E4031对慢性心衰大鼠离体心脏功能和心肌细胞内静息Ca^2＋水平的影响

目的：研究具有钠钙交换（NCX）激动作用的药物E 4031对慢性心衰大鼠离体心脏功能和心肌细胞内静息Ca2＋水平的影响。方法：通过腹主动脉缩窄建立大鼠慢性心力衰竭模型;利用Langendorff装置进行离体心脏灌流,检测大鼠心功能及E 4031对血流动力学指标的影响;急性分离心衰大鼠心肌细胞,与钙荧光指示剂fluo3/AM共同孵育后,用激光共聚焦显微镜系统观察E 4031对心肌细胞内荧光强度的影响。结果：缩窄大鼠腹主动脉12周后,langendorff离体灌流检测显示大鼠心功能明显降低;在灌流液中加入10μmol/L E 4031可以使心衰大鼠心脏左室发展压（LVDP）和左室收缩/舒张最大速率（±dp/dtmax）提高;与正常组和伪手术组相比,心衰大鼠心肌细胞内静息钙荧光强度明显升高,和10μmol/L E 4031共孵育后,心衰大鼠心肌细胞静息钙荧光强度呈现短期先升后降过程,然后在较低的水平保持稳定。结论：E 4031可以增强慢性心衰大鼠离体心功能,可能与其增强心肌细胞膜NCX活动,稳定细胞内Ca2＋水平有关。     

温度和胞内钠、ATP及酸碱度对心室肌细胞钠钙交换电流的调节

心肌缺血损伤过程中,胞内Na^＋、ATP及pH都出现明显变化。钠／钙交换对心肌细胞的钙平衡起重要的调节作用。本实验采用膜片钳全细胞记录豚鼠心室肌细胞钠／钙交换电流,研究温度和胞内Na^＋、ATP及pH对钠／钙交换双向电流的影响。结果表明,温度从22℃升至34℃,钠／钙交换电流增大约4倍,而pH值的改变对钠／钙交换双向电流没有明显的影响。在22～24℃时,同时耗竭胞内ATP和胞内酸化对钠／钙交换双向转运功能影响程度小;而在34—37℃时,同时耗竭胞内ATP和胞内酸化能抑制钠／钙交换双向电流的外向和内向成分,且内向成分抑制程度高于外向成分抑制程度。表明同时耗竭胞内ATP和胞内酸化对钠／钙交换的作用具有温度依赖性。胞内Na^＋超载能使钠／钙交换电流的外向成分增加,但不增加或减少内向电流（即正向转运）成分。因此,胞内酸化及耗竭胞内ATP损伤细胞排钙机制和胞内钠超载通过钠／钙反向交换引起钙内流是引起心肌细胞钙超载的两个独立的重要因素。     

膜钠钙交换蛋白和过氧化氢交互作用对细胞内钙稳态调控

本研究旨在阐明过氧化氢（H2O2）和膜钠钙交换蛋白相互作用对胞浆钙[Ca^2 ],的调控。在稳定表达钠钙交换蛋白CK1．4细胞上,用^45Ca同位素液闪计数法测定钠钙交换蛋白的活性;用fura-2荧光探针和340／380nm双兴奋波长荧光影像技术测定钙释放和[Ca^2 ]i。两因素两水平和三因素两水平正交分析表明10mmol/L H2O2与150mmol/L细胞外钠（[Na^ ]o,1mmol/L细胞外钙[Ca^2 ],相互作用或10mmol/L H2O2分别与150mmol/L[Na ]。或1[Na^ ]。激活钠钙交换蛋白,排出细胞内钙离子,降低[Ca2 ]i。当[Na^ ]。递减至0mmol/L时,10mmol/L H2O2直接抑制钠钙交换蛋白的活性,增加钙释放和升高[Ca2 ]i.在不同[Na^=},梯度中,10mmol/LH2O2对膜的钠钙交换活动和[Ca2 ],起双重调节作用,即抑制或增加钙内流和[Ca^2=]i.10mmol/L H2O2与膜钠钙交换蛋白和[Ca2 ]。相互作用对钠钙交换活动方向,钙释放和[Ca^2_]起负反馈谳节作用。     

河豚毒素停搏液对缺血／再灌注心肌细胞钠超载的影响

目的：观察Na^＋通道阻滞剂河豚毒素（TTX）极化心脏停搏液对离体大鼠心肌细胞内游离Na^＋浓度（[Na^＋]i）的影响。方法：成年Wistar大鼠心脏,用酶解法分离成具有搏动性的单个心室肌细胞悬液,随机分成基础组、STH2组（缺血／再灌注损伤对照组）和TTX组（实验组）,STH2组和TTX组分别应用St．ThomasⅡ号停搏液和TTX停搏液处理,建立模拟缺血／再灌注损伤的停搏／复搏细胞模型,激光扫描共聚焦显微镜（LSCM）测定各组细胞不同时期的[Na^＋]。倒置显微镜观察细胞形态学变化。结果：TTX组和STH2组细胞复搏后[Na^＋];均明显高于基础组（P〈0．01）,但TTX组明显低于STH2组（P〈0．01）;在停搏期间,TTX组细胞[Na^＋]i上升速度和幅度明显低于STH2组;形态学观察,TTX组复搏后具有正常活力的杆形心肌细胞比例高于STH2组（P〈0．01）。结论：河豚毒素心脏停搏液较去极化心脏停搏液能减轻心肌细胞Na^＋超载和缺血／再灌注损伤。     

抗钠-钙交换体α-1重复序列(117-137)抗体对心肌钠-钙交换电流及钙电流的抑制作用(英文)

本研究旨在探讨抗钠-钙交换体(Na+-Ca2+ exchanger,NCX)α-1重复序列(117-137)抗体对大鼠心肌细胞钠-钙交换电流的影响。实验用合成的α-1重复序列(117-137)肽免疫大鼠制备抗α-1重复序列(117-137)抗体;应用全细胞膜片钳技术,在急性分离的心肌细胞上观察抗α-1重复序列(117-137)抗体对大鼠心肌细胞钠-钙交换电流的影响,同时还观察了其对大鼠心肌细胞L型钙通道、电压门控钠通道和豚鼠心肌延迟整流钾通道电流的影响;最后,采用EMBOSS Pairwise Alignment Algorithms软件对NCXα-1重复序列和L型钙通道(1076～1096)的氨基酸序列进行了比较。结果显示,抗α-1重复序列(117-137)抗体对大鼠心肌细胞Na+-Ca2+交换电流呈现剂量依赖性的抑制作用。在钳制电压为+50和-100mV时,其抑制外向和内向钠-钙交换电流的IC50分别为18.9和22.4nmol/L。此外,该抗体还对L型钙通道电流具有抑制效应(IC50=22.7nmol/L),对电压门控钠通道和延迟整流钾通道电流则无明显影响。通过氨基酸序列比对发现,NCX α-1重复序列(117-137)与L型钙通道IIIS5-S6序列(孔环)之间具有23.8%的相似性。以上结果表明,抗NCX α-1重复序列(117-137)抗体是心肌NCX的一种抑制性抗体,同时也可以抑制L型钙通道。     

L型钙流和反向钠-钙交换在豚鼠心室肌细胞兴奋-收缩偶联中的作用

目的 :比较和探讨L型钙流 [ICa(L) ]和反向钠—钙交换 (NCX)在触发豚鼠心室肌细胞兴奋—收缩偶联中的作用。方法 :以分离的豚鼠单个心室肌细胞为对象 ,采用膜片钳和单细胞收缩测量技术 ,给予 35℃的各种含药物细胞外液快速灌流 ,同时记录ICa(L) 和细胞收缩。结果 :①在 +10mV的钳制电压 ,使用硝苯地平 (Nif) 10～ 10 0 μmol/L和Nif 30 μmol/L +Cd2 +30 μmol/L ,阻滞ICa(L) 越多 ,细胞收缩被阻滞得越多 ,呈线性相关。②在 +5 0mV的钳制电压 ,Nif 10 0 μmol/L以及Nif 30 μmol/L +Cd2 +3 0 μmol/L仅能抑制部分细胞收缩 ,但剩余的细胞收缩起始时间明显延迟 ,且能被 5mmol/LNi2 +所阻滞。③在 +10 0mV的钳制电压 ,细胞收缩起始时间较 +5 0mV明显延迟 ,且不能被Nif 10 0 μmol/L和Nif 30 μmol/L +Cd2 +30 μmol/L所阻滞。结论 :在生理条件下 ,ICa(L) 是触发心室肌细胞兴奋—收缩偶联的主要途径 ,但在膜电位 >+5 0mV时 ,反向NCX也参与兴奋—收缩偶联。     

心肌细胞发育过程中胞浆内钙稳态的调控

Ca^2＋信号是细胞和各器官生长发育、行使其生理功能的基础,维持心肌细胞的钙稳态是保持正常心脏功能的先决条件。作为在胚胎发育过程中最早出现并行使功能的器官,胚胎期心脏的形态结构发生了明显的变化,泵血功能不断增强,以适应不断增强的机体的生理需求。从胚胎到成年,心肌细胞的功能有非常大的改变,各钙离子通道的表达也发生明显变化。因此,发育早期心肌细胞的钙稳态调控与成熟心肌细胞有明显的不同,在发育过程中引起细胞收缩的Ca^2＋来源也有明显的变化。随着分子和细胞生物学研究的发展,以及胚胎干细胞体外分化模型的应用,人们对心肌细胞发育过程中钙稳态的调控有了进一步的认识。本文综述了早期心肌细胞发育过程中胞浆内钙稳态的变化,总结了早期心肌细胞钙稳态调控机制的最新研究进展。     

耐钙心肌细胞的分离和电生理特性观察

用快速、恒压的无钙和胶原酶Ｔｙｒｏｄｅ液相继灌流豚鼠心脏冠脉系统后,再经无钙液室温浸泡心脏和用改变的Ｋ－Ｂ液帮助分离细胞的恢复,可获得耐钙的游离心肌细胞。全细胞电流记录：静息电位为－７２±９ｍＶ（ｎ＝１２）,并显示出快内向电流（ＩＮａ）,可被异搏定阻断的慢钙离子流和时间依赖性外向钾流（Ｉｋ）;单通道记录分别显示了Ｎａ＋Ｃａ２＋和Ｋ＋通道的电压依赖性等特征。结果表明了用此法分离的细胞具有耐钙性和正常电生理特性。     

β-肾上腺素受体调控心肌细胞兴奋收缩耦联的分子过程研究

兴奋收缩耦联是肌细胞兴奋期间由动作电位触发肌质网释放钙离子,从而导致收缩的过程。心肌细胞的兴奋收缩耦联是通过“钙致钙释放（Ca^2＋-induced Ca^2＋ release）的机制完成的。兴奋期间,细胞膜电位的去极化导致电压依赖性的L．型钙通道（LCC）开放,细胞外钙离子通过LCC流入细胞,激活了肌质网膜上称为ryanodine受体（RyR）的钙释放通道,后者从肌质网钙库中释放钙离子,使细胞质游离钙浓度迅速上升。细胞质钙浓度的升高一方面启动细胞收缩,另一方面激活了肌质网钙泵和细胞膜钠钙交换,二者分别将钙离子运回肌质网或细胞外,使细胞质钙浓度很快回落,从而完成了一次“钙瞬变（Ca^2＋ transient）”。钙瞬变在每个心动周期发生一次,是直接控制细胞收缩的细胞内信号。     

成年大鼠心肌细胞分离培养及兴奋-收缩耦联表征

目的：比较两种细胞分离液分离成年大鼠心肌细胞,进一步表征成年大鼠心室肌细胞兴奋-收缩耦联。方法 Langendorff装置进行主动脉逆流灌流,分别用两种细胞分离液分离成年大鼠心肌细胞,无血清培养并进行腺病毒感染。显微镜下观察单个心肌细胞的形态学特点,荧光显微镜下检测病毒感染。采用IonOptix仪器检测心肌细胞肌节收缩-舒张指标以及心肌细胞钙离子摄入-排出指标。结果两种分离液均可获得70％横纹清晰的长杆状心肌细胞,培养可存活7 d以上。腺病毒感染48 h,绿色荧光蛋白持续表达7 d以上。分离液一获得的心肌细胞不能很好地随电场刺激产生收缩,分离液二获得的细胞可用于检测兴奋-收缩耦联特性,心肌细胞肌节缩短分数为11.61％±2.15％,舒张时间为（0.177±0.031） s,钙瞬变幅度为30.79％±9.74％,钙瞬变衰减时间为（0.300±0.074） s。结论两种分离液均可用于分离和培养成年大鼠心肌细胞,并用于腺病毒转染等长时程研究。分离液二更适用于检测成年大鼠心肌细胞的兴奋-收缩耦联特性。     

Effects of Na+/Ca2+ exchange induced by SR Ca2+ release on action potentials and afterdepolarizations in guinea pig ventricular myocytes

In cardiac cells, evoked Ca2+ releases or spontaneous Ca2+ waves activate the inward Na+/Ca2+ exchange current (INaCa), which may modulate membrane excitability and arrhythmogenesis. In this study, we examined changes in membrane potential due to INaCa elicited by sarcoplasmic reticulum (SR) Ca2+ release in guinea pig ventricular myocytes using whole cell current clamp, fluorescence, and confocal microscopy. Inhibition of INaCa by Na+-free, Li+-containing Tyrode solution reversibly abbreviated the action potential duration at 90% repolarization (APD90) by 50% and caused SR Ca2+ overload. APD90 was similarly abbreviated in myocytes exposed to the Na+/Ca2+ exchange inhibitor KB-R7943 (5 microM) or after inhibition of SR Ca2+ release with ryanodine (20 microM). In the absence of extracellular Na+, spontaneous SR Ca2+ releases caused minimal changes in resting membrane potential. After the myocytes were returned to Na+-containing solution, the potentiated intracellular Ca2+ concentration ([Ca2+]i) transients dramatically prolonged APD90 and [Ca2+]i oscillations caused delayed and early afterdepolarizations (DADs and EADs). Laser-flash photolysis of caged Ca2+ mimicked the effects of spontaneous [Ca2+]i oscillations, confirming that APD prolongation, DADs, and EADs could be ascribed to intracellular Ca2+ release. These results suggest that Na+/Ca2+ exchange is a major physiological determinant of APD and that INaCa activation by spontaneous SR Ca2+ release/oscillations, depending on the timing, can account for both DADs and EADs during SR Ca2+ overload.     

Electrophysiological demonstration of Na+/Ca2+ exchange in bovine articular chondrocytes

Altered fluxes of Ca2+ across the chondrocyte membrane have been proposed as one pathway by which mechanical load can modulate cartilage turnover. In many cells, Na+/Ca2+ exchange (NCX) plays a key role in Ca2+ homeostasis, and recent studies have suggested it is operative in articular chondrocytes. In this study, an electrophysiological characterisation of NCX in articular bovine chondrocytes has been performed, using the whole-cell patch clamp technique, and the effects of inhibitors and the transmembrane electrochemical gradients of Na+ and Ca2+ on NCX function have been assessed. A Ni2+-sensitive current (I(NCX)) which exhibited outward rectification, was elicited by a voltage ramp protocol. The current was also attenuated by the NCX inhibitors benzamil and KBR7943, without significant differences between the effect of these two compounds upon outward and inward currents. The Ni2+-sensitive current was modulated by changes in extracellular and pipette Na+ and Ca2+ in a manner characteristic of I(NCX). Measured values for the reversal potential differed significantly from those predicted for an exchanger stoichiometry of 3Na+ : 1Ca2+, implying that accumulation of intracellular Ca2+ (from influx or release from stores) or more than one transport mode is occurring. These results demonstrate the operation of NCX in articular chondrocytes and suggest that changes in its turnover rate, as might occur in response to mechanical load, may modify cell composition and thereby dictate cartilage turnover.     

Postnatal developmental changes in Ca2+ homeostasis in supraoptic magnocellular neurons

Supraoptic magnocellular neurons (SMNs) undergo dramatic changes in morphological and electrical properties during postnatal development. We investigated the developmental change in Ca2+ homeostasis in SMNs. The decay rate of Ca2+ transients markedly increased during the third postnatal week (PW3) to an adult level. This increase in the Ca2+ decay rate was paralleled by hypertrophy of the SMN somata. Activity of Na+/Ca2+ exchanger (Na/CaX) and sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) was quantified as a decrement in the Ca2+ decay rate caused by extracellular [Na+] reduction and that by thapsigargin, respectively. SERCA activity was negligible during PW2, and markedly increased during PW3. SERCA activity and soma size remained stable thereafter. Na/CaX activity was a major Ca2+-clearance mechanism (CCM) during PW2, increased further during PW3, but was negligible in mature SMNs (PW10). In parallel with the decrease in Na/CaX activity, endogenous Ca2+ buffering capacity declined, resulting that the apparent Ca2+ decay rate remained relatively constant between PW4 and PW10. Replacement of intracellular K+ with Li+ had no effect on Na/CaX activity, suggesting that NCX rather than NCKX comprises Na/CaX. These findings indicate a developmental shift in the balance of CCMs from Ca2+ extrusion via NCX toward Ca2+ sequestration into endoplasmic reticulum via SERCA.     

Excitation-contraction coupling in isolated locomotor muscle fibres from the pelagic tunicate Doliolum which lack both sarcoplasmic reticulum and transverse tubular system

In the locomotor muscle of the pelagic tunicate Doliolum, both the sarcoplasmic reticulum (SR) and the transverse-tubular (T-tubular) system are absent. The mechanism of excitation-contraction (E-C) coupling was studied in single muscle fibres enzymatically dissociated from Doliolum denticulatum. Whole cell voltage clamp experiments demonstrated an inward ionic current associated with membrane depolarisation. This current was blocked by 5 mmol.l(-1)Co(2+), a calcium current blocker, and suppressed by nifedipine, a specific L-type calcium channel blocker. An increase in the external K(+) concentration to 200 mmol.l(-1) (K(+)-depolarisation) induced a rise in the intracellular Ca(2+) level detected with fluo-3, a Ca(2+)-sensitive dye. However, when 5-10 mmol.l(-1) Co(2+) or 10-15 micro mol.l(-1) nifedipine was present in the external solution, K(+)-depolarisation did not induce a rise in the intracellular Ca(2+) level. Externally applied 5-10 mmol.l(-1) caffeine or 20 micro mol.l(-1) ryanodine had no effect on the intracellular Ca(2+) level. K(+)-depolarisation induced a rise in the intracellular Ca(2+) level in the presence of caffeine or ryanodine. Replacement of external Na(+) with Li(+) increased intracellular Ca(2+) levels. Our results show that contraction of the locomotor muscle in Doliolum is solely due to the influx of Ca(2+) through L-type calcium channels, and that relaxation is due to extrusion of Ca(2+) by Na(+)/Ca(2+) exchange across the sarcolemma.     

SEA0400, a novel Na+/Ca2+ exchanger inhibitor, reduces calcium overload induced by ischemia and reperfusion in mouse ventricular myocytes

Given the potential clinical benefit of inhibiting Na+/Ca2+ exchanger (NCX) activity during myocardial ischemia reperfusion (I/R), pharmacological approaches have been pursued to both inhibit and clarify the importance of this exchanger. SEA0400 was reported to have a potent NCX selectivity. Thus, we examined the effect of SEA0400 on NCX currents and I/R induced intracellular Ca2+ overload in mouse ventricular myocytes using patch clamp techniques and fluorescence measurements. Ischemia significantly inhibited inward and outward NCX current (from -0.04+/-0.01 nA to 0 nA at -100 mV; from 0.23+/-0.08 nA to 0.11+/-0.03 nA at +50 mV, n=7), Subsequent reperfusion not only restored the current rapidly but enhanced the current amplitude obviously, especially the outward currents (from 0.23+/-0.08 nA to 0.49+/-0.12 nA at +50 mV, n=7). [Ca2+]i, expressed as the ratio of Fura-2 fluorescence intensity, increased to 138+/-7% (P<0.01) during ischemia and to 210+/-11% (P<0.01) after reperfusion. The change of NCX current and the increase of [Ca2+]i during I/R can be blocked by SEA0400 in a dose-dependent manner with an EC50 value of 31 nM and 28 nM for the inward and outward NCX current, respectively. The results suggested that SEA0400 is a potent NCX inhibitor, which can protect mouse cardiac myocytes from Ca2+ overload during I/R injuries.     

Fluorescent Na+-Ca+ exchangers: electrophysiological and optical characterization

The cardiac sarcolemmal Na+-Ca2+ exchanger (NCX1) influences cardiac contractility by extruding Ca2+ from myocytes. As a Ca2+ efflux mechanism, the exchanger plays a prominent role in Ca2+ homeostasis. To track NCX1 and study changes in conformation, NCX1 was tagged with derivatives of green fluorescent protein. Cyan (CFP) and yellow (YFP) fluorescent proteins were used for both visualization of the protein in HEK cells and fluorescent resonance energy transfer (FRET). CFP or YFP was inserted at position 266, 371, 467, or 548 of the large intracellular loop of NCX1 located between transmembrane segments 5 and 6. These constructs were tested for functional activity and visualized for cell surface expression. All constructs were targeted to the plasma membrane. Transport properties were assessed by both 45Ca2+ uptake and electrophysiological measurements. The fluorescent-tagged exchangers had similar biophysical properties to the wild type NCX1. Unexpectedly, all constructs retain their sensitivity to regulation by cytoplasmic Na+ and Ca2+ ions. FRET analysis indicates the proximity of NCX1 to plasma membrane phosphatidylinositol 4,5-bisphosphate. These results indicate that insertion of CFP or YFP into the large intracellular loop of NCX1 protein does not impair exchanger properties. These constructs will be useful to further characterize the biological properties of the exchanger in intact cells.     

Cytoplasmic tail of phospholemman interacts with the intracellular loop of the cardiac Na+/Ca2+ exchanger

Phospholemman (PLM), a member of the FXYD family of small ion transport regulators, inhibits cardiac Na+/Ca2+ exchanger (NCX1). NCX1 is made up of N-terminal domain consisting of the first five transmembrane segments (residues 1-217), a large intracellular loop (residues 218-764), and a C-terminal domain comprising the last four transmembrane segments (residues 765-938). Using glutathione S-transferase (GST) pull-down assay, we demonstrated that the intracellular loop, but not the N- or C-terminal transmembrane domains of NCX1, was associated with PLM. Further analysis using protein constructs of GST fused to various segments of the intracellular loop of NCX1 suggest that PLM bound to residues 218-371 and 508-764 but not 371-508. Split Na+/Ca2+ exchangers consisting of N- or C-terminal domains with different lengths of the intracellular loop were co-expressed with PLM in HEK293 cells that are devoid of endogenous PLM and NCX1. Although expression of N-terminal but not C-terminal domain alone resulted in correct membrane targeting, co-expression of both N- and C-terminal domains was required for correct membrane targeting and functional exchange activity. NCX1 current measurements indicate that PLM decreased NCX1 current only when the split exchangers contained residues 218-358 of the intracellular loop. Co-immunoprecipitation experiments with PLM and split exchangers suggest that PLM associated with the N-terminal domain of NCX1 when it contained intracellular loop residues 218-358. TM43, a PLM mutant with its cytoplasmic tail truncated, did not co-immunoprecipitate with wild-type NCX1 when co-expressed in HEK293 cells, confirming little to no interaction between the transmembrane domains of PLM and NCX1. We conclude that PLM interacted with the intracellular loop of NCX1, most likely at residues 218-358.     

Possible role of Na+ ions in intracellular Ca2+ release in frog auricular trabeculae

Membrane potential-current and mechanical tension of frog atrial muscle were studied in a Ca and Mg-free solution containing 1 mmol/l EGTA (Ca-free solution). Exposure to Ca-free solution resulted in a shortening of action potential duration within 1.5 min and a subsequent lengthening which were paralleled by changes in magnitude and duration of the contraction. Similarly, the slow inward current quickly disappeared and progressively reappeared with a quite slower inactivation time-course. Its reversal potential varied with [Na]0 as for a pure Na current. By 12 min in Ca-free solution, the tension-voltage relation could be interpreted as the sum of two components correlated with the slow inward current and the membrane potential respectively. Contractures in response to sustained large depolarizations had similar time courses in Ca-free solution and Ringer's containing Na-Ca exchange blockers (Mn2+ 15 mmol/l or La3+ 3 mmol/l). Intracellular Na loading by voltage-clamp depolarizations (40 mV from the resting potential for 100 ms, at 0.2 Hz) in the presence of Veratrine (7.5 X 10(-6) g/ml) caused a large progressive increase in tonic tension. An intracellular Ca2+ release is invoked, partly related to Na+ entry and partly to membrane potential changes. The potential dependent part could be influenced by intracellular Na+.     

Inhibitory effects of purified antibody against α-1 repeat (117-137) on Na(+)-Ca(2+) exchange and L-type Ca(2+) currents in rat cardiomyocytes

Considering that α-1 repeat region may be involved in the ion binding and translocation of Na(+)-Ca(2+) exchanger (NCX), it is possible that the antibodies against NCX α-1 repeat may have a crucial action on NCX activity. The aim of the present study is to investigate the effect of antibody against α-1 repeat (117-137), designated as α-1(117-137), on NCX activity. The antibody against the synthesized α-1(117-137) was prepared and affinity-purified. Whole-cell patch clamp technique was used to study the change of Na(+)-Ca(2+) exchange current (I(Na/Ca)) in adult rat cardiomyocytes. To evaluate the functional specificity of this antibody, its effects on L-type Ca(2+) current (I(Ca,L)), voltage-gated Na(+) current (I(Na)) and delayed rectifier K(+) current (I(K)) were also observed. The amino acid sequences of α-1(117-137) in NCX and residues 1?076-1?096 within L-type Ca(2+) channel were compared using EMBOSS Pairwise Alignment Algorithms. The results showed that outward and inward I(Na/Ca) were decreased by the antibody against α-1(117-137) dose-dependently in the concentration range from 10 to 160 nmol/L, with IC(50) values of 18.9 nmol/L and 22.4 nmol/L, respectively. Meanwhile, the antibody also decreased I(Ca,L) in a concentration-dependent manner with IC(50) of 22.7 nmol/L. No obvious effects of the antibody on I(Na) and I(K) were observed. Moreover, comparison of the amino acid sequences showed there was 23.8% sequence similarity between NCX α-1(117-137) and residues 1?076-1?096 within L-type Ca(2+) channel. These results suggest that antibody against α-1(117-137) is a blocking antibody to NCX and can also decrease I(Ca,L) in a concentration-dependent manner, while it does not have obvious effects on I(Na) and I(K).