Molecular characterization of L-type calcium channel splice variants expressed in human T lymphocytes

Calcium (Ca²⁺) influx is a fundamental intracellular signal that is required to initiate and sustain T lymphocyte activation. Dihydropyridine-sensitive, L-type Ca²⁺ channels appear to play a significant role in Ca²⁺ mobilization during T cell activation, but very little is known about the molecular structure of these channels in T lymphocytes. Here we identify two novel splice variants of the Ca_V1.4 (_1F) L-type Ca²⁺ channel that are expressed in human T lymphocytes, and also demonstrate expression of the Ca_V1.4 protein in the human Jurkat T cell leukemia line and human peripheral blood T lymphocytes (PBTs). The carboxy-termini of both Ca_V1.4 splice isoforms contain unique exon usages distinct from the Ca_V1.4 channel isolated from human retina that may render these channel variants insensitive to changes in membrane depolarization. Additional evidence of the importance of these new splice variants comes from the demonstration that the mRNA expression of the Ca_V1.4 splice isoforms is regulated by TCR-induced activation in Jurkat T cells, and to a lesser extent in human PBTs. Overall these results provide the first evidence that structurally unique L-type Ca²⁺ channels exist in T lymphocytes, which can contribute to a Ca²⁺ influx during T lymphocyte activation.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

L-type calcium channel blockade on haloperidol-induced c-Fos expression in the striatum

Haloperidol-induced c-Fos expression in the lateral part of the neostriatum has been correlated with motor side effects while c-Fos induction in the medial part of the neostriatum and the nucleus accumbens is thought to be associated with the therapeutic effects of the drug. Induction of c-Fos in the striatum by haloperidol involves dopamine D(2) (DA D(2)) receptor antagonism and is dependent on activation of N-methyl-d-aspartate (NMDA) receptors and L-type Ca(2+) channels. In the current study, pretreatment with L-type Ca(2+) channel blockers suppressed haloperidol-induced c-Fos throughout the neostriatum and the nucleus accumbens at 2 h postinjection. However, elevated c-Fos protein expression was observed only in the lateral part of the neostriatum at 5 h postinjection of haloperidol following pretreatment of L-type Ca(2+) channel blocker compared with rats pretreated with vehicle alone. In addition, pretreatment prolonged the duration of haloperidol-induced catalepsy in rats. Infusions of L-type Ca(2+) channel blockers directly into the neostriatum mimicked similar patterns of changes in haloperidol-induced c-Fos expression. Prolonged expression of c-Fos was not observed following coadministration of nifedipine and a dopamine D(1) (DA D(1)) receptor agonist, SKF 81297, but could be mimicked by the DA D(2/3) receptor antagonist raclopride, suggesting that the phenomenon is likely related to DA D(2) receptor antagonism. Moreover, the expression levels of haloperidol-induced zif 268 and haloperidol-induced phosphorylated CREB and phosphorylated Elk-1 were also substantially elevated for a prolonged period of time in the lateral, but not the medial part of the neostriatum, following blockade of L-type Ca(2+) channels. Collectively, the results suggest that coadministration of L-type Ca(2+) channel blockers affects haloperidol signaling in the lateral part of the neostriatum and may exacerbate the development of acute motor side effects.     

Expression and functional characterization of the cardiac L-type calcium channel carrying a skeletal muscle DHP-receptor mutation causing hypokalaemic periodic paralysis

A histidine substitution for the outermost arginine in II/S4 of the ₁ subunit of the human skeletal muscle dihydropyridine (DHP) receptor has been reported to cause hypokalaemic periodic paralysis (HypoPP). This mutation shifts the voltage dependence of L-type Ca current inactivation in myotubes from HypoPP patients by –40 mV without affecting activation. Based on the strong homology of II/S4 in cardiac and skeletal muscle ₁, we introduced the corresponding mutation into the rabbit cardiac ₁ subunit (R650H). Wild type (WT) and mutant constructs were transiently transfected in HEK cells together with and ₂ subunits and Ca and Ba currents were studied using the whole-cell patch-clamp technique. In contrast to the results obtained from human myotubes, R650H produced a small (–5 mV) but significant shift of both the steady-state activation and inactivation curves. When external pH was increased from 7.4 to 8.4 in order to favour deprotonization of H650, the only difference between WT and mutant channels was a slightly reduced steepness of the inactivation curve. Additional cotransfection of the subunit which is only found in skeletal but not in heart muscle, shifted the inactivation curves of both WT and R650H by –20 mV. We conclude that R650 plays a different role in voltage-dependent gating of the cardiac L-type Ca channel than the corresponding residue in the human skeletal muscle L-type channel, since a distinct and selective effect on the midpoint voltage of steady-state inactivation could not be found for R650H.     

Regional expression of L-type calcium channel subunits during cardiac development.

The contraction of cardiomyocytes is initiated by the entrance of extracellular calcium through specific calcium channels. Within the myocardium, L-type calcium channels are most abundant. In the heart, the main pore-forming subunit is the alpha1C, although there is a larger heterogeneity on auxiliary beta subunits. We have analyzed the distribution pattern of different alpha1C and beta subunits during cardiac development by immunohistochemistry. We observed homogeneous expression of alpha1C and beta subunits within the early tubular heart, whereas regional differences are observed during the late embryogenesis. beta2 and beta4 show differential expression within the embryonic myocardium. alpha1CD1 displays only a transient enhanced expression in the ventricular conduction system. In adult heart, the expression of the different calcium channel subunits analyzed is homogeneous along the entire myocardium except for alpha1CD1 that is practically undetectable. These findings suggest that beta subunits might play a major role in conferring calcium handling heterogeneity within the developing embryonic myocardium, while alpha1C subunits might contribute just transiently.     

氯化镧对下丘脑神经元L型钙通道的影响

目的和方法：采用膜片钳技术（ 细胞贴附式） 研究不同浓度氯化镧（ＬａＣｌ３） 对培养下丘脑神经元上Ｌ－ 型钙通道单通道电流的影响。结果：与加药前相比,１ μｍｏｌ／Ｌ 和１０ μｍｏｌ／Ｌ ＬａＣｌ３ 分别使Ｌ－ 型钙通道的开放下降７５ ％ （ Ｐ＜０－０１） ,开放时间缩短４４ ％ （ Ｐ＜ ０－０１） ,关闭时间延长５３ ％ （ Ｐ＜ ０－０１） ;而０－１ μｍｏｌ／ＬＬａＣｌ３ 分别使Ｌ－ 型Ｃａ２＋ 通道的开放概率增加３８ ％ （ Ｐ＜ ０－０５） ,开放时间延长４８ ％ （ Ｐ＜ ０－０１） ,关闭时间缩短２６ ％ （ Ｐ＜ ０－０５） 。结论：低浓度ＬａＣｌ３ 可兴奋培养下丘脑神经细胞Ｌ 型钙通道     

Inhibition of type A GABA receptors by L-type calcium channel blockers

Modulation of type A GABA receptors (GABAA) by L-type Ca++ channel blockers was investigated. The dihydropyridines nifedipine and nitrendipine, and the phenylalkylamine verapamil inhibited recombinant rat alpha1beta2gamma2 receptors recorded from human embryonic kidney (HEK) 293 cells; nifedipine at low concentrations also elicited modest stimulatory effects on GABA-gated current. The IC50 for GABA current inhibition was lowest for nitrendipine (17.3 +/- 1.3 microM), so subsequent studies were focused on further exploring its mechanism and possible site of action. When co-applied with GABA, nitrendipine had minimal effects on initial current amplitude, but significantly enhanced current decay rate. Nitrendipine-mediated inhibition was subunit-selective, as its IC50 was 10-fold lower in alpha1beta2 receptors. Nitrendipine's effect in recombinant human alpha1beta2gamma2 receptors was similar (IC50=23.0 +/- 1.3 microM) to that observed in rat receptors of the same configuration, indicating the site of action is conserved in the two species. The inhibitory effects were dependent on channel gating, were independent of transmembrane voltage, and were also observed in GABAA receptors recorded from hypothalamic brain slices. The pharmacologic mechanism of inhibition by nitrendipine was non-competitive, indicating it does not act at the GABA binding site. Nitrendipine block was retained in the presence of the benzodiazepine antagonist flumazenil, indicating it does not interact at the benzodiazepine site. The actions of nitrendipine were not affected by a mutation (beta2T246F) that confers resistance to the channel blocker picrotoxin, and they were not altered in the presence of the picrotoxin site antagonist alpha-isopropyl-alpha-methyl-gamma-butyrolactone, demonstrating nitrendipine does not act at the picrotoxin site of the GABAA receptor. Possible interaction of nitrendipine with the Zn++ site was also eliminated, as mutation of beta2 H267 to A, which confers resistance to Zn++, had no effect on nitrendipine-mediated inhibition. Our data suggest some of the central effects of dihydropyridines may be due to actions at GABAA receptors. Moreover, the effects may be mediated through interaction with a novel modulatory site on the GABAA receptor.     

心肌L型钙通道/电流及其在心肌肥大和心力衰竭时的变化

心肌细胞电活动的基础就是各种通道的离子流。有两类离子流左右心肌细胞的电活动 :一类是内向离子流 ,包括Ｉｎａ、Ｉｎａ-ｂ、Ｉｆ、Ｉｃａ-Ｌ、Ｉｃａ-Ｔ;另一类是外向离子流 ,包括Ｉｔｏ(Ｉｔｏ1 、Ｉｔｏ2 )、ＩＫ(ＩＫｒ、ＩＫｓ、Ｉｋｐ)、ＩＫ1 、ＩＫ -ＡＴＰ、ＩＫ -Ａｃｈ等。心肌细胞的钙通道 /电流属内向电流 ,可分为两大类 :(1 )Ｌ型Ｃａ2 通道 /电流(ＩＣａ-Ｌ) ,它在决定心肌细胞动作电位平台期的内向电流和启动心肌细胞兴奋 -收缩耦联都发挥极其重要的作用。 (2 )Ｔ型Ｃａ2 通道 /电流 (Ｉｃａ-Ｔ) ,它可能在心脏起搏细胞…     

L-type calcium channels: the low down

L-type calcium channels couple membrane depolarization in neurons to numerous processes including gene expression, synaptic efficacy, and cell survival. To establish the contribution of L-type calcium channels to various signaling cascades, investigators have relied on their unique pharmacological sensitivity to dihydropyridines. The traditional view of dihydropyridine-sensitive L-type calcium channels is that they are high-voltage-activating and have slow activation kinetics. These properties limit the involvement of L-type calcium channels to neuronal functions triggered by strong and sustained depolarizations. This review highlights literature, both long-standing and recent, that points to significant functional diversity among L-type calcium channels expressed in neurons and other excitable cells. Past literature contains several reports of low-voltage-activated neuronal L-type calcium channels that parallel the unique properties of recently cloned CaV1.3 L-type channels. The fast kinetics and low activation thresholds of CaV1.3 channels stand in stark contrast to criteria currently used to describe L-type calcium channels. A more accurate view of neuronal L-type calcium channels encompasses a broad range of activation thresholds and recognizes their potential contribution to signaling cascades triggered by subthreshold depolarizations.     

Evidence for a specific site for modulation of calcium channel activation by external calcium ions

We have investigated the effect of permeant ion species on activation of transiently expressed neuronal _1A Ca channels. Equimolar replacement of Ba with Ca resulted in a consistent depolarizing shift of the half-activation potential whose magnitude (10 mV) was constant over a range of 2 to 100 mM permeant ion, suggesting that the effects of Ca ions were fully developed at concentrations below 2 mM and indicating that Ba and Ca screened surface charges equally. In mixtures of Ba and Ca at constant divalent cation concentration the voltage-shift, as a function of Ca mole fraction, was well described by a model in which Ba and Ca compete for a single site but only Ca ions produce a gating effect. Overall, our data are consistent with Ca ions exerting their effects on activation via a specific regulatory site.     

Effect of L-type calcium channel antagonists on spermine-induced CNS excitation in vivo

The ability of nitrendipine, nisoldipine, verapamil and gabapentin to inhibit the development of CNS excitation induced by spermine was assessed in mice. Injection of an excitotoxic dose of spermine (100 μg, i.c.v.) in mice results in worsening tremor that culminates in the development of a fatal tonic convulsion within 8 h of spermine administration. The dihydropyridines, nitrendipine and nisoldipine, which are L-type calcium channel antagonists acting at the 1 subunit, inhibited the development of spermine-induced effects. Verapamil, which also acts at the 1 subunit of the L-type calcium channel, also inhibited the development of spermine-induced CNS excitation. Gabapentin, a postulated L-type calcium channel antagonist interacting at the 2δ subunit, did not inhibit the development of spermine-induced effects. These results show that antagonists of the 1 subunit of L-type calcium channels can effectively inhibit the effects of spermine in vivo. This may highlight the importance of L-type calcium channels in spermine action.     

Interleukin-6 inhibits L-type calcium channel activity of cultured cerebellar granule neurons

Our previous work has shown that interleukin-6 (IL-6) implements its neuroprotective effect by inhibiting the intracellular Ca²⁺ overload in neurons. Here, we examined whether regulation of L-type calcium channels (LCCs) activities is involved in the neuroprotective action of IL-6. In cultured cerebellar granule neurons (CGNs), patch-clamp recording showed that the whole-cell Ca²⁺ current and LCC current were significantly reduced by IL-6 pretreatment (120 ng/ml, for 24 h). Calcium imaging data indicated that IL-6 significantly suppressed high K⁺-induced intracellular Ca²⁺ overload and LCC Ca²⁺ influx. Moreover, expression of the LCC subunit, Ca_v1.2, was remarkably downregulated by IL-6 in cultured CGNs. These findings suggest that IL-6 exerts a neurotrophic effect by preventing Ca²⁺ overload, at least partly through inhibition of LCC activity in cultured CGNs.     

抗大鼠心室肌细胞L-型钙通道亚单位抗体的制备与验证

目的：用人工合成的大鼠心室肌细胞L-型钙通道4个亚单位的抗原表位短肽免疫家兔，产生可识别该通道不同亚单位的特异性抗体，并进行验证。方法：从大鼠心室肌细胞L-型钙通道4个亚单位的氨基酸序列中筛选出三段短肽，进行人工合成。用化学合成的多肽与破伤风类毒素（TT）以戊二醛法连接，并以此作为抗原免疫家兔制备抗大鼠心室肌细胞L-型钙通道的免疫血清，经分离纯化获得抗体。抗体的验证通过ELISA，Western blot，免疫荧光组织化学技术进行检测。结果：所制备的抗大鼠心室肌细胞L-型钙通道α1c、β2和α2/δ俗亚单位的抗体滴度分别为1：51200—1：102400、1：1280和1：1280，3个抗体结合到大鼠心肌细胞膜蛋白的分子量分别为235、89和162kD。免疫荧光组织化学实验显示，抗体均能特异性的结合在培养大鼠心室肌细胞膜上。结论：所制备的抗大鼠心室肌细胞L-型钙通道亚单位抗体能特异性识别大鼠心室肌细胞膜上L-型钙通道的不同亚单位。     

增龄犬左心房肌细胞动作电位和L型钙通道的变化

背景：增龄是心房颤动发生的独立危险因素,心房肌细胞电生理特性的变化是触发和维持心房颤动的重要因素。 目的：观察不同年龄犬左心房肌细胞动作电位和L型钙通道的改变。 方法：实验纳入7只成年犬和10只老年犬,用Ⅱ型胶原酶分离成年犬和老年犬的左心房肌细胞,用全细胞膜片钳方法记录成年犬和老年犬左心房肌细胞动作电位和L型钙通道电流;采用实时荧光定量PCR和Western blot检测左心房肌L型钙通道α1c亚基mRNA和蛋白的表达。 结果与结论：与成年犬相比,老年犬左心房肌细胞动作电位平台期的幅度明显降低(P < 0.05),动作电位时程明显延长(P < 0.05),而最大舒张电位和动作电位幅度在老年犬和成年犬间差异无显著性意义(P > 0.05);同时老年犬左心房肌细胞的L型钙通道电流密度较成年犬明显降低(P < 0.05),而L型钙通道电流动力学参数在成年犬和老年犬间差异无显著性意义(P > 0.05);此外,与成年犬相比,老年犬左心房肌L型钙通道α1c亚基mRNA和蛋白的表达明显降低(P < 0.05)。由此认为,左心房肌细胞电生理学特性存在增龄性改变。     

乙醇对豚鼠单个心房肌细胞L-型钙通道的影响

目的： 采用膜片钳全细胞记录方法，观察乙醇对分离的豚鼠单个心房肌细胞L-型钙电流（ICa.L）的影响。方法： 使用酶解方法获得豚鼠单个心房肌细胞，采用全细胞膜片钳记录技术观察不同浓度乙醇急性干预对ICa.L电流-电压曲线、激活曲线、失活曲线的影响。结果： 急性乙醇干预下，ICa.L的I-V曲线发生了变化，而失活曲线未发生变化，激活曲线仅在80mmol/L的时候发生了变化。在乙醇浓度≤45 mmol/L时可抑制ICa.L，而在≥50 mmol/L时可明显增大ICa.L，在45 mmol/L-50 mmol/L之间这两种作用发生了突然的转换。结论： 乙醇对于ICa.L的影响作用基本为非电压依赖性，但具有浓度依赖性，不同浓度的乙醇可对ICa.L产生不同甚至相反的作用，这有助于解释房颤和房扑之间的关系。