上皮细胞K+通道的生理学意义与临床应用:跨上皮细胞转运的驱动力生成和K+循环

K^＋通道在上皮细胞内以极化的方式表达,形成一个庞大的膜蛋白家族。出于对主要依赖Na^＋-K^＋-ATPase而维持的细胞内跨膜K^＋梯度的考虑,K^＋通道在跨上皮细胞转运中的主要作用为：膜电位生成和K^＋循环。本文以肾近端小管和胃壁上皮细胞转运为例简要阐述了K^＋通道的作用。在这两个组织中,K^＋通道活性限速跨上皮细胞转运,调节细胞体积。近年来,药理学工具和转基因动物的实验证实了对K^＋通道的原先认知,并将研究深入到分子水平。K^＋通道的分子结构挑战高亲和力药物分子的设计,及其多组织同时表达的两个典型特征阻碍了高活性、组织特异性小分子治疗的进展。然而,抑制K^＋通道能阻断胃酸分泌等病理生理机制的深入研究,促进K^＋通道药物用于胃病治疗和作为肾脏转运抑制剂用于肾脏相关疾病治疗。     

Enhanced Orai1-mediated store-operated Ca2+ channel/calpain signaling contributes to high glucose-induced podocyte injury

Podocyte injury induced by hyperglycemia is the main cause of kidney dysfunction in diabetic nephropathy. However, the underlying mechanism is unclear. Store-operated Ca²⁺ entry (SOCE) regulates a diversity of cellular processes in a variety of cell types. Calpain, a Ca²⁺-dependent cysteine protease, was recently shown to be involved in podocyte injury. In the present study, we sought to determine whether increased SOCE contributed to high glucose (HG)–induced podocyte injury through activation of the calpain pathway. In cultured human podocytes, whole-cell patch clamp indicated the presence of functional store-operated Ca²⁺ channels, which are composed of Orai1 proteins and mediate SOCE. Western blots showed that HG treatment increased the protein abundance of Orai1 in a dose-dependent manner. Consistently, calcium imaging experiments revealed that SOCE was significantly enhanced in podocytes following HG treatment. Furthermore, HG treatment caused overt podocyte F-actin disorganization as well as a significant decrease in nephrin protein abundance, both of which are indications of podocyte injury. These podocyte injury responses were significantly blunted by both pharmacological inhibition of Orai1 using the small molecule inhibitor BTP2 or by genetic deletion of Orai1 using CRISPR-Cas9 lentivirus. Moreover, activation of SOCE by thapsigargin, an inhibitor of Ca²⁺ pump on the endoplasmic/sarcoplasmic reticulum membrane, significantly increased the activity of calpain, which was inhibited by BTP2. Finally, the calpain-1/calpain-2 inhibitor calpeptin significantly blunted the nephrin protein reduction induced by HG treatment. Taken together, our results suggest that enhanced signaling via an Orai1/SOCE/Calpain axis contributes to HG-induced podocyte injury.     

Subcellular dynamics and functional activity of the cleaved intracellular domain of the Na+ channel β1 subunit

The voltage-gated Na⁺ channel β1 subunit, encoded by SCN1B, regulates cell surface expression and gating of α subunits and participates in cell adhesion. β1 is cleaved by α/β and γ-secretases, releasing an extracellular domain and intracellular domain (ICD), respectively. Abnormal SCN1B expression/function is linked to pathologies including epilepsy, cardiac arrhythmia, and cancer. In this study, we sought to determine the effect of secretase cleavage on β1 function in breast cancer cells. Using a series of GFP-tagged β1 constructs, we show that β1-GFP is mainly retained intracellularly, particularly in the endoplasmic reticulum and endolysosomal pathway, and accumulates in the nucleus. Reduction in endosomal β1-GFP levels occurred following γ-secretase inhibition, implicating endosomes and/or the preceding plasma membrane as important sites for secretase processing. Using live-cell imaging, we also report β1ICD-GFP accumulation in the nucleus. Furthermore, β1-GFP and β1ICD-GFP both increased Na⁺ current, whereas β1STOP-GFP, which lacks the ICD, did not, thus highlighting that the β1-ICD is necessary and sufficient to increase Na⁺ current measured at the plasma membrane. Importantly, although the endogenous Na⁺ current expressed in MDA-MB-231 cells is tetrodotoxin (TTX)-resistant (carried by Na_v1.5), the Na⁺ current increased by β1-GFP or β1ICD-GFP was TTX-sensitive. Finally, we found β1-GFP increased mRNA levels of the TTX-sensitive α subunits SCN1A/Na_v1.1 and SCN9A/Na_v1.7. Taken together, this work suggests that the β1-ICD is a critical regulator of α subunit function in cancer cells. Our data further highlight that γ-secretase may play a key role in regulating β1 function in breast cancer.     

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。     

河蟹眼柄神经分泌细胞离子通道的膜片钳研究

采用全细胞膜片钳技术对培养12－24小时不同形态河蟹眼柄视节端髓X器官（MTXO）神经分泌细胞离子通道进行了研究。结果表明,河蟹眼柄MTXO中分布的A、B、C三种类型神经分泌细胞均可记录到由向电流和外向电流组成的正常全细胞电流。内向电流由高电压激活钙离子通道电流（Lca)和对TTX敏感钠离子通道电流（INa）组成。ICa的激活电压为－30mV,在0－ 20mV电压下达到峰值,在－40mV和－70mV保持电压下记录的ICa激活阈值、初始峰值及I－V曲线无明显差别。外向电流明显,幅值较大,包括对4－AP敏感的快速激活、快速失活钾离子通道电流（IA）和对TEA敏感的缓慢激活、缓慢失活钾离子通道电流（IK）。正常蟹种、二龄成蟹和早熟蟹种MTXO神经分泌细胞均表达电压门控钠、钾、钙离子通道,通道电流和电压特征无明显区别.     

A novel homozygous 10 nucleotide deletion in BBS10 causes Bardet–Biedl syndrome in a Pakistani family

Bardet–Biedl Syndrome is a multisystem autosomal recessive disorder characterized by central obesity, polydactyly, hypogonadism, learning difficulties, rod-cone dystrophy and renal dysplasia. Bardet–Biedl Syndrome has a prevalence rate ranging from 1 in 100,000 to 1 in 160,000 births although there are communities where Bardet–Biedl Syndrome is found at a higher frequency due to consanguinity. We report here a Pakistani consanguineous family with two affected sons with typical clinical features of Bardet–Biedl Syndrome, in addition to abnormal liver functioning and bilateral basal ganglia calcification, the latter feature being typical of Fahr's disease. Homozygous regions obtained from SNP array depicted three known genes BBS10, BBS14 and BBS2. Bidirectional sequencing of all coding exons by traditional sequencing of all these three genes showed a homozygous deletion of 10 nucleotides (c.1958_1967del), in BBS10 in both affected brothers. The segregation analysis revealed that the parents, paternal grandfather, maternal grandmother and an unaffected sister were heterozygous for the deletion. Such a large deletion in BBS10 has not been reported previously in any population and is likely to be contributing to the phenotype of Bardet–Biedl Syndrome in this family.     

海马脑片盲法膜片钳全细胞记录技术

本文较为详细地介绍了海马脑片盲法膜片钳全细胞记录技术,对其关键步骤和需要注意的问题进行了重点说明,同时对CA1区锥体神经元突触活动的特点,电压门控性Ca^2 通道以及谷氨酸（glutamate,Glu)γ－氨基丁酸（GABA）受体通道电流性质等进行了观察和分析,实验结果为采用海马脑片盲法膜片钳全细胞记录技术研究海马神经元离子通道动力学性质和中枢神经系统药物对突触活动的影响提供了可靠的依据。     

芦苇型湿地生态系统的潜水水质状态研究

以白洋淀芦苇滩地为实验地,研究了湿地生态系统中潜水的水质状况.苇地潜水运动受到芦苇根孔的强烈影响.受芦苇根区生物呼吸作用的影响,潜水pH值(6.94士0.14)显著低于淀水(9.17).潜水中CO2分压约为淀水的85～1039倍.潜水水质类型在离淀2.7至6.7m发生变化,逐渐由ClN3转变为CCa.自苇地边缘至中央,HT(总硬度),Ar(总碱度)与∑C(主要离子总量)逐渐升高.潜水中Mg2+/Ca2+的平均摩尔比值为0.60,明显低于淀水(1.31).淀水和潜水Cl-,SO2-,Na++K+含量无显著差异,平均值分别是176,116和112mg/L.苇地濒水区为一过渡带,对磷氮污染物具备首先的过滤作用.在离淀0.7m处TN,TP和活性磷酸盐的减少量分别为63.3%,84.6%,80.8%.     

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.