobenzamil on the response of cardiac cell aggregates to epidermal growth factor

The purpose of this study was to examine the effect of epidermal growth factor (EGF) on cardiac function and to explore ionic mechanisms as potential explanations for EGF-induced changes in cardiac contractile frequency. Cardiac cell aggregates were prepared from 7-day-old chick embryo hearts and were maintained in culture. EGF over a concentration range of 5 to 20 ng/ml produced a dose-dependent increase in cardiac contractile frequency. Inhibition of Na(+)-H+ exchange by amiloride antagonized the action of EGF. Inhibition of Na(+)-Ca2+ exchange by dichlorobenzamil prevented the effects of EGF. Inhibition of voltage-dependent calcium influx by diltiazem also antagonized the effect of EGF. The positive chronotropic action of EGF was significantly enhanced when the concentration of Na+ or Ca2+ was increased in the medium. These data indicate that EGF has a definite dose-dependent effect on the cardiac contractile frequency that is operative through ionic transport mechanisms that include increased calcium entry through voltage-dependent calcium channels and stimulation of Na(+)-H+ and Na(+)-Ca2+ exchange. The similarity in the effects of inhibition of these three ionic mechanisms suggests they are interrelated so that interference at any step in the process inhibits the action of EGF on cardiac myocytes.     

ANEPIII, a new recombinant neurotoxic polypeptide derived from scorpion peptide, inhibits delayed rectifier, but not A-type potassium currents in rat primary cultured hippocampal and cortical neurons

A new recombinant neurotoxic polypeptide ANEPIII (BmK ANEPIII) derived from Scorpion peptide, which was demonstrated with antineuroexcitation properties in animal models, was examined for its action on K+ currents in primary cultured rat hippocampal and cortical neurons using the patch clamp technique in the whole-cell configuration. The delayed rectifier K+ current (I(k)) was inhibited by externally applied recombinant BmK ANEPIII, while the transient A-current (I(A)) remained virtually unaffected. BmK ANEPIII 3 microM, reduced the delayed rectifier current by 28.2% and 23.6% in cultured rat hippocampal and cortical neurons, respectively. The concentration of half-maximal block was 155.1 nM for hippocampal neurons and 227.2 nM for cortical neurons, respectively. These results suggest that BmK ANEPIII affect K+ currents, which may lead to a reduction in neuronal excitability.