Ionic channels,ion transport and plant cell membranes: Potential applications of the patch-clamp technique

Conclusion Exciting innovations in the methodologies available for the study of ionic channels (notably in animal cells) have allowed hitherto impossible advances in the comprehension of both structure and function. In using channels like the Na channel and the AChR as examples of these strategies, we have tried to give a concise but up to date account of the current possibilities (in particular, the patch-clamp) for research in membrane physiology. That few of these techniques have been applied to plant cell membranes simply indicates the scope for advancement in the understanding of some problems fundamental to plant physiology. The mechanisms of transport involved in processes known to be important for the life of plant cells (e.g., regulation of cytoplasmic and vacuolar potential differences and pH, maintenance of vacuolar turgor pressure, accumulation of metabolites and their counterions, response to environmental stimuli) are relatively speaking, poorly characterized. In that ion fluxes through plasmalemma and tonoplast membranes are at least in part likely to be via ionic channels for all of these processes, an important step forward would be the application of patch-clamp techniques for the direct demonstration of a channel mechanism and the subsequent elucidation of their role.     

Mitotic inheritance of the Golgi complex and its role in cell division

The Golgi apparatus plays essential roles in the processing and sorting of proteins and lipids, but it can also act as a signalling hub and a microtubule‐nucleation centre. The Golgi complex (GC) of mammalian cells is composed of stacks connected by tubular bridges to form a continuous membranous system. In spite of this structural complexity, the GC is highly dynamic, and this feature becomes particularly evident during mitosis, when the GC undergoes a multi‐step disassembly process that allows its correct partitioning and inheritance by daughter cells. Strikingly, different steps of Golgi disassembly control mitotic entry and progression, indicating that cells actively monitor Golgi integrity during cell division. Here, we summarise the basic mechanisms and the molecular players that are involved in Golgi disassembly, focussing in particular on recent studies that have revealed the fundamental signalling pathways that connect Golgi inheritance to mitotic entry and progression.     

Interdependence of cell cycle events inSchizosaccharomyces pombe. Terminal phenotypes of cell division cycle mutants arrested during dna synthesis and nuclear division

Summary Temperature-sensitive cell division cycle (cdc) mutants of the fission yeastSchizosaccharomyces pombe, previously characterized as defective in nuclear division were examined by thin section electron microscopy. All of the mutants failed to enter mitosis, rather they accumulated at one of four distinct terminal phenotypes. Class one were arrested with a nucleus rectangular in cross-section and a laterally situated spindle pole body (SPB). The second group had spherical or rectangular nuclei with a single SPB. The sole member of the third group wascdc 27. K 3, which had a spherical crenated nucleus with a single SPB from which microtubules emerged and extended into the cytoplasm. Allelic variants ofcdc 25 comprised the fourth group all of which displayed aberrant nuclear morphologies. Utilizing this ultrastructural data together with a knowledge of the transition points of these mutants a model for the interdependence of certain cell cycle event is proposed in which the initiation of DNA synthesis is uncoupled from the replication and separation of the SPB. This paper also provides new information on SPB structure inS. pombe. This is discussed in connection with the transient assembly of both spindle and cytoplasmic microtubules.     

Functional ion channels in stem cells

Bioelectrical signals generated by ion channels play crucial roles in excitation genesis and impulse conduction in excitable cells as well as in cell proliferation,migration and apoptosis in proliferative cells.Recent studies have demonstrated that multiple ion channels are heterogeneously present in different stem cells;however,patterns and phenotypes of ion channels are species-and/or origin-dependent.This editorial review focuses on the recent findings related to the expression of functional ion channels and the roles of these channels in regulation of cell proliferation in stem cells.Additional effort is required in the future to clarify the ion channel expression in different types of stem cells;special attention should be paid to the relationship between ion channels and stem cell proliferation,migration and differentiation.     

Pharmacological modulation of ion channels and transporters

Ion channels and transporter proteins are prerequisites for formation and conduction of cardiac electrical impulses. Acting in concert, these proteins maintain cellular Na(+) and Ca(2+) homeostasis. Since intracellular Ca(2+) concentration determines contractile activation, we expect the majority of agents that modulate activity of ion channels and transporters not only to influence cellular action potentials but also contractile force. Drugs which block ion channels usually possess antiarrhythmic properties, those inhibiting the Na(+) pump have predominantly inotropic effects and those affecting Na(+),Ca(2+)- or Na(+),H(+)-exchanger protect against ischaemic cell damage. However, irrespective of their primary indication, all compounds targeted against ion channels and transporter proteins possess potential proarrhythmic activity.     

Differential regulation of ion channels function by proteolysis

Ion channels are pore-forming protein complexes in membranes that play essential roles in a diverse array of biological activities. Ion channel activity is strictly regulated at multiple levels and by numerous cellular events to selectively activate downstream effectors involved in specific biological activities. For example, ions, binding proteins, nucleotides, phosphorylation, the redox state, channel subunit composition have all been shown to regulate channel activity and subsequently allow channels to participate in distinct cellular events. While these forms of modulation are well documented and have been extensively reviewed, in this article, we will first review and summarize channel proteolysis as a novel and quite widespread mechanism for altering channel activity. We will then highlight the recent findings demonstrating that proteolysis profoundly alters Inositol 1,4,5 trisphosphate receptor activity, and then discuss its potential functional ramifications in various developmental and pathological conditions.     

Cell proliferation, calcium influx and calcium channels

Both increases in the basal cytosolic calcium concentration ([Ca²⁺]_cyt) and [Ca²⁺]_cyt transients play major roles in cell cycle progression, cell proliferation and division. Calcium transients are observed at various stages of cell cycle and more specifically during late G₁ phase, before and during mitosis. These calcium transients are mainly due to calcium release and reuptake by the endoplasmic reticulum (ER) and are observed over periods of hours in oocytes and mammalian cells. Calcium entry sustains the ER Ca²⁺ load and thereby helps to maintain these calcium transients for such a long period. Calcium influx also controls cell growth and proliferation in several cell types. Various calcium channels are involved in this process and the tight relation between the expression and activity of cyclins and calcium channels also suggests that calcium entry may be needed only at particular stages of the cell cycle. Consistent with this idea, the expression of l-type and T-type calcium channels and SOCE amplitude fluctuate along the cell cycle. But, as calcium influx regulates several other transduction pathways, the presence of a specific connection to trigger activation of proliferation and cell division in mammalian cells will be discussed in this review.     

Evolutionary origins of mechanosensitive ion channels

According to the recent revision, the universal phylogenetic tree is composed of three domains: Eukarya (eukaryotes), Bacteria (eubacteria) and Archaea (archaebacteria). Mechanosensitive (MS) ion channels have been documented in cells belonging to all three domains suggesting their very early appearance during evolution of life on Earth. The channels show great diversity in conductance, selectivity and voltage dependence, while sharing the property of being gated by mechanical stimuli exerted on cell membranes. In prokaryotes, MS channels were first documented in Bacteria followed by their discovery in Archaea. The finding of MS channels in archaeal cells helped to recognize and establish the evolutionary relationship between bacterial and archaeal MS channels and to show that this relationship extends to eukaryotic Fungi (Schizosaccharomyces pombe) and Plants (Arabidopsis thaliana). Similar to their bacterial and archaeal homologues, MS channels in eukaryotic cell-walled Fungi and Plants may serve in protecting the cellular plasma membrane from excessive dilation and rupture that may occur during osmotic stress. This review summarizes briefly some of the recent developments in the MS channel research field that may ultimately lead to elucidation of the biophysical and evolutionary principles underlying the mechanosensory transduction in living cells.     

The influence of cell volume changes on tumour cell proliferation

Ion channels and cell volume control participate in a wide variety of cellular functions, including cell proliferation. According to the pump-leak model or the double Donnan system, the cell volume is constant in physiological medium so long as the cell metabolism and the Na-K pump are not inhibited and the passive Na⁺ permeability is not dramatically increased. At short term, this model has been supported by a large number of experiments made on different cell types. However, at long term, it may be insufficient to describe the volume control because it does not take into account the fact that cells possess a large number of membrane transporters and interconnected volume regulatory mechanisms. In this review, we present recent results indicating that, in physiological conditions, ion channels may have important roles in cell volume control. Furthermore, we emphasize that cell proliferation and volume are phenomenologically correlated. On the basis of the macromolecular crowding theory, the possibility that the cell osmolyte and water content mediates this correlation is discussed.Abbreviations 4-AP 4-aminopyridine - NPPB 5-nitro-2-(3-phenylpropylamino)benzoic acid - TEA tetraethylammonium - TOR target of rapamycin Presented at the Biophysical Society Meeting on Ion channels—from structure to desease held in May 2003, Rennes, France     

Flow-activated ion channels in vascular endothelium

The ability of vascular endothelial, cells (ECs) to respond to fluid mechanical forces associated with blood flow is essential for flow-mediated vasoregulation and arterial wall remodeling. Abnormalities in endothelial responses to flow also play a role in the development of atherosclerosis. Although our understanding of the endothelial signaling pathways stimulated by flow has greatly increased over the past two decades, the mechanisms by which ECs sense flow remain largely unknown. Activation of flow-sensitive ion channels is among the fastest known endothelial responses to flow; therefore, these ion channels have been proposed as candidate flow sensors. This review focuses on: 1) describing the various types of flow-sensitive ion channels that have been reported in ECs, 2) discussing the implications of activation of these ion channels for endothelial function, and 3) proposing candidate mechanisms for activation of flow-sensitive ion channels.     

A carboxy-terminal fragment of colicin Ia forms ion channels

A carboxy-terminal, 18 kD fragment of colicin Ia, a bacterial toxin, forms ion channels in artificial phospholipid bilayers. This fragment, which comprises a quarter of the intact 70 kD molecule, is resistant to extensive protease digestion and probably constitutes a structural domain of the protein. The ion channels formed by the 18 kD fragment are functionally heterogeneous, having conductances that range from 15 to 30 pS at positive voltages and from 70 to 250 pS at negative voltages, and open lifetimes that range from at least 25 msec to 5 sec. In contrast, ion channels formed by whole colicin Ia open only at negative voltages, at which their conductances range from 6 to 30 pS, and their open lifetimes range from 1 sec to 3 min. Additionally, the open state of the 18 kD fragment channel is characterized by noisy fluctuations in current, while the open state of the whole molecule ion channel is often marked by numerous, stable subconductance states. Since the properties of the fragment channel differ substantially from those of the whole molecule channel, we suggest that portions of the molecule outside of the 18 kD fragment are involved in forming the whole molecule ion channel.     

Functional implication of human serine/threonine kinase, hAIK, in cell cycle progression

Protein phosphorylation is involved in many biological activities and plays important roles in cell cycle progression. In the present study, we identified a serine/threonine kinase, hAIK, from human hepatic cells using degenerated polymerase chain reactions with a pair of primers derived from the highly conserved sequence in the catalytic domain of kinases. The full-length hAIK cDNA was then obtained, which contained 403 amino acids and was homologous to Drosophila Aurora2 and yeast Ipl1 proteins. Northern blotting analysis revealed that hAIK was highly expressed in the testis but not in other tissues. Expressions of hAIK drastically increased in cancer tissues/cell lines but not in fibroblasts or nontumorigenic cell lines. The recombinant hAIK protein phosphorylated itself and histone H1; this phosphorylation activity was totally abolished after a point mutation at the catalytic domain (hAIKm). During the interphase cell, hAIK was found mainly in the cytoplasm; during mitosis hAIK accumulated at the centrosomes. In addition, overexpression of hAIK in cancer cell lines (HEK293T and HeLa) appeared to inhibit cell cycle progression. None of these phenomena were observed in hAIKm whose kinase activity was rendered inactive. Our results suggest that hAIK protein/activity might modulate cell cycle progression by interacting with the centrosomes and/or proteins associated with these structures.     

The human small glutamine-rich TPR-containing protein is required for progress through cell division

Eukaryotic organisms from yeast to human harbor genes encoding the small glutamine-rich tetratricopeptide repeat-containing (SGT) protein. Work presented here demonstrated the presence of human SGT (hSGT) protein in a panel of human cell lines and throughout the cell cycle. To identify cellular processes in which hSGT is involved, knock down populations were analyzed which were generated through transfection of hsgt-specific small interfering RNA. Most strikingly, depletion of hSGT led to reduced proliferation of the affected cell populations while the mitotic index was increased. Time-lapse video microscopy revealed that cells from hSGT-depleted populations were unable to complete cell division due to mitotic arrest which was frequently followed by cell death. Further evidence for a role in cell division was given by the accumulation of hSGT in the midzone and the midbody, and by a mitosis-specific migration pattern of hSGT as detected by Western blotting after SDS-PAGE or two-dimensional gel electrophoresis. In conclusion, results obtained in this study demonstrate that hSGT protein is a constitutive component of all human cell lines tested and that this protein is essential for successful completion of cell division.     

Some aspects of the physiological role of ion channels in the nervous system

Recent analyses of the genomes of several animal species, including man, have revealed that a large number of ion channels are present in the nervous system. Our understanding of the physiological role of these channels in the nervous system has followed the evolution of biophysical techniques during the last century. The observation and the quantification of the electrical events associated with the operation of the ionic channels has been, and still is, one of the best tools to analyse the various aspects of their contribution to nerve function. For this reason, we have chosen to use electrophysiological recordings to illustrate some of the main functions of these channels. The properties and the roles of Na+ and K+ channels in neuronal resting and action potentials are illustrated in the case of the giant axons of the squid and the cockroach. The nature and role of the calcium currents in the bursting behaviour of the neurons are illustrated for Aplysia giant neurons. The relationship between presynaptic calcium currents and synaptic transmission is shown for the squid giant synapse. The involvement of calcium channels in survival and neurite outgrowth of cultured neurons is exemplified using embryonic cockroach brain neurons. This same neuronal preparation is used to illustrate ion channel noise and single-channel events associated with the binding of agonists to nicotinic receptors. Some features of the synaptic activity in the central nervous system are shown, with examples from the cercal nerve giant-axon preparation of the cockroach. The interplay of different ion conductances involved in the oscillatory behaviour of the Xenopus spinal motoneurons is illustrated and discussed. The last part of this review deals with ionic homeostasis in the brain and the function of glial cells, with examples from Necturus and squids.     

Small ubiquitin-related modifier ligase activity of Mms21 is required for maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in Saccharomyces cerevisiae

The SUMO ligase activity of Mms21/Nse2, a conserved member of the Smc5/6 complex, is required for resisting extrinsically induced genotoxic stress. We report that the Mms21 SUMO ligase activity is also required during the unchallenged mitotic cell cycle in Saccharomyces cerevisiae. SUMO ligase-defective cells were slow growing and spontaneously incurred DNA damage. These cells required caffeine-sensitive Mec1 kinase-dependent checkpoint signaling for survival even in the absence of extrinsically induced genotoxic stress. SUMO ligase-defective cells were sensitive to replication stress and displayed synthetic growth defects with DNA damage checkpoint-defective mutants such as mec1, rad9, and rad24. MMS21 SUMO ligase and mediator of replication checkpoint 1 gene (MRC1) were epistatic with respect to hydroxyurea-induced replication stress or methyl methanesulfonate-induced DNA damage sensitivity. Subjecting Mms21 SUMO ligase-deficient cells to transient replication stress resulted in enhancement of cell cycle progression defects such as mitotic delay and accumulation of hyperploid cells. Consistent with the spontaneous activation of the DNA damage checkpoint pathway observed in the Mms21-mediated sumoylation-deficient cells, enhanced frequency of chromosome breakage and loss was detected in these mutant cells. A mutation in the conserved cysteine 221 that is engaged in coordination of the zinc ion in Loop 2 of the Mms21 SPL-RING E3 ligase catalytic domain resulted in strong replication stress sensitivity and also conferred slow growth and Mec1 dependence to unchallenged mitotically dividing cells. Our findings establish Mms21-mediated sumoylation as a determinant of cell cycle progression and maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in budding yeast.     

离子通道与人类遗传病

细胞膜离子通道结构和功能正常是细胞进行生理活动的基础,对离子通道功能具有决定性意义的特定位点的突变导致其开放、关闭或激活、失活功能异常,引起组织机能紊乱,形成各种遗传性疾病。本文从水通道蛋白,钙通道,钠通道,钾通道等多种通道蛋白引起的遗传病的现象以及机理做较深入的阐述。     

Redox active calcium ion channels and cell death

Calcium plays a key role in both apoptotic and necrotic cell death. Emptying of intracellular calcium stores and/or alteration in intracellular calcium levels can modulate cell death in almost all cell types. These calcium fluxes are determined by the activity of membrane channels normally under tight control. The channels may be ligand activated or voltage dependent as well as being under the control of affector molecules such as calmodulin. It has become increasingly apparent that many calcium channels are affected by reactive oxygen or reactive nitrogen species; ROS/RNS. This may be part of the normal signaling pathways in the cell or by the action of exogenously generated ROS or RNS often by toxins. This review covers the recent literature on the activity of these redox active channels as related to cell death.     

Regulation of epithelial ion channels by Rab GTPases

Epithelial ion channels are crucial to many of life's processes and disruption of their functions can lead to several disorders. Cystic fibrosis, an autosomal recessive disorder, is caused by defects in the biosynthesis or function of the CFTR chloride channel. Similarly, mutations in certain ENaC genes leading to increased or reduced channel activity cause diseases such as Liddle's syndrome or PHA. In order for ion channel proteins to be functional they need to be expressed on the plasma membrane. Thus, molecules that modulate the trafficking of ion channels to and from the membrane are of utmost significance. Among the numerous factors that regulate their functioning is a family of small GTPases known as Rab proteins. While Rabs have always played a pivotal role in membrane trafficking, their diversity of functions and plethora of interacting partners have lately been brought to light. Recent studies reveal that multiple Rab isoforms physically interact with and/or modulate the activity of several ion channels. Rab proteins have the ability to serve as molecular switches and many of the ion channels are regulated differentially by the GTP- or GDP-bound Rab isoforms. This review examines the role of Rab GTPases in the trafficking of ion channels, including CFTR, ENaC, TRPV5/6, and aquaporins, based on recent evidence.     

Potassium channels in cell cycle and cell proliferation

Normal cell-cycle progression is a crucial task for every multicellular organism, as it determines body size and shape, tissue renewal and senescence, and is also crucial for reproduction. On the other hand, dysregulation of the cell-cycle progression leading to uncontrolled cell proliferation is the hallmark of cancer. Therefore, it is not surprising that it is a tightly regulated process, with multifaceted and very complex control mechanisms. It is now well established that one of those mechanisms relies on ion channels, and in many cases specifically on potassium channels. Here, we summarize the possible mechanisms underlying the importance of potassium channels in cell-cycle control and briefly review some of the identified channels that illustrate the multiple ways in which this group of proteins can influence cell proliferation and modulate cell-cycle progression.     

Dynamic localization and functional implications of Aurora-C kinase during male mouse meiosis

Aurora-C was first identified during screening for kinases expressed in mouse sperm and eggs. Herein, we report for the first time the precise subcellular localization of endogenous Aurora-C during male meiotic division. The localization of Aurora-C was analyzed by immunofluorescence staining on chromosome spreads of mouse spermatocytes or in squashed seminiferous tubules. Aurora-C was first detected at clusters of chromocenters in diplotene spermatocytes and was concentrated at centromeres in metaphase I and II. Interestingly, Aurora-C was also found along the chromosome axes, including both the regions of centromeres and the chromosome arms in diakinesis. During the anaphase I/telophase I and anaphase II/telophase II transitions, Aurora-C was relocalized to the spindle midzone and midbody. A similar distribution pattern was also observed for Aurora-B during male meiotic divisions. Surprisingly, we detected no Aurora-C in mitotic spermatogonia. Furthermore, immunoprecipitation analyses revealed that INCENP associated with Aurora-C in the male testis. We propose that INCENP recruits Aurora-C (or some other factor(s) recruit INCENP and Aurora-C) to meiotic chromosomes, while Aurora-C may either work alone or cooperate with Aurora-B to regulate chromosome segregation during male meiosis.