Functional properties of ion channels and transporters in tumour vascularization

Vascularization is crucial for solid tumour growth and invasion, providing metabolic support and sustaining metastatic dissemination. It is now accepted that ion channels and transporters play a significant role in driving the cancer growth at all stages. They may represent novel therapeutic, diagnostic and prognostic targets for anti-cancer therapies. On the other hand, although the expression and role of ion channels and transporters in the vascular endothelium is well recognized and subject of recent reviews, only recently has their involvement in tumour vascularization been recognized. Here, we review the current literature on ion channels and transporters directly involved in the angiogenic process. Particular interest will be focused on tumour angiogenesis in vivo as well as in the different steps that drive this process in vitro, such as endothelial cell proliferation, migration, adhesion and tubulogenesis. Moreover, we compare the ‘transportome’ system of tumour vascular network with the physiological one.     

Effect of mineralocorticoid deficiency on ion and urea transporters and aquaporin water channels in the rat

Mineralocorticoid deficiency is associated with impaired urinary concentration and dilution. The present investigation was undertaken to determine the effects of selective mineralocorticoid deficiency on renal sodium and urea transporters and aquaporin water channels and whether these perturbations can be reversed by maintenance of extracellular fluid volume. Mineralocorticoid deficiency was induced by bilateral adrenalectomies with glucocorticoid replacement. Mineralocorticoid deficient rats receiving plain drinking water (MDW) were compared with mineralocorticoid deficient rats receiving saline-drinking water (MDS) in order to maintain extracellular fluid volume, and with controls (CTL). In MDW rats, there was a significant decrease in renal outer medulla Na-K-2Cl co-transporter and outer medulla Na-K-ATPase as well as an increase in inner medulla aquaporins 2 and 3. There were no significant changes in aquaporin-1, aquaporin-4, or urea transporters. These alterations were reversed with maintenance of extracellular fluid volume in MDS rats. Our findings indicate that mineralocorticoid deficiency in the rat is associated with alterations in factors involved in the countercurrent concentrating mechanism (Na-K-2Cl, Na-K-ATPase) and osmotic water equilibration in the collecting duct (AQP2, AQP3). Maintenance of sodium balance and extracellular fluid volume is associated with normalization of these perturbations.     

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.     

Defective interactions of protein partner with ion channels and transporters as alternative mechanisms of membrane channelopathies

The past twenty years have revealed the existence of numerous ion channel mutations resulting in human pathology. Ion channels provide the basis of diverse cellular functions, ranging from hormone secretion, excitation–contraction coupling, cell signaling, immune response, and trans-epithelial transport. Therefore, the regulation of biophysical properties of channels is vital in human physiology. Only within the last decade has the role of non-ion channel components come to light in regard to ion channel spatial, temporal, and biophysical regulation in physiology. A growing number of auxiliary components have been determined to play elemental roles in excitable cell physiology, with dysfunction resulting in disorders and related manifestations. This review focuses on the broad implications of such dysfunction, focusing on disease-causing mutations that alter interactions between ion channels and auxiliary ion channel components in a diverse set of human excitable cell disease. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé     

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.     

Characterization of isolated ventricular myocytes from adult zebrafish (Danio rerio)

The zebrafish is widely used for human related disease studies. Surprisingly, there is no information about the electrical activity of single myocytes freshly isolated from adult zebrafish ventricle. In this study, we present an enzymatic method to isolate ventricular myocytes from zebrafish heart that yield a large number of calcium tolerant cells. Ventricular myocytes from zebrafish were imaged using light and confocal microscopy. Myocytes were mostly rod shaped and responded by vigorous contraction to field electrical stimulation. Whole cell configuration of the patch clamp technique was used to record electrophysiological characteristics of myocytes. Action potentials present a long duration and a plateau phase and action potential duration decreases when increasing stimulation frequency (as observed in larger mammals). Together these results indicate that zebrafish is a species ideally suited for investigation of ion channels related mutation screening of cardiac alteration important in human.     

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.     

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.     

Stretch-activated channels in the heart: contributions to length-dependence and to cardiomyopathy

The stretch-induced increase in force production of ventricular muscle is biphasic. An abrupt increase in force coincides with the stretch, which is then followed by a slower response that develops over minutes (the slow force response or SFR). The SFR is accompanied by a slow increase in the magnitude of the intracellular Ca2+ transient, but the stretch-dependent mechanisms that give rise to this remain controversial. We characterized the SFR using right ventricular trabeculae from mouse hearts. Application of three different blockers of stretch-activated non-selective cation channels (SAC NSC) reduced the magnitude of the SFR 60s after stretch (400 microM streptomycin: from 86+/-25% to 38+/-14%, P<0.01, n=9; 10 microM GdCl3: from 65+/-21%, to 12+/-7%, P<0.01, n=7; 10 microM GsMTx-4 from 122+/-40% to 15+/-8%, P<0.05, n=6). Streptomycin also decreased the increase in Ca2+ transient amplitude 60s after the stretch from 43.5+/-12.7% to 5.7+/-3.5% (P<0.05, n=4), and reduced the stretch-dependent increase in intracellular Ca2+ in quiescent muscles when stretched. The transient receptor potential, canonical channels TRPC1 and TRPC6 are mechano-sensitive, non-selective cation channels. They are expressed in mouse ventricular muscle, and could therefore be responsible for stretch-dependent influx of Na+ and/or Ca2+ during the SFR. Expression of TRPC1 was investigated in the mdx heart, a mouse model of Duchenne's muscular dystrophy. Resting Ca2+ was raised in isolated myocytes from old mdx animals, which was blocked by application of SAC blockers. Expression of TRPC1 was increased in the older mdx animals, which have developed a dilated cardiomyopathy, and might therefore contribute to the dilated cardiomyopathy.     

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.     

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.     

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.     

Intracellular regions of potassium channels: Kv2.1 and heag

Intracellular regions of voltage-gated potassium channels often comprise the largest part of the channel protein, and yet the functional role of these regions is not fully understood. For the Kv2.1 channel, although there are differences in activation kinetics between rat and human channels, there are, for instance, no differences in movement of the S4 region between the two channels, and indeed our mutagenesis studies have identified interacting residues in both the N- and C -terminal intracellular regions that are responsible for these functional effects. Furthermore, using FRET with fluorescent-tagged Kv2.1 channels, we have shown movement of the C-termini relative to the N-termini during activation. Such interactions and movements of the intracellular regions of the channel appear to form part of the channel gating machinery. Heag1 and heag2 channels also display differing activation properties, despite their considerable homology. By a chimeric approach, we have shown that these differences in activation kinetics are determined by multiple interacting regions in the N-terminus and membrane-spanning regions. Furthermore, alanine mutations of many residues in the C-terminal cyclic nucleotide binding domain affect activation kinetics. The data again suggest interacting regions between N- and C- termini that participate in the conformational changes during channel activation. Using a mass-spectrometry approach, we have identified α-tubulin and a heat shock protein as binding to the C-terminus of the heag2 channel, and α-tubulin itself has functional effects on channel activation kinetics. Clearly, the intracellular regions of these ion channels (and most likely many other ion channels too) are important regions in determining channel function. EBSA Satellite Meeting: Ion channels, Leeds, July 2007.     

Voltage sensor of ion channels and enzymes

Placed in the cell membrane (a two-dimensional environment), ion channels and enzymes are able to sense voltage. How these proteins are able to detect the difference in the voltage across membranes has attracted much attention, and at times, heated debate during the last few years. Sodium, Ca²⁺ and K⁺ voltage-dependent channels have a conserved positively charged transmembrane (S4) segment that moves in response to changes in membrane voltage. In voltage-dependent channels, S4 forms part of a domain that crystallizes as a well-defined structure consisting of the first four transmembrane (S1–S4) segments of the channel-forming protein, which is defined as the voltage sensor domain (VSD). The VSD is tied to a pore domain and VSD movements are allosterically coupled to the pore opening to various degrees, depending on the type of channel. How many charges are moved during channel activation, how much they move, and which are the molecular determinants that mediate the electromechanical coupling between the VSD and the pore domains are some of the questions that we discuss here. The VSD can function, however, as a bona fide proton channel itself, and, furthermore, the VSD can also be a functional part of a voltage-dependent phosphatase.     

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.     

Molecular regions underlying the activation of low- and high-voltage activating calcium channels

We have studied two aspects of calcium channel activation. First, we investigated the molecular regions that are important in determining differences in activation between low- and high-voltage activated channels. For this, we made chimeras between the low-voltage activating Ca_V3.1 channel and the high-voltage activating Ca_V1.2 channel. Chimeras were expressed in oocytes, and calcium channel currents recorded by voltage clamp. For domain I, we found that the molecular region that is important in determining the voltage dependence of activation comprises the pore regions S5-P as well as P-S6, but surprisingly not the voltage sensor S1–S4 region, which might have been expected to play a major part. By contrast, the smaller, but still significant, modulating effects of domain II on activation properties were due to effects involving both S1–S4 and S5–S6 but not the I/II linker. Second, during channel activation we studied movement of the S4 segment in domain I of one of the chimeras, using cysteine-scanning mutagenesis. The reagent parachloromercuribenzensulfonate inhibited currents for mutants V263, A265, L266 and A268, but not for F269 and V271, and voltage dependence of inhibition for residue V263 indicated S4 movement, which occurred before channel opening. The data indicate movement outwards upon depolarisation so as to expose amino acids up to residue 268 in S4.Junying Li and Louisa Stevens contributed equally to this work.     

Flexoelectric effects in model and native membranes containing ion channels

An experimental study of flexoelectricity in model membranes containing ion pores and native membranes containing ion channels has been undertaken with the objective of determining the relationship, if any, between flexoelectricity and ion transport. Model membrane patches containing ion pores induced by a bluegreen algal toxin, microcystin-LR, and locust muscle membrane patches containing potassium channels were studied using patch-clamp techniques. A correspondence was established between the presence of open channels and pores and the amplitude of the 1st harmonic of the total membrane current when the membranes or patches were subjected to pressure oscillations. The 2nd harmonic of the membrane current provided a measure of the amplitude of a membrane curvature induced by pressure, thus making it possible to determine the membrane flexoelectric coefficient. This study shows that flexoelectricity could be an effective driving force for ion transport through membrane pores and channels, thus further highlighting the possible biological significance of this mechano-electric phenomenon.Correspondence to: P. N. R. Usherwood     

IRBIT: A regulator of ion channels and ion transporters

IRBIT (also called AHCYL1) was originally identified as a binding protein of the intracellular Ca^2 + channel inositol 1,4,5-trisphosphate (IP₃) receptor and functions as an inhibitory regulator of this receptor. Unexpectedly, many functions have subsequently been identified for IRBIT including the activation of multiple ion channels and ion transporters, such as the Na⁺/HCO₃⁻ co-transporter NBCe1-B, the Na⁺/H⁺ exchanger NHE3, the Cl⁻ channel cystic fibrosis transmembrane conductance regulator (CFTR), and the Cl⁻/HCO₃⁻ exchanger Slc26a6. The characteristic serine-rich region in IRBIT plays a critical role in the functions of this protein. In this review, we describe the evolution, domain structure, expression pattern, and physiological roles of IRBIT and discuss the potential molecular mechanisms underlying the coordinated regulation of these diverse ion channels/transporters through IRBIT. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.