甘丙肽在离体新生大鼠三叉神经主核神经元引发的两种不同膜电位和膜电流

运用细胞内记录和全细胞膜片钳技术 ,在 7～ 16d大鼠带三叉神经残根的 4 0 0 μm厚脑桥切片上进行研究。细胞内电流钳记录显示三叉神经主核灌流甘丙肽引发两种膜电位变化 :54.2 %细胞超极化 (3.2±1.2 )mV ;35.4 %细胞去极化 (2 .9± 1.3)mV ,两者均伴有兴奋性突触后电位抑制和膜电阻减小。电压钳实验显示甘丙肽也引致两种膜电流变化 :57.9%细胞出现外向性电流 (11.6± 6 .1)pA ,该电流可被四乙基胺所抑制 ,其反转电位为 (- 77.1± 5.3)mV ;2 7.3%细胞出现内向性电流 (10 .9± 5.9)pA ,该电流可被细胞外低钠所抑制而被细胞外高钾所增强 ,其反转电位为 (- 2 9.2± 4 .3)mV。两电流均可被甘丙肽激动剂M16所模拟 ,为甘丙肽阻断剂M35和M4 0所阻抑。上述结果提示 ,甘丙肽在突触后膜引发两种不同的膜电活动 :钾离子外流引发超极化和外向性电流 ;钠离子内流和钾离子外流引发去极化和内向性电流 ,两种反应均能抑制三叉神经主核神经元的突触传递。     

他莫昔芬对SHG-44胶质瘤细胞氯通道的抑制作用

目的研究氯通道阻断剂他莫昔芬（tamoxifen）对SHG-44胶质瘤细胞电压依赖性氯通道电流的作用。方法采用全细胞膜片钳方法记录SHG-44细胞的电压依赖性氯通道电流，并观察施用不同浓度的他莫昔芬后电流的变化。结果该电流特性为外向整流、不失活，他莫昔芬能够明显阻断该电流，该阻断具有剂量依赖性及电压依赖性。在＋100mV时，1μM及5μM他莫昔芬对氯电流抑制率分别为48％及89％。结论他莫昔芬可明显阻断SHG-44胶质瘤细胞上的电压依赖性氯通道。     

老龄房颤犬模型静脉肌袖细胞分离及起搏电流记录

背景：电生理改变是老化心脏表现的主要特征之一。 目的：构建老龄房颤犬模型，观察静脉肌袖细胞动作电位和起搏电流的特征。 设计、时间及地点：分组对照动物实验，于2005-03/2007-12在解放军总医院实验动物中心和老年心血管病研究所完成。 材料：7~9岁健康杂种老龄犬12只，体质量15~20 kg，雌雄兼有。台氏液成分(mmol/L)：NaCl 135, KCl 5.4, CaCl2 1.8, MgCl2 1, NaH2PO4 0.33, HEPES 10, 葡萄糖10, pH 用NaOH 调至 7.4。 方法：取老龄犬6只，快速起搏8周制备房颤模型，余6只犬未经快速起搏处理作对照。分离静脉肌袖细胞，利用全细胞膜片钳在电流钳模式下记录动作电位，在电压钳模式下记录起搏电流。 主要观察指标：老龄犬肺静脉肌袖细胞动作电位的特征、起搏电流、起搏电流的Ⅰ~Ⅴ曲线及起搏电流稳态激活的特征。 结果：约82.6％的老龄犬静脉肌袖细胞记录到自动除极和自发性动作电位，而未经快速起搏处理犬的细胞约有40%记录到工作心肌动作电位特征。在-120 mV时，房颤模型细胞起搏电流的电流密度为(-2.66±0.4) pA/pF，而对照组细胞的电流密度为(-1.24±0.21) pA/pF。从Ⅰ~Ⅴ曲线可见，起搏电流的电流密度具有电压依赖性特征，且病理模型犬的肺静脉肌袖细胞起搏电流电流随超极化电位增加更为明显。在-120 mV时，房颤犬静脉肌袖细胞电流的半激活电压为(-84.3±4.9)mV，激活曲线斜率为(+12.1±2.6) mV，而未处理细胞的半激活电压为(-98.0±7.2) mV，激活曲线斜率为(+9.5±1.8) mV，明显向更正的电位方向移动，提示老龄房颤犬静脉肌袖起搏电流更易激活。 结论：老龄房颤犬静脉肌袖细胞更易出现自动除极电位，起搏电流的密度更高，起搏电流激活也更容易，提示起搏电流可能参与老年房颤异位起搏的发生。     

老龄心房颤动犬模型肺静脉肌袖细胞起搏电流在异丙肾上腺素作用下的变化*

背景：心房颤动是老年人常见的心律失常疾病,有研究表明肺静脉肌袖细胞起搏电流能增加快速心律失常特别是心房颤动发生的风险。 目的：观察异丙肾上腺素对老龄犬肺静脉肌袖细胞起搏电流的作用。 方法：选择14只老龄健康犬,7只快速起搏10周制备心房颤动模型,其余未经快速起搏处理的犬作对照。分离各组肺静脉肌袖细胞,利用全细胞膜片钳在电压钳模式下记录起搏电流。 结果与结论：在0.1~10.0 μmol/L的浓度范围内异丙肾上腺素均使细胞膜起搏电流电流升高,且作用呈现出浓度依赖性,半数有效量为1.7 μmol/L (95%可信区间为1.2~2.6 μmol/L)。房颤组肺静脉肌袖细胞给予1.0 μmol/L异丙肾上腺素时,起搏电流显著增加,在-120 mV的测试电位时,使其电流密度升高(P < 0.01)。1.0 μmol/L异丙肾上腺素使房颤组肺静脉肌袖细胞起搏电流的稳态激活曲线向正方向移动,使半激活电压下降,更接近静息电位,同时也使激活曲线斜率(P < 0.01)。结果证实,异丙肾上腺素可能通过升高肺静脉肌袖细胞起搏电流促进心房颤动的发生。     

星形胶质细胞诱导成年大鼠骨髓基质细胞分化前后的电生理功能鉴定

目的 探讨骨髓基质细胞向神经元方向分化后是否具备神经元的电生理功能。方法 应用膜片钳技术,釆用全细胞记录方式.对由星形胶质细胞诱导的分化前、分化3d、分化7d的成年大鼠骨髓基质细胞进行电生理功能测定。结果分化前后的骨髓基质细胞未记录到内向Na~+电流,但成功记录到了静息膜电位,分別为:(-12.71±3.25)mV(n=7)、(-26.29±10.34)mV(n=7)、(-45.0±8.46)mV(n=6);刺激脉冲为40 mV时三组的外向延迟整流K~+电流分別为:(0.31±0.17)nA(n=7)、(1.34±0.59)nA(n=8)、(1.99±0.97)nA(n=7);刺激脉冲为-40 mV时的内向整流K~+电流为:(79±58)pA(n=7)、(260±150)pA(n=8)、(420±120)pA(n=7),伴随着分化时间的逐渐延长,两种K~+电流及膜电位逐渐增高。结论(1)骨髓基质细胞表达弱的外向延迟整流K~+电流及内向整流K~+电流可能是其特征性的电生理指标。(2)骨髓基质细胞经过星形胶质细胞的诱导能够向功能性神经元方向分化。     

超极化激活环化核苷酸门控通道基因转染猪骨髓间充质干细胞

背景：研究证实超极化激活环化核苷酸门控通道（hyperpolarization-activated cyclic nucleotide-gated,HCN）电流在调控心脏的自发搏动中起着非常重要的作用。 目的：观察HCN2基因重组腺病毒转染后猪骨髓间充质干细胞目的基因的表达及电生理特征。 设计、时间及地点：细胞-基因学体外观察,于2007-07/2008-03在解放军第二军医大学胸心外科实验室完成。 材料：Yorkshire猪由解放军第二军医大学动物所提供,HCN2质粒由意大利Dario DiFrancesco教授惠赠,重组腺病毒Ad.HCN2由本实验室采用Ad5系统构建并保存。 方法：Percoll密度梯度离心+贴壁法体外分离纯化猪骨髓间充质干细胞,以感染复数=50进行Ad.HCN2转染,同时设立未转染组和转染Ad.Null组。 主要观察指标：通过RT-PCR、免疫荧光染色检测各组细胞HCN2 mRNA和蛋白的表达,用全细胞膜片钳检测各组细胞电生理学变化。 结果：未转染组和转染Ad.Null组均未见扩增片段,而Ad.HCN2扩增后在250~500 bp可见扩增片段,与携带HCN2基因的质粒扩增出的片段位置相同。转染后细胞核染色强度明显弱于胞膜和胞浆,与HCN2蛋白的分布相符合,未转染组及转染Ad.Null组无HCN2蛋白阳性表达。全细胞膜片钳可记录到起搏电流,其激活电位约为-60 mV,完全激活电位-140 mV,呈电压依赖性,当给予4 mmol/L CsCl后,内向的起搏电流即被明显抑制;未转染组及转染Ad.Null组细胞均无起搏电流。 结论：通过起搏基因HCN2重组腺病毒载体可成功转染猪骨髓间充质干细胞,使其能够表达HCN2通道蛋白,全细胞膜片钳可检测到起搏电流。     

Sombati癫癎细胞模型电压依赖性钾电流的变化

目的：采用全细胞膜片钳记录技术研究Sombati癫癎细胞模型电压依赖性K＋电流的变化。方法：取SD新生乳鼠双侧海马,体外原代培养海马神经元,培养至第9天的部分海马神经元经无镁液处理3h制备癫癎细胞模型,采用全细胞膜片钳技术分别记录正常组（未经无镁液处理的海马神经元）和致癎组（经无镁液处理3h后更换为正常维持培养液的海马神经元）海马神经元的电压依赖性外向K＋电流。结果：致癎组外向K＋电流比正常组增大,峰值电流由（596.62±79.23）pA增至（978.68±53.23）pA,两组间比较差异有统计学意义（P=0.000）。与正常组相比,致癎组的I-V曲线明显左移,I-V曲线形态无显著改变。结论：Sombati癫癎细胞模型中,电压依赖性外向K＋电流显著增加可能是癫癎细胞模型癎性放电后为了保持细胞稳态的代偿性保护作用。     

硬脑膜炎性刺激对三叉神经节小直径神经元电压门控钾电流的影响

目的通过炎性介质(IM)和降钙素基因相关肽(CGRP)诱发偏头痛反复发作,运用全细胞膜片钳的方法观察大鼠三叉神经节小直径神经元电压门控性钾电流的变化。方法雄性SD大鼠15只,分为空白组(不做任何干预)、生理盐水组和IM+CGRP组(大鼠硬脑膜上埋置PE-10管,连续7 d给予等量生理盐水和IM+CGRP)。用Von Frey毛测定大鼠眶周皮肤机械痛阈。在急性分离的三叉神经节小直径神经元上,通过全细胞膜片钳方法记录延迟外向钾电流(IK)和瞬时外向钾电流(IA)的变化。结果给药7 d后,IM+CGRP组大鼠的眶周机械痛阈明显降低,生理盐水组眶周机械痛阈无明显改变。生理盐水组三叉神经节神经元膜上总钾电流、IK、IA与空白组比较无明显差异;IM+CGRP组三叉神经节神经元膜上总钾电流、IK、IA与空白组和生理盐水组比较明显减小。结论 IM和CGRP诱发偏头痛反复发作模型大鼠的三叉神经节中急性分离神经元的IK和IA明显降低,提示电压门控性钾通道可能参与了外周机械痛阈的降低。     

依托咪酯激活大鼠骶髓后连合核神经元GABAA门控氯通道电流

目的　 研究依托咪酯 (etomidate ,ET)在急性分离的大鼠骶髓后连合核 (sacraldorsalcommissuralnucleus,SDCN)神经元的药理学特性。方法　采用制霉菌素穿孔膜片钳全细胞记录。结果　在大鼠SDCN神经元 ,ET(10～ 30 0 0 μmol/L)在钳制电位为 - 40mV时 ,可引起内向电流 (IET) ,EC50 为 (33.35± 3.0 7) μmol/L ,该电流可被GABAA受体拮抗剂荷包牡丹碱 (bicuculline)及氯通道阻滞剂印防己毒素 (picrotoxin)以浓度依赖方式所阻断。结论　ET在大鼠SDCN神经元可通过作用于GABAA受体而诱发氯通道电流 ,此作用可能和全麻状态下脊髓水平的麻醉效应有关     

大鼠背根神经节细胞的分离及特性探讨

目的探讨大鼠背根神经节（dorsal root ganglion，DRG）细胞的分离方法以及细胞形态和电生理特征。方法采用显微外科技术获取大鼠DRG体，用双酶法急性分离大鼠DRG获得DRG细胞，全细胞膜片钳技术记录动作电位和钠电流。结果本实验能得到完整圆形或椭圆长条形的大鼠DRG体。正常的单个DRG细胞呈圆形或椭圆形，大小不等，胞膜清晰，折光性好，隐约可见细胞核。在DRG细胞上记录的动作电位都具有从0期到4期，呈正立锐角三角形，静息电位小，动作电位时程短。DRG细胞的钠通道最大电流密度在-30mV左右，几乎能被，TTX完全抑制，具有可逆性恢复。结论本实验采用分离方法简单易行，DRG细胞容易获得和辨认，适合膜片钳技术要求，电生理特征明确可靠，值得推崇。     

Fibrotic scar model and TGF‐β1 differently modulate action potential firing and voltage‐dependent ion currents in hippocampal neurons in primary culture

Traumatic injury of the central nervous system is accompanied by various functional and morphological changes. Animal models of traumatic brain injury are commonly used to investigate changes in behaviour, morphology, in the expression of various proteins around the site of the injury, or the expression of diagnostically important biomarkers. Excitability of a single neuron at, or close to, the site of injury was rarely investigated. Several in vitro models were developed which allow such investigation. In the present work, we employed a fibrotic scar model according to Kimura‐Kuroda and coauthors to analyse altered excitability of rat hippocampal neurons under the conditions mimicking traumatic brain injury. Hippocampal neurons from newborn rats were cultured either on a fibrotic scar model or in the presence of TGF‐β1, a cytokine secreted at a brain injury site that may have both neuroprotective and neurodegenerative function. Fibrotic scar facilitated ability of neonatal hippocampal neurons to fire action potential series by increasing the density of voltage activated sodium and potassium currents. Chondroitin sulphate proteoglycans played substantial role in these effects, as proven by their full reversion after administration of Chondroitinase ABC. In contrast, TGF‐β1 did not contribute to them. An application of TGF‐β1 itself attenuated generation of action potentials, inhibited sodium current and potentiated potassium currents. Main alteration of electrophysiological parameters of neonatal hippocampal neurons caused by a fibrotic scar model is enhanced excitability. TGF‐β1 may have predominantly neuroprotective role in injured rat hippocampus.     

Voltage-dependent ionic channels in differentiating neural precursor cells collected from adult mouse brains six hours post-mortem

A novel type of adult neural precursor cells (NPCs) has been isolated from the subventricular zone of the mouse 6 hr after animal death (T6-NPCs). This condition is supposed to select hypoxia-resistant cells of scientific and clinical interest. Ionic channels are ultimately the expression of the functional maturation of neurons, so the aim of this research was to characterize the pattern of the main voltage-dependent ionic channels in T6-NPCs differentiating to a neuronal phenotype, comparing it with NPCs isolated soon after death (T0-NPCs). T6- and T0-NPCs grow in medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Differentiation was performed in small wells without the addition of growth factors, in the presence of adhesion molecules, fetal bovine serum, and leukemia inhibitory factor. Ionic currents, recorded by means of whole-cell patch-clamp, namely, I(Ca2+) HVA, both L- and non-L-type, I(K+) delayed rectifying, I(K+) inward rectifier, transient I(K+A) , and TTX-sensitive I(Na+) have been found, although Na(+) currents were found in only a small percentage of cells and after the fifth week of differentiation. No significant differences in current types, density, orcell capacitance were observed between T6-NPCs and T0-NPCs. The sequence in which the markers appear in new neural cells is not necessarily a fixed program, but the discrepancies in morphological, biochemical, and electrophysiological maturation of mouse NPCs to neurons, possibly different in vivo, suggest that the various steps of the differentiation are independently regulated. Therefore, in addition to morphological and biochemical data, functional tests should be considered for characterizing the maturation of neurons.     

The voltage‐dependent conductances of rat neocortical layer I neurons

Whole cell patch-clamp techniques were used to study voltage-dependent sodium (Na⁺), calcium (Ca²⁺), and potassium (K⁺) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of γ-aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca²⁺-binding protein calretinin in a subset of cells, indicated the presence of Cajal–Retzius (C-R) cells. All investigated cells displayed a rather homogeneous profile of voltage-dependent membrane currents. A fast Na⁺ current activated at about −45 mV, was half-maximal steady-state inactivated at −66.6 mV, and recovery from inactivation followed a two-exponential process (τ₁ = 8.4 ms and τ₂ = 858.8 ms). Na⁺ currents declined rapidly with two voltage-dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (I_fast) activated ≈–40 mV, declined rapidly with a voltage-insensitive time constant (τ≈ 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mm ). I_fast was separated into two components based on their sensitivity to 4-aminopyridine (4-AP): one was blocked by low concentrations (40 μm ) and a second by high concentrations (6 mm ). After elimination of I_fast by a conditioning prepulse (50 ms to −50 mV), a slow K⁺ current (I_KV) could be studied in isolation. I_KV was only moderately affected by 4-AP (6 mm ), while TEA (20 mm ) blocked most (> 80%) of the current. I_KV activated at about −40 mV, declined monoexponentially in a voltage-dependent manner (τ≈ 850 ms at −30 mV), and revealed an incomplete steady-state inactivation. In addition to I_fast and I_KV, indications of a Ca²⁺-dependent outward current component were found. When Na⁺ currents, I_fast, and I_KV were blocked by tetrodotoxin (TTX, 1 μm ), 4-AP (6 mm ) and TEA (20 mm ) an inward current carried by Ca²⁺ was found. Ca²⁺ currents activated at depolarized potentials at about −30 mV, were completely blocked by 50 μm cadmium (Cd²⁺), were sensitive to verapamil (≈ 40% block by 10 μm ), and were not affected by nickel (50 μm ). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (≈ 2 ms, measured at half maximal amplitude) of relative small amplitude (≈ 83 mV, measured from the action potential threshold).     

Developmental expression of Na+ currents in mouse Purkinje neurons

As Purkinje neurons mature during postnatal development, they change from electrically quiescent to active and exhibit high frequency spontaneous action potentials. This change in electrical activity is determined by both alteration in ion channel expression and the acquisition of synaptic input. To gain a better understanding of the development the intrinsic electrical properties of these neurons, acutely isolated Purkinje neurons from mice aged postnatal day 4 (P4) to P18 were examined. This included recording action potential frequency, threshold, height and slope, and input resistance and capacitance. Changes in a number of these properties were observed, suggesting significant changes in voltage-gated Na(+) currents. Because voltage-gated Na(+) currents, including the transient, resurgent and persistent currents, are known to play important roles in generating spontaneous action potentials, the developmental changes in these currents were examined. A large increase in the density of transient current, resurgent current and persistent current was observed at times corresponding with changes in action potential properties. Interestingly, the developmental up-regulation of the persistent current and resurgent current occurred at rate which was faster than the up-regulation of the transient current. Moreover, the relative amplitudes of the persistent and resurgent currents increased in parallel, suggesting that they share a common basis. The data indicate that developmental up-regulation of Na(+) currents plays a key role in the acquisition of Purkinje neuron excitability.     

Ionic basis of the membrane potential responses of rat dorsal vagal motoneurones to HEPES buffer

The effects of 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) buffered artificial cerebrospinal fluid (aCSF) on membrane potential and the action potential were studied in 93 dorsal vagal motoneurones (DVMs) using an in vitro slice preparation of the rat medulla. Changing from bicarbonate/CO₂ aCSF to HEPES aCSF resulted in a depolarisation of 6.0 ± 0.6 V and an increase in input resistance (R_In; N = 61). In the presence of 5 mM 4-AP, HEPES either had little effect (n = 9) or hyperpolarised the membrane (n = 10). Mn²⁺ (3 mM) or Ni²⁺ (200 μm) abolished the hyperpolarisation and its associated increase in R_In. In voltage-clamp studies 5 mM 4-AP eliminated a transient outward current and Ni²⁺ blocked an inactivating inward current. It is concluded that HEPES buffer reduces the contribution of the A current to resting membrane potential and also reduces a Ni²⁺-sensitive transient I_Ca.     

Delayed potassium current and non-specific outward current in pyramidal neurones acutely dissociated from rat hippocampus

The electrical property of delayed K+ currents (IKD) was studied in pyramidal neurones freshly isolated from the rat hippocampal CA1 region. The IKD was separated pharmacologically from other membrane currents. Activation and inactivation processes of the IKD were highly voltage-dependent in the potential range between -30 and +20 mV. The steady-state inactivation of IKD was observed at -100 mV or more positive potentials. The potential for half steady-state inactivation was -65 mV. The IKD was fully inactivated around -20 mV. Reactivation of IKD consisted of two exponential components. After pharmacological suppression of IKD, the small amount of residual voltage-dependent outward current (one-fifteenth to one-twentieth of IKD amplitude) was observed. The current kinetics was similar to that of IKD and greatly reduced by substitution of internal K+ with N-methyl-D-glucamine+. It was concluded that the properties of IKD was basically similar to those of IKD in other excitable tissues and that the residual current might be non-specific outward current.     

Effects of insulin-like growth factor 1 on voltage-gated ion channels in cultured rat hippocampal neurons

Insulin-like growth factor 1 (IGF-1) has important functions in the brain, including metabolic, neurotrophic, neuromodulatory, and neuroendocrine actions, and it is also prevents amyloid beta-induced death of hippocampal neurons. However, its functions on the voltage-gated ion channels in hippocampus remain uncertain. In the present study, we investigated the effects of IGF-1 on voltage-gated potassium, sodium, and calcium channels in the cultured rat hippocampal neurons using the whole-cell patch clamp recordings. Following incubation with different doses of IGF-1 for 24 h, a block of the peak transient A-type K+ currents amplitude (IC50: 4.425 ng/ml, Hill coefficient: 0.621) was observed. In addition, after the application of IGF-1, the amplitude of high-voltage activated Ca2+ currents significantly increased but activation kinetics did not significantly alter (V1/2: -33.45 +/- 1.32 mV, k = 6.16 +/- 1.05) compared to control conditions (V1/2: -33.19 +/- 2.28 mV, k = 7.26 +/- 1.71). However, the amplitude of Na+, K+, and low-voltage activated Ca2+ currents was not affected by the application of IGF-1. These data suggest that IGF-1 inhibits transient A-type K+ currents and enhances high-voltage-activated Ca2+ currents, but has no effects on Na+ and low-voltage-activated Ca2+ currents.     

Membrane Currents Influencing Action Potential Latency in Granule Neurons of the Rat Cochlear Nucleus

Granule cells are the most numerous neurons in the cochlear nucleus, but, because of their small size, little information on their membrane properties and ionic currents is available. We used an in vitro slice preparation of the rat ventral cochlear nucleus to make whole-cell recordings from these cells. Under current clamp, some granule neurons fired spontaneous action potentials and all generated a train of action potentials on depolarization (threshold current, 10–35 pA). Hyperpolarization increased the latency to the first action potential evoked during a subsequent depolarization. We examined which voltage-gated currents might underlie this latency shift. In addition to a fast inward Na⁺ current, depolarization activated two outward potassium currents. A transient current was rapidly inactivated by membrane potentials positive to -60 mV, while a second, more slowly inactivating current was observed following the decay of the transient current. No hyperpolarization-activated conductances were observed in these cells. Modelling of the currents suggests that removal of inactivation on hyperpolarization accounts for the increased action potential latency in granule cells. Such a mechanism could account for the 'pauser'-type firing patterns of the fusiform cells which receive a prominent projection from the granule cells in the dorsal cochlear nucleus.     

A tetrodotoxin- and Mn2(+)-insensitive Na+ current in Duchenne muscular dystrophy

Muscle myotube cultures were obtained from normal and Duchenne muscular dystrophy (DMD) biopsies by using an explant technique. The current-voltage (I/V) curve of the whole sodium (Na+) current (INa) in normal myotubes was similar to that obtained from DMD myotubes. However, the inactivation curve of the whole INa was different in normal myotubes when compared to that obtained from DMD myotubes. Addition of 10(-4) M tetrodotoxin (TTX, a fast INa blocker) decreased the whole INa in both preparations. The inorganic calcium (Ca2+) blocker manganese (Mn2+) completely blocked the remaining TTX-resistant INa of normal myotubes and decreased this current in DMD myotubes leaving behind a TTX- and Mn2(+)-insensitive INa that was insensitive to the Ca2+ blocker desmetoxyverapamil ((-)D888). The slow inward barium current (IBa) of both normal and DMD myotubes was blocked by Mn2+ and (-)D888. However the kinetics of the slow channel in normal myotubes was different from that of DMD myotubes. This study demonstrates the presence of a TTX- and Mn2(+)-insensitive INa in DMD myotubes. This channel may contribute to the increase of intracellular Na+ [( Na]i) in DMD and allow Ca2+ to enter the cells through the Na(+)-Ca2+ exchanger, thus contributing to calcium loading.