连接蛋白43磷酸化状态与心脏缝隙连接通道功能的关系

缝隙连接是介导相邻细胞间直接通讯的特殊膜结构。心室肌细胞间的缝隙连接通道主要由连接蛋白43(Cx43)构成。Cx43的磷酸化状态除了快速调节通道的开放/闭合状态(单通道传导性和通道开放概率),还可通过影响Cx43的合成、转运、聚集/解聚和降解等不同环节,改变活化通道数量,最终实现对缝隙连接功能的调控。迄今,已发现多种激酶和蛋白磷酸酶可直接和(或)间接调控Cx43羧基末端的丝氨酸残基和酪氨酸残基的磷酸化状态,从而影响缝隙连接通道的功能。磷酸化/去磷酸化影响Cx43和缝隙连接通道功能的确切作用和具体机制未明。本文就Cx43磷酸化状态与心脏缝隙连接通道功能的关系作一综述。     

连接蛋白43磷酸化状态与心脏缝隙连接通道功能的关系

缝隙连接是介导相邻细胞间直接通讯的特殊膜结构.心室肌细胞间的缝隙连接通道主要由连接蛋白43(Cx43)构成.Cx43的磷酸化状态除了快速调节通道的开放/闭合状态(单通道传导性和通道开放概率),还可通过影响Cx43的合成、转运、聚集/解聚和降解等不同环节,改变活化通道数量,最终实现对缝隙连接功能的调控.迄今,已发现多种激酶和蛋白磷酸酶可直接和(或)间接调控Cx43羧基末端的丝氨酸残基和酪氨酸残基的磷酸化状态,从而影响缝隙连接通道的功能.磷酸化/去磷酸化影响Cx43和缝隙连接通道功能的确切作用和具体机制未明.本文就Cx43磷酸化状态与心脏缝隙连接通道功能的关系作一综述.     

磷酸化IKK和磷酸化IκBα在糖尿病大鼠心肌组织中的表达

雄性Wistar大鼠腹腔注射STZ以建立糖尿病模型成膜后4周、8周、12周测量大鼠心肌细胞面积并检测磷酸化IKK和磷酸化IκBα在糖尿病大鼠心肌组织中的表达水平等。结果：各时间点磷酸化IKK和磷酸化IκBα比正常对照组大鼠显著增2D（P〈0．01）。结论：磷酸化IKK和磷酸化IκBα伴随着心脏病理学改变而表达增加，可能在糖尿病心肌病中发挥重要作用。     

磷酸化IKK和磷酸化IκBα在糖尿病大鼠心肌组织中的表达

雄性Wistar大鼠腹腔注射STZ以建立糖尿病模型成膜后4周、8周、12周测量大鼠心肌细胞面积并检测磷酸化IKK和磷酸化IκBα在糖尿病大鼠心肌组织中的表达水平等。结果：各时间点磷酸化IKK和磷酸化IκBα比正常对照组大鼠显著增2D（P〈0．01）。结论：磷酸化IKK和磷酸化IκBα伴随着心脏病理学改变而表达增加，可能在糖尿病心肌病中发挥重要作用。     

磷酸化对HERG钾通道的调控作用

HERG(human ether-a-go-go-related gene)编码的钾通道介导快速激活延迟整流钾电流(IKr).IKr在心肌动作电位复极中发挥极其关键的作用,IKr的减弱可延长心肌动作电位时程,导致长QT综合征(LQTS).HERG突变和HERG钾通道的药物性阻滞是LQTS的常见原因之一.心脏HERG钾通道功能受众多因素的调控,该通道的磷酸化是重要的调控因素之一.现有报道证实HERG钾通道的调节由蛋白激酶(protein kinases A、C和protein tyrosine kinases Src等)介导完成.     

磷酸化对HERG钾通道的调控作用

HERG（human ether—a—go—go—related gene）编码的钾通道介导快速激活延迟整流钾电流（IKr）。IKr在心肌动作电位复极中发挥极其关键的作用,IKr的减弱可延长心肌动作电位时程,导致长QT综合征（LQTS）。HERG突变和HERG钾通道的药物性阻滞是LQTS的常见原因之一。心脏HERG钾通道功能受众多因素的调控,该通道的磷酸化是重要的调控因素之一。现有报道证实HERG钾通道的调节由蛋白激酶（protein kinasesA、C和protein tyrosine kinases Src等）介导完成。     

缝隙连接蛋白Cx43的磷酸化对缝隙连接通讯的调控

缝隙连接介导细胞间通讯。磷酸化通过影响缝隙连接蛋白生命周期中的各个过程而调节细胞间通讯,这些过程涉及到缝隙连接蛋白的转运、组装/解离、降解以及通道的门控等。本文将探讨缝隙连接蛋白Cx43的磷酸化对缝隙连接通讯的调控,继而讨论Cx43蛋白的磷酸化特异性抗体,有助于我们深入研究特定位点的磷酸化事件对缝隙连接通道功能的影响。     

cAMP反应元件结合蛋白磷酸化与肝糖输出调控基因表达关系的研究

目的 观察CREB磷酸化对肝糖输出调控基因表达的影响.方法 利用cAMP激动剂forskolin处理原代培养的大鼠肝脏细胞,以Western blot法检测肝脏细胞处理前后CREB和磷酸化CREB蛋白的表达变化,以RT-PCR方法检测肝糖输出调控基因PGC-1α、磷酸烯醇式丙酮酸羧激酶(PEPCK)和葡萄糖6磷酸酶(G6Pase)mRNA的表达.结果 以cAMP激动剂Forskolin作用的肝细胞CREB蛋白的磷酸化水平明显增高,肝糖输出调控基因PGC-1α、PEPCK、G6Pase mRNA的表达也增高.结论 CREB磷酸化后可以调控糖异生关键酶的表达.     

心脏肌钙蛋白I的磷酸化调控与心衰

心脏肌钙蛋白I(cTnI)是心肌收缩/舒张的关键性调控蛋白.多种激酶调控的cTnI磷酸化通过改变心肌纤维性状影响心脏功能.生理条件下,PKA依赖的cTnI Ser23/24磷酸化与PKC依赖的Ser43/45磷酸化相互拮抗,相互制衡,共同调控心脏的功能.心衰时,动态平衡被打破,cTnI出现磷酸化紊乱,Ser23/24"...     

2型猪链球菌二元信号系统孤儿调控因子CovR磷酸化位点鉴定

目的 利用Escherichia coli BL21原核表达系统共表达2型猪链球菌丝氨酸/苏氨酸激酶STK与孤儿调控因子CovR蛋白,通过质谱鉴定CovR蛋白磷酸化位点,探究STK对CovR的磷酸化作用。方法 构建CovR蛋白表达载体pCDFDuet-1::covR及pCDFDuet-1::covR/stk,重组表达蛋白分别命名为CovR1与CovR2。通过蛋白磷酸化质谱技术检验CovR2蛋白的关键磷酸化位点。分别构建CovR苏氨酸突变体CovRT、丝氨酸突变体CovRS、酪氨酸突变体CovRY及丝/苏/酪氨酸全突变体CovRA,与STK共表达后分析其磷酸化状态,确定CovR磷酸化靶点。结果 在E. coli BL21表达系统中成功表达并纯化CovR1与CovR2蛋白,Western blot分析发现CovR1无磷酸化信号,CovR2可以被磷酸化。进一步对CovR2磷酸化质谱检测发现其第45、148、150、159、168、194、219位苏氨酸,第40、172、215位丝氨酸以及第225位酪氨酸为关键磷酸化位点。对CovRT、CovRS、CovRY及CovRA突变体磷酸化状态检测,结果表明CovR2的丝/苏/酪氨酸均可发生磷酸化。结论 在E. coli BL21中共表达STK与CovR可导致CovR丝/苏/酪氨酸磷酸化,通过质谱技术鉴定了CovR的磷酸化位点,提示CovR可能是STK的磷酸化调控靶点。     

Toward a Molecular Understanding of Voltage-Gated Potassium Channels

Voltage-Gated Potassium Channels . Many different types of potassium (K⁺) channels exist and they play a central role in the fine tuning of excitability properties. Of the distinct subpopulations of K⁺ channels expressed in different cells, voltage-gated K⁺ channels have been studied most thoroughly at a molecular level. Over the last few years, the joint application of recombinant DNA technology together with electrophysiology, such as the voltage clamp and the patch clamp techniques, has produced a wealth of information. We have begun to unravel the genetic basis of ion channel diversity. In particular, the Xenopus oocyte expression system has turned out to be of enormous experimental value. Oocytes microinjected with “cloned” mRNA have been used to gain insight into biophysical and pharmacologic properties of voltage-gated K⁺, Na⁺, and Ca²⁺ channels. Here, we will review our understanding of K⁺ channel diversity based upon the fact that ion channels are encoded as a large multigene family. We have caught a first glimpse at possible molecular mechanisms underlying several biophysical properties characteristic for voltage-gated ion channels: voltage dependence of activation and inactivation, and ion permeation and selectivity. We will discuss molecular mechanisms of K⁺ channel activation and inactivation. We will also describe experiments that led to the identification of the “pore region,” and we will present a model of a potassium selective ion channel pore.     

Ethanol Actions on Multiple Ion Channels: Which Are Important?

BACKGROUND: This review is based on a plenary lecture presented at the 1999 meeting of the Research Society on Alcoholism. It provides an overview of the search for sites of action for ethanol in the brain. Initial studies were directed at interaction of ethanol with membrane lipids, but during the past decade, emphasis has been shifted to protein sites, particularly those on ion channels. Molecular biological techniques have provided the opportunity to study isolated channels in cellular expression systems and also provide the opportunity to manipulate these channels in mutant mice. CONCLUSIONS: There is now compelling evidence that multiple ion channels are affected by ethanol and growing support for the idea that ethanol interacts directly with specific sites on ion channels. The key, and unanswered, question is which of these channels are responsible for alcohol-induced behaviors such as intoxication, tolerance, dependence, or craving. Mutant mice will likely give (some) answers to these questions during the next decade.     

Calcium-activated potassium channels, cardiogenesis of pluripotent stem cells, and enrichment of pacemaker-like cells

Potassium channels represent the largest group of pore proteins regulating K(+)efflux from the K(+)-rich inner cell to the extracellular compartment, thereby inducing changes in the membrane potential. Activity is regulated either by voltage or calcium concentrations, thus the nomenclature of voltage- and calcium-activated potassium channels. The critical role of potassium ion channels in developmental processes remains enigmatic, although it is well accepted that cell differentiation and maturation affect the expression patterns of certain ion channels. Recently, a series of studies delineated the precise function of calcium-activated potassium channels during cardiac, particularly pacemaker, cell development using human and mouse pluripotent stem cell models. It has become evident that this protein family not only regulates proliferation, apoptosis, and cell metabolism but also drives critical events during organ development such as the heart. This review summarizes the literature on calcium-activated potassium channels, their role in cardiac stem cell differentiation and development, and provides an outlook on how this process could be mechanistically regulated.     

The molecular basis of cardiac arrhythmias in patients with cardiomyopathy

Cardiac arrhythmias are a leading cause of mortality and morbidity in Western society. In some specific instances, these arrhythmias are caused by abnormalities of cardiac ion channels, such as sodium, calcium, and potassium channels, which carry ionic currents and are fundamental determinants of cardiac excitability. Abnormalities of these ion channels are attributed to mutations in the genes encoding the channel protein and cause altered function of channels, which can predispose to arrhythmias. During heart failure, many channels also malfunction because of altered expression, resulting in lethal arrhythmias.     

Ion channels

Insulin secretion by the pancreatic Beta cell is dependent upon transmembrane ion fluxes gated by the ATP-regulated potassium channel and the voltage regulated, L-type calcium channel. This work group examined major recent advances in the structure and modulation of ion channels and how those advances may pertain to the physiology of insulin secretion and the pharmacological treatment of Type 2 (non-insulin-dependent) diabetes mellitus. Structural studies have revealed that voltage gated ion channels are related, complex, and comprised of multiple components: sodium channels consist of three distinct subunits. L-type calcium channels, crucial to the insulin secretory response are structurally related to the sodium channel but contain additional subunits. Potassium channels are less closely related and appear to function as homotetramers. Modulation of ion channel activity is similarly complex: site specific phosphorylation by multiple protein kinases under the control of several intracellular second messenger systems may increase or decrease conductance. Subunit composition and relatively stable changes in the modal state of ion channels also appear to be critical to ion channel gating properties. Functional studies of the Beta-cell ATP-regulated potassium channel suggest two distinct nucleotide binding sites which link this channel to the metabolic state of the Beta cell. The multiple paths of ion channel modulation provide multiple targets for therapeutic intervention. Where detailed characterisation of ion channel structure has been achieved, those targets are being used for specific drug design. Such complete characterisation has not yet been achieved for Beta-cell ion channels and this presents a major goal for diabetes research.     

Ethanol interactions with calcium-dependent potassium channels

In most neurons and other excitable cells, calcium-activated potassium channels of small (SK) and large conductance (BK; MaxiK) control excitability and neurotransmitter release. The spontaneous activity of dopamine neurons of the ventral tegmental area is increased by ethanol. This ethanol excitation is potentiated by selective blockade of SK, indicating that SK channels modulate ethanol stimulation of neurons that are critical in reward and reinforcement. On the other hand, ethanol directly modulates BK channel activity in a variety of systems, including rat neurohypophysial nerve endings, primary sensory dorsal root ganglia, nucleus accumbens neurons, Caenorhabditis elegans type-IV dopaminergic CEP neurons, and nonneuronal preparations, such as rat pituitary cells, cerebrovascular myocytes and human umbilical vein endothelial cells. Ethanol action on BK channels can modify neuropeptide and growth hormone release, nociception, cerebrovascular tone, and endothelial proliferation. Ethanol modulates BK channels even when the drug is evaluated using recombinant BK channel-forming alpha (slo) subunits or channel reconstitution in artificial, binary lipid bilayers, indicating that the slo subunit and its immediate lipid microenvironment are the essential targets of ethanol. Consistent with this, single amino acid slo channel mutants display altered ethanol sensitivity. Furthermore, C. elegans slo1 null mutants are resistant to ethanol-induced motor incoordination. On the other hand, Drosophila melanogaster slo null mutants fail to acquire acute tolerance to ethanol sedation. Ethanol action on slo channels, however, may be tuned by a variety of factors, including posttranslational modification of slo subunits, coexpression of channel accessory subunits, and the lipid microenvironment, resulting in increase, refractoriness, or even decrease in channel activity. In brief, both SK and BK channels are important targets of ethanol throughout the body, and interference with ethanol effects on these channels could form the basis for novel pharmacotherapies to ameliorate the actions or consequences of alcohol abuse.     

Controlling potassium channel activities: Interplay between the membrane and intracellular factors

Neural signaling is based on the regulated timing and extent of channel opening; therefore, it is important to understand how ion channels open and close in response to neurotransmitters and intracellular messengers. Here, we examine this question for potassium channels, an extraordinarily diverse group of ion channels. Voltage-gated potassium (Kv) channels control action-potential waveforms and neuronal firing patterns by opening and closing in response to membrane-potential changes. These effects can be strongly modulated by cytoplasmic factors such as kinases, phosphatases, and small GTPases. A Kv alpha subunit contains six transmembrane segments, including an intrinsic voltage sensor. In contrast, inwardly rectifying potassium (Kir) channels have just two transmembrane segments in each of its four pore-lining alpha subunits. A variety of intracellular second messengers mediate transmitter and metabolic regulation of Kir channels. For example, Kir3 (GIRK) channels open on binding to the G protein betagamma subunits, thereby mediating slow inhibitory postsynaptic potentials in the brain. Our structure-based functional analysis on the cytoplasmic N-terminal tetramerization domain T1 of the voltage-gated channel, Kv1.2, uncovered a new function for this domain, modulation of voltage gating, and suggested a possible means of communication between second messenger pathways and Kv channels. A yeast screen for active Kir3.2 channels subjected to random mutagenesis has identified residues in the transmembrane segments that are crucial for controlling the opening of Kir3.2 channels. The identification of structural elements involved in potassium channel gating in these systems highlights principles that may be important in the regulation of other types of channels.     

Small potassium ion channel proteins encoded by chlorella viruses

Kcv, a 94-aa protein encoded by Paramecium bursaria chlorella virus 1, is the smallest known protein to form a functional potassium ion channel and basically corresponds to the "pore module" of potassium channels. Both viral replication and channel activity are inhibited by the ion channel blockers barium and amantadine but not by cesium. Genes encoding Kcv-like proteins were isolated from 40 additional chlorella viruses. Differences in 16 of the 94 amino acids were detected, producing six Kcv-like proteins with amino acid substitutions occurring in most of the functional domains of the protein (N terminus, transmembrane 1, pore helix, selectivity filter, and transmembrane 2). The six proteins form functional potassium selective channels in Xenopus oocytes with different properties including altered current kinetics and inhibition by cesium. The amino acid changes together with the different properties observed in the six Kcv-like channels will be used to guide site-directed mutations, either singularly or in combination, to identify key amino acids that confer specific properties to Kcv.     

Deletion of the Ca2+-activated potassium (BK) alpha-subunit but not the BKbeta1-subunit leads to progressive hearing loss

The large conductance voltage- and Ca2+-activated potassium (BK) channel has been suggested to play an important role in the signal transduction process of cochlear inner hair cells. BK channels have been shown to be composed of the pore-forming alpha-subunit coexpressed with the auxiliary beta1-subunit. Analyzing the hearing function and cochlear phenotype of BK channel alpha-(BKalpha-/-) and beta1-subunit (BKbeta1-/-) knockout mice, we demonstrate normal hearing function and cochlear structure of BKbeta1-/- mice. During the first 4 postnatal weeks also, BKalpha-/- mice most surprisingly did not show any obvious hearing deficits. High-frequency hearing loss developed in BKalpha-/- mice only from approximately 8 weeks postnatally onward and was accompanied by a lack of distortion product otoacoustic emissions, suggesting outer hair cell (OHC) dysfunction. Hearing loss was linked to a loss of the KCNQ4 potassium channel in membranes of OHCs in the basal and midbasal cochlear turn, preceding hair cell degeneration and leading to a similar phenotype as elicited by pharmacologic blockade of KCNQ4 channels. Although the actual link between BK gene deletion, loss of KCNQ4 in OHCs, and OHC degeneration requires further investigation, data already suggest human BK-coding slo1 gene mutation as a susceptibility factor for progressive deafness, similar to KCNQ4 potassium channel mutations.     

Voltage-dependent sodium and potassium channels in mammalian cultured Schwann cells.

Cultured Schwann cells from sciatic nerves of newborn rabbits and rats have been examined with patch-clamp techniques. In rabbit cells, single sodium and potassium channels have been detected with single channel conductances of 20 pS and 19 pS, respectively. Single sodium channels have a reversal potential within 15 mV of ENa, are blocked by tetrodotoxin, and have rapid and voltage-independent inactivation kinetics. Single potassium channels show current reversal close to EK and are blocked by 4-aminopyridine. From these results, and from comparisons of single-channel and whole-cell data, we show that these Schwann cells contain voltage-dependent sodium and potassium channels that are similar in most respects to the corresponding channels in mammalian axonal membranes. Cultured rat Schwann cells also have sodium channels, but at a density about 1/10th that of rabbit cells, a result in agreement with saxitoxin binding experiments on axon-free sectioned nerves. Saxitoxin binding to cultured cells suggests that there are up to 25,000 sodium channels in a single rabbit Schwann cell. We speculate that in vivo Schwann cells in myelinated axons might act as a local source for sodium channels at the nodal axolemma.