Effect of clonidine on potential-dependent sodium currents in sensory neurons

In experiments on rat pups we studied the effect of clonidine on potential-dependent Na⁺ currents in dorsal root ganglia by the voltage clamp method. Clonidine decreased the amplitude of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium currents. The range of acting concentrations and the absence of modulatory effect of norepinephrine on the efficiency of clonidine-induced blockade of sodium currents suggest that this blockade results from a direct interaction of clonidine with sodium channels.__________This revised version was published online in August 2005 with the addition of the issue titleTranslated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 1, pp. 44–48, January, 2005     

电压门控钠离子通道与癫痫

电压门控钠离子通道(voltage-gated sodium channels,VGSCs)在动作电位产生和传导中至关重要.近年来研究发现,VGSCs与癫痫发病机制有着密切关系,许多癫痫综合征的发生已被证明是由VGSCs相关突变引起,且VGSCs的两种亚基(α和β)的相关基因发生突变均可以引起癫痫发作.其中与癫痫相关的α亚基主要有Nav1.1,Nav1.2,Nav1.3,Nav1.6,Nav1.7以及Na等几种亚型.本文就电压门控钠离子通道相关基因突变致癫痫的研究进展进行综述,旨在提高对癫痫的认识.     

电压-门控Na+通道基因突变与癫痫

电压-门控Na+通道和癫痫的关系十分密切，许多癫痫综合征的发生已被证明是由Na+通道基因突变引起，且编码α和/或β亚单位的基因发生突变均可以引起癫痫。基因突变后产生的异常Na+通道蛋白引起癫痫的发病机制仍不明确。基因和细胞治疗为治疗离子通道基因突变引起的癫痫提供了新的思路。     

Use-dependent block of cardiac sodium channels by quaternary derivatives of lidocaine

Modulation of the reduction of fast inward sodium current by local anesthetics due to changes in electrical activity has been termed use-dependent block (Courtney 1975). To determine the mechanisms responsible for use-dependent block of cardiac sodium channels and to compare use-dependent block in cardiac and nerve preparations, we investigated use-dependent block of cardiac sodium channels by the quaternary lidocaine analogues QX-314 and QX-222 (two agents previously studied in nerve). We used canine cardiac Purkinje fibers, and assessed changes in the fast inward sodium current using changes in the maximum rate of rise of the action potential upstroke ( ). Two microelectrode voltage clamp and current clamp techniques were used to control membrane potential prior to stimulated upstrokes. Use-dependent block was not affected by shortening the action potential duration during rapid stimulation. Partial recovery from use-dependent block was observed during rapid stimulation with brief depolarizing prepulses terminating immediately prior to the upstroke. Similar prepulses also prevented the development of use-dependent block following an abrupt increase in the stimulation rate. Hyperpolarizing prepulses during rapid stimulation caused recovery from use-dependent block; recovery was greater and more rapid with increasingly negative prepulses. Hyperpolarization during periods of electrical quiescence also caused greater recovery. These results, interpreted using the modulated receptor hypothesis (Hille 1977; Hondeghem and Katzung 1977), suggest that use-dependent block of cardiac sodium channels by quaternary local anesthetics is due to drug association with the inactivated sodium channel receptor which occurs only after these drugs gain access to the receptor site through open sodium channels.Supported in part by grants HL-08508-18 and HL-12738-13 from the NHLBI     

Developmental exposure to lead causes inherent changes on voltage-gated sodium channels in rat hippocampal CA1 neurons

In this study, the effects of chronic lead (Pb(2+)) exposure, during day 0 of gestation (E0) to postnatal day 15 (P15), on voltage-gated sodium channel currents (I(Na)) were investigated in CA1 field of the hippocampus (CA1) neurons using the conventional whole-cell patch-clamp technique on rat hippocampal slices. We found that developmental lead exposure increased the activation threshold and the voltage at which the maximum I(Na) current was evoked, caused positive shifts of I(Na) steady-state activation curve, and enlarged I(Na) tail-currents; Pb(2+) delayed the activation of I(Na) in a voltage-dependent manner, prolonged the time course of the fast inactivation of sodium channels; Pb(2+) induced a right shift of the steady-state inactivation curve, accelerated the activity-dependent attenuation of I(Na), but made no significant effects on the time course of the recovery of I(Na) from inactivation and the fraction of inactivated channels. In addition, the co-treatment with alpha-tocopherol (VE), an effective antioxidant and free radical scavenger, completely prevented the aforementioned changes on I(Na). The alterations on I(Na) properties induced by developmental lead exposure were partly different from that in previous acute experiments under the conditions closer to physiological situation, and the process was considered related to the participating of lead in lipid peroxidation reaction, which has been reported to change the conformation and biophysical functions of membrane proteins.     

Ion channels in lymphocytes

The advent of the gigaohm-seal recording technique has enabled the study of the electrical properties of small cells, such as individual lymphocytes. Recent studies using this technique in combination with standard immunological and biochemical techniques indicate that cells of the immune system may utilize ion channels, similar in properties to those described in nerve and muscle, in the process of activation. For example, potassium channels may be required for T-lymphocyte mitogenesis and calcium channels for antibody production. This article summarizes these recent reports.     

Morphine decreases the voltage sensitivity of slow sodium channels

Cell membrane recordings were made in conditions of voltage clamping with tight attachment of the microelectrode—patch clamping— to study the effects of morphine on tetrodotoxin-resistant (TTX_r) sodium channels in rat spinal ganglion neurons in culture. The effects of a number of biologically active substances which regulate the receptor-mediated actions of morphine were studied. The effects of morphine were found to involve a chain of sequential reactions leading to decreases in the transfer of effective charge (Z_eff) by the activatory gate system of TTX_r sodium channels, depending on the concentration of agonist in the extracellular solution. A value of 8 nM was obtained forK _D , with a Hill coefficient of X=0.5. Non-specific antagonists of opioid receptors blocked the actions of morphine; these included ouabain at a concentration of 100 μM. An inhibitor, and activator, and a blocker of G-proteins had no effect on the effective charge. These data provide evidence that morphine decreases the voltage sensitivity of TTX_r sodium channels. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 85, No. 2, pp. 225–236, February, 1999.     

Signaling complexes of voltage-gated sodium and calcium channels

Membrane depolarization and intracellular Ca²⁺ transients generated by activation of voltage-gated Na⁺ and Ca²⁺ channels are local signals, which initiate physiological processes such as action potential conduction, synaptic transmission, and excitation–contraction coupling. Targeting of effector proteins and regulatory proteins to ion channels is an important mechanism to ensure speed, specificity, and precise regulation of signaling events in response to local stimuli. This article reviews experimental results showing that Na⁺ and Ca²⁺ channels form local signaling complexes, in which effector proteins, anchoring proteins, and regulatory proteins interact directly with ion channels. The intracellular domains of these channels serve as signaling platforms, mediating their participation in intracellular signaling processes. These protein–protein interactions are important for regulation of cellular plasticity through modulation of Na⁺ channel function in brain neurons, for short-term synaptic plasticity through modulation of presynaptic Ca_V2 channels, and for the fight-or-flight response through regulation of postsynaptic Ca_V1 channels in skeletal and cardiac muscle. These localized signaling complexes are essential for normal function and regulation of electrical excitability, synaptic transmission, and excitation–contraction coupling.     

Tetrodotoxin-sensitive inactivation-resistant sodium channels in pacemaker cells influence heart rate

There is currently some uncertainty about whether cardiac pacemaker cells contain tetrodotoxin (TTX)-sensitive Na⁺ channels although TTX is known to slow heart rate. We have recorded transient and persistent single-channel currents activated by depolarization in myocytes isolated from the toad sinus venosus. The myocytes were identified as pacemaker cells by their characteristic morphology, spontaneous action potentials that were blocked by cobalt but not by TTX, and lack of an inwardly rectifying K⁺ current. The voltage dependence of the single-channel currents, their presence in solutions containing no K⁺ or Ca²⁺, or in solutions to which Cs⁺ and Co²⁺ had been added, their dependence on [Na⁺] and their sensitivity to TTX indicated that they were Na⁺ channel currents. The persistent Na⁺ channel currents were resistant to inactivation and were activated over the range of potentials that occur during diastole in pacemaker cells: they would therefore contribute to the pacemaker current that sets heart rate. It was concluded that TTX slows heart rate by blocking these channels in pacemaker cells. Received: 1 September 1995/Received after revision: 7 November 1995/Accepted: 13 November 1995     

Differences in action of Ca++ ions on cumulative blockade of sodium channels induced by tertiary and quaternary amines

Bulletin of Experimental Biology and Medicine -     

Mechanisms of the inhibition of Shaker potassium channels by protons

Potassium channels are regulated by protons in various ways and, in most cases, acidification results in potassium current reduction. To elucidate the mechanisms of proton-channel interactions we investigated N-terminally truncated Shaker potassium channels (K_v1 channels) expressed in Xenopus oocytes, varying pH at the intracellular and the extracellular face of the membrane. Intracellular acidification resulted in rapid and reversible channel block. The block was half-maximal at pH 6.48, thus even physiological excursions of intracellular pH will have an impact on K⁺ current. The block displayed only very weak voltage dependence and C-type inactivation and activation were not affected. Extracellular acidification (up to pH 4) did not block the channel, indicating that protons are effectively excluded from the selectivity filter. Channel current, however, was reduced greatly due to marked acceleration of C-type inactivation at low pH. In contrast, inactivation was not affected in the T449V mutant channel, in which C-type inactivation is impaired. The pH effect on inactivation of the wild-type channel had an apparent pK of 4.7, suggesting that protonation of extracellular acidic residues in Kv channels makes them subject to pH regulation.     

Ischemic poison lysophosphatidylcholine modifies heart sodium channels gating inducing long-lasting bursts of openings

The effects of lysophosphatidylcholine (LPC) on Na channels in inside-out patches of adult rat ventricular cells using the patch-clamp technique have been investigated. Application of LPC (9–25 M) from the inner side of membrane for 4–15 min caused a reduction of averaged Na current (I_Na) peak and prolonged the time course of inactivation in the potential range of -50 to -10 mV. Analysis of single channel behaviour revealed that after 30–50 min of exposure, in addition to normally functioning Na channels with short openings, LPC induced longlasting bursts of Na channel openings (up to the 300 ms duration of the test pulses). This resulted in an appearance of noninactivated component of I_Na. The slope conductance of these modified channels remained the same as in control (11.3 pS — control; 11.6 pS — LPC-treated). The dwell time for modified channels increased significantly.     

Voltage-gated Na+ channels confer invasive properties on human prostate cancer cells

Prostate cancer is the second leading cause of cancer deaths in American males, resulting in an estimated 37,000 deaths annually, typically the result of metastatic disease. A consequence of the unsuccessful androgen ablation therapy used initially to treat metastatic disease is the emergence of androgen-insensitive prostate cancer, for which there is currently no prescribed therapy. Here, three related human prostate cancer cell lines that serve as a model for this dominant form of prostate cancer metastasis were studied to determine the correlation between voltage-gated sodium channel expression/function and prostate cancer metastatic (invasive) potential: the non-metastatic, androgen-dependent LNCaP LC cell line and two increasingly tumorogenic, androgen-independent daughter cell lines, C4 and C4-2. Fluorometric in vitro invasion assays indicated that C4 and C4-2 cells are more invasive than LC cells. Immunoblot analysis showed that voltage-gated sodium channel expression increases with the invasive potential of the cell line, and this increased invasive potential can be blocked by treatment with the specific voltage-gated sodium channel inhibitor, tetrodotoxin (TTX). These data indicate that increased voltage-gated sodium channel expression and function are necessary for the increased invasive potential of these human prostate cancer cells. When the human adult skeletal muscle sodium channel Na_v1.4 was expressed transiently in each cell line, there was a highly significant increase in the numbers of invading LC, C4, and C4-2 cells. This increased invasive potential was reduced to control levels by treatment with TTX. These data are the first to indicate that the expression of voltage-gated sodium channels alone is sufficient to increase the invasive potential of non-metastatic (LC cells) as well as more aggressive cells (i.e., C4 and C4-2 cells). Together, the data suggest that increased voltage-gated sodium channel expression alone is necessary and sufficient to increase the invasive potential of a set of human prostate cancer cell lines that serve as a model for prostate cancer metastasis.     

Interaction of DDT with sodium channels in squid giant axon membranes

The effects of DDT on the sodium channel gating mechanism were studied with internally perfused squid giant axons under voltage clamp conditions. In contrast to earlier studies, DDT has pronounced effects on the sodium conductance system of squid axons. Internal application of 1 × 10^?4M DDT had no effect on the activation time constant (τ_m) or on the peak current, but the falling phase of the sodium current and the tail current upon repolarization were slowed dramatically (τ_tail= 3–13 ms). The sodium inactivation process became second order after DDT treatment. The time constant of the fast phase (τ₁) was identical to τ_h recorded in the control axon, while the time constant of the slow phase (τ₂) was more than 10 times greater. The voltage dependence of τ₂ was different from τ_h, increasing with depolarization. The steady-state inactivation curve was shifted in the direction of hyperpolarization by 6–8 mV, but even after prolonged depolarizing pulses, the sodium conductance did not inactivate completely.These data are consistent with the hypothesis that a population of sodium channels modified by DDT activates normally but is then retained in a second open state which inactivates slowly.     

研究电压门控性钠离子通道的3种细胞处理方法比较

目的 建立适用于膜片钳技术记录电压门控性钠离子通道的3种细胞处理方法并进行比较.方法 采用急性分离技术、分离脑片技术、pCMV_SCN1A质粒钙转染HEK293细胞3种方式处理细胞,镜下观察细胞形态,膜片钳记录钠通道激活情况.结果 急性分离的神经元形态正常,有较长突起;膜片钳记录封接成功率达50％以上,记录到电流的细胞膜电容为(7.56±3.47)pF(n=20),串联电阻为(10.18±4.82)MΩ(n=20).分离脑片的神经元形态正常饱满;膜片钳记录封接成功率达30％以上,记录到电流的细胞膜电容为(9.45±4.26)pF(n=10),串联电阻为(12.53±3.87)MΩ(n=10).钙转染处理的细胞完整,有立体感;膜片钳记录封接成功率约20％,记录到电流的细胞膜电容为(8.79±4.54)pF(n=10),串联电阻为(14.12±4.26)MΩ(n=10).3种细胞处理方法均能提供研究所需的钠通道电流.结论 3种细胞处理方法均可以简单、快速记录电压门控性钠离子通道,各有优缺点,适用于不同的研究需求.     

Riluzole specifically blocks inactivated Na channels in myelinated nerve fibre

The effects of 0.15–250 M riluzole, a novel psychotropic agent with anticonvulsant properties, were studied on voltage-clamped nodes of Ranvier of isolated nerve fibres of the frog. When added to the external solution, the drug rapidly and reversibly inhibited both K and Na currents with an apparent dissociation constant of 0.09 mM. The riluzole-induced decrease of these currents was not use-dependent. At concentrations up to 100M, the drug had no noticeable effect on the time course of Na current inactivation nor on the shape and the position along voltage axis of the Na conductance/voltage relationship. On the other hand, it induced substantial shifts towards negative voltages of the steady-state Na inactivation/voltage curve. From these results, according to the modulated-receptor model, an apparent dissociation constant of 0.29 M could be calculated for riluzole-induced blockage of inactivated Na channels. The recovery from Na current inactivation was also affected by the drug. It is concluded that riluzole is a highly specific blocker of inactivated Na channels, which is more than 300 times more effective on these channels than on K or resting Na channels.     

Decreased expression of apical Na+ channels and basolateral Na+, K+-ATPase in ulcerative colitis

Impaired absorption of sodium (Na+) and water is a major factor in the pathogenesis of diarrhoea in ulcerative colitis (UC). Electrogenic Na+ absorption, present mainly in human distal colon and rectum, is defective in UC, but the molecular basis for this is unclear. The effect of UC on the expression of apical Na+ channels (ENaC) and basolateral Na+, K+-ATPase, the critical determinants of electrogenic Na+ transport, was therefore investigated in this study. Sigmoid colonic and/or proximal rectal mucosal biopsies were obtained from patients with mild to moderate UC, and patients with functional abdominal pain (controls). ENaC subunit expression was studied by immunohistochemistry, western blot analysis, and in situ hybridization, and Na+, K+-ATPase isoform expression was studied by immunohistochemistry, western blotting, and northern blot analysis. UC was associated with substantial decreases in the expression of the ENaC beta- and gamma-subunit proteins and mRNAs, whereas the decrease in ENaC alpha-subunit protein detected by immunolocalization was less marked. The levels of expression of Na+, K+-ATPase alpha1- and beta1-isoform proteins were also lower in UC patients than in controls, although there were no differences in Na+, K+-ATPase alpha1- and beta1-isoform mRNA levels between the two groups. Taken together, these results show that UC results mainly in decreased expression of the apical ENaC beta- and gamma-subunits, as well as the basolateral Na+, K+-ATPase alpha1- and beta1-isoforms. In conclusion, these changes provide a basis for the low/negligible levels of electrogenic Na+ absorption seen in the distal colon and rectum of UC patients, which contribute to the pathogenesis of diarrhoea in this disease.     

Batrachotoxin protects sodium channels from the blocking action of oenanthotoxin

1. The blocking action of oenanthotoxin (OETX) and butanol on Na⁺ channels was studied in voltage clamp experiments on single myelinated nerve fibres treated by batrachotoxin (BTX).
2. OETX (40 μM) blocked Na⁺ currents through normal channels but did not affect significantly the BTX modified Na⁺ current.
3. BTX removed the depolarization-induced charge immobilization and slowed down significantly the OFF charge movement. However, the maximum charge displaced, as well as the kinetics of the ON charge movement during a strong membrane depolarization, remained unchanged.
4. OETX blocked the charge movement in normal Na⁺ channels but did not affect noticeably the charge movement modified by BTX.
5. BTX did not modify the K⁺ currents and did not protect them from the blocking action of OETX.
6. Butanol (0.01–0.1 M) decreased almost identically and reversibly both normal and BTX-modified Na⁺ currents.
7. It is concluded that binding of BTX to its receptor protects the Na⁺ channel from interaction with OETX but left it accessible to butanol.

     

血小板氯通道

Chloride channels distribute widely in the body, and participate in many physiological actions and regulatory processes. Based on their physiological roles and molecular structures, six kinds of chloride channels have been identified: (1) The chloride channels family; (2) Cystic fibrosis transmembrane conductance regulator; (3) Swelling- activated chloride channels; (4) Calcium- activated chloride channels; (5) The p64 (CLIC) gene family; (6) γ- aminobutyric acid and glycine receptors. The chloride channels do exist in platelets, and their appearances are dependent on the presence of intracellular calcium. Blocking agents of chloride channels inhibit the thrombin - activated platelet aggregation and the elevation of the intracellular calcium concentration in a dose - dependent manner. It is suggested that chloride channels play a role in the activation of platelets. In addition, chloride channels act on both the cell volume regulation and the intracellular pH regulation in platelets.