Remodelling of Ca2+ transport in cancer: how it contributes to cancer hallmarks?

Cancer involves defects in the mechanisms underlying cell proliferation, death and migration. Calcium ions are central to these phenomena, serving as major signalling agents with spatial localization, magnitude and temporal characteristics of calcium signals ultimately determining cell''s fate. Cellular Ca²⁺ signalling is determined by the concerted action of a molecular Ca²⁺-handling toolkit which includes: active energy-dependent Ca²⁺ transporters, Ca²⁺-permeable ion channels, Ca²⁺-binding and storage proteins, Ca²⁺-dependent effectors. In cancer, because of mutations, aberrant expression, regulation and/or subcellular targeting of Ca²⁺-handling/transport protein(s) normal relationships among extracellular, cytosolic, endoplasmic reticulum and mitochondrial Ca²⁺ concentrations or spatio-temporal patterns of Ca²⁺ signalling become distorted. This causes deregulation of Ca²⁺-dependent effectors that control signalling pathways determining cell''s behaviour in a way to promote pathophysiological cancer hallmarks such as enhanced proliferation, survival and invasion. Despite the progress in our understanding of Ca²⁺ homeostasis remodelling in cancer cells as well as in identification of the key Ca²⁺-transport molecules promoting certain malignant phenotypes, there is still a lot of work to be done to transform fundamental findings and concepts into new Ca²⁺ transport-targeting tools for cancer diagnosis and treatment.     

Ion channels as pharmacologic receptors: The chirality of drug interactions

Ion channels are pharmocologic receptors and as such exhibit stereoselective interactions with drugs. Ion channels are conformationally mobile transmembrane proteins existing in a number of open and closed states. Drug interactions with these different states may differ quantitatively and qualitatively. Stereoselectivity may not be a constant factor and may change according to channel state as determined by stimulus mode or experimental conditions. Selected examples are cited for Na⁺ and Ca²⁺ channels. © 1996 Wiley-Liss, Inc.     

Heat shock protein 90: The cancer chaperone

Heat shock protein 90 (Hsp90) is a molecular chaperone required for the stability and function of a number of conditionally activated and/or expressed signalling proteins, as well as multiple mutated, chimeric, and/or over-expressed signalling proteins, that promote cancer cell growth and/or survival. Hsp90 inhibitors are unique in that, although they are directed towards a specific molecular target, they simultaneously inhibit multiple cellular signalling pathways. By inhibiting nodal points in multiple overlapping survival pathways utilized by cancer cells, combination of an Hsp90 inhibitor with standard chemotherapeutic agents may dramatically increase the in vivo efficacy of the standard agent. Hsp90 inhibitors may circumvent the characteristic genetic plasticity that has allowed cancer cells to eventually evade the toxic effects of most molecularly targeted agents. The mechanism-based use of Hsp90 inhibitors, both alone and in combination with other drugs, should be effective toward multiple forms of cancer. Further, because Hsp90 inhibitors also induce Hsf-1-dependent expression of Hsp70, and because certain mutated Hsp90 client proteins are neurotoxic, these drugs display ameliorative properties in several neurodegenerative disease models, suggesting a novel role for Hsp90 inhibitors in treating multiple pathologies involving neurodegeneration.     

Signalling in ciliates: long‐ and short‐range signals and molecular determinants for cellular dynamics

In ciliates, unicellular representatives of the bikont branch of evolution, inter‐ and intracellular signalling pathways have been analysed mainly in Paramecium tetraurelia, Paramecium multimicronucleatum and Tetrahymena thermophila and in part also in Euplotes raikovi. Electrophysiology of ciliary activity in Paramecium spp. is a most successful example. Established signalling mechanisms include plasmalemmal ion channels, recently established intracellular Ca²⁺‐release channels, as well as signalling by cyclic nucleotides and Ca²⁺. Ca²⁺‐binding proteins (calmodulin, centrin) and Ca²⁺‐activated enzymes (kinases, phosphatases) are involved. Many organelles are endowed with specific molecules cooperating in signalling for intracellular transport and targeted delivery. Among them are recently specified soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs), monomeric GTPases, H⁺‐ATPase/pump, actin, etc. Little specification is available for some key signal transducers including mechanosensitive Ca²⁺‐channels, exocyst complexes and Ca²⁺‐sensor proteins for vesicle–vesicle/membrane interactions. The existence of heterotrimeric G‐proteins and of G‐protein‐coupled receptors is still under considerable debate. Serine/threonine kinases dominate by far over tyrosine kinases (some predicted by phosphoproteomic analyses). Besides short‐range signalling, long‐range signalling also exists, e.g. as firmly installed microtubular transport rails within epigenetically determined patterns, thus facilitating targeted vesicle delivery. By envisaging widely different phenomena of signalling and subcellular dynamics, it will be shown (i) that important pathways of signalling and cellular dynamics are established already in ciliates, (ii) that some mechanisms diverge from higher eukaryotes and (iii) that considerable uncertainties still exist about some essential aspects of signalling.     

Increased Ca++ uptake by erythrocytes infected with malaria parasites: Evidence for exported proteins and novel inhibitors

Malaria parasites export many proteins into their host erythrocytes and increase membrane permeability to diverse solutes. Although most solutes use a broad‐selectivity channel known as the plasmodial surface anion channel, increased Ca⁺⁺ uptake is mediated by a distinct, poorly characterised mechanism that appears to be essential for the intracellular parasite. Here, we examined infected cell Ca⁺⁺ uptake with a kinetic fluorescence assay and the virulent human pathogen, Plasmodium falciparum. Cell surface labelling with N‐hydroxysulfosuccinimide esters revealed differing effects on transport into infected and uninfected cells, indicating that Ca⁺⁺ uptake at the infected cell surface is mediated by new or altered proteins at the host membrane. Conditional knockdown of PTEX, a translocon for export of parasite proteins into the host cell, significantly reduced infected cell Ca⁺⁺ permeability, suggesting involvement of parasite‐encoded proteins trafficked to the host membrane. A high‐throughput chemical screen identified the first Ca⁺⁺ transport inhibitors active against Plasmodium‐infected cells. These novel chemical scaffolds inhibit both uptake and parasite growth; improved in vitro potency at reduced free [Ca⁺⁺] is consistent with parasite killing specifically via action on one or more Ca⁺⁺ transporters. These inhibitors should provide mechanistic insights into malaria parasite Ca⁺⁺ transport and may be starting points for new antimalarial drugs.     

Annexin A6-regulator of the EGFR/Ras signalling pathway and cholesterol homeostasis

Annexin A6 (AnxA6) belongs to the highly conserved annexin protein family. Like other annexins, the function of AnxA6 is linked to its ability to bind phospholipids in a Ca²⁺-dependent manner, thereby interacting with cellular membranes in a dynamic, reversible and regulated fashion. Upon cell activation, AnxA6 is recruited to the plasma membrane, endosomes and caveolae/membrane rafts to interact with signalling proteins, the endocytic machinery and actin cytoskeleton to inhibit epidermal growth factor receptor and Ras signalling. In addition, AnxA6 associates with late endosomes to regulate cholesterol export leading to reduced cytoplasmic phospholipase A2 activity and caveolae formation. Accordingly, AnxA6 may function as an organizer of membrane domains (i) to create a scaffold for the formation of multifactorial signalling complexes, (ii) to regulate transient membrane–actin interactions during endocytic transport, and (iii) to modulate intracellular cholesterol homeostasis. Altogether, this will regulate critical physiological processes including proliferation, differentiation, inflammation and cell migration.     

The chloride anion as a signalling effector

The specific role of the chloride anion (Cl^?) as a signalling effector or second messenger has been increasingly recognized in recent years. It could represent a key factor in the regulation of cellular homeostasis. Changes in intracellular Cl^? concentration affect diverse cellular functions such as gene and protein expression and activities, post‐translational modifications of proteins, cellular volume, cell cycle, cell proliferation and differentiation, membrane potential, reactive oxygen species levels, and intracellular/extracellular pH. Cl^? also modulates functions in different organelles, including endosomes, phagosomes, lysosomes, endoplasmic reticulum, and mitochondria. A better knowledge of Cl^? signalling could help in understanding the molecular and metabolic changes seen in pathologies with altered Cl^? transport or under physiological conditions. Here we review relevant evidence supporting the role of Cl^? as a signalling effector.     

Calcium signalling and secretory epithelia

Ca²⁺ is now firmly established as the most important intracellular regulator of physiological and pathological events in a vast number of different cell types, including secretory epithelia. In these tissues, Ca²⁺ signalling is crucially important for the control of both fluid secretion and electrolyte secretion as well as the regulation of macromolecule secretion. In this overview article, I shall attempt to give some general background to the concepts underlying our current thinking about Ca²⁺ signalling in epithelia and its roles in regulating secretion. It is outside the scope of this review to provide a comprehensive account of Ca²⁺ signalling and the many different processes in the many different secretory epithelia that are controlled by Ca²⁺ signals. It is my aim to draw attention to some general features of Ca²⁺ signalling processes in secretory epithelia, which are rather different from those in, for example, endocrine glands. The principal examples will be taken from studies of exocrine cells and, in particular, pancreatic acinar cells, as they are the pioneer cells with regard to investigations of Ca²⁺ signalling due to primary intracellular Ca²⁺ release. They also represent the cell type which has been characterized in most detail with regard to Ca²⁺ transport events and mechanisms.     

Adaptation of plants to salt stress: the role of the ion transporters

Adaptation to high salinity is achieved by cellular ion homeostasis which involves regulation of toxic sodium ion (Na⁺) and Chloride ion (Cl⁻) uptake, preventing the transport of these ions to the aerial parts of the plants and vacuolar sequestration of these toxic ions. Ion transporters have long been known to play roles in maintaining ion homeostasis. Na⁺ enters the cell through various voltage dependent selective and non-selective ion channels. High Na⁺ concentration in the plasma membrane is balanced either by uptake of potassium ion (K⁺) by various potassium importing channels, by salt exclusion mechanism or by sequestration of Na⁺ in the vacuoles. Therefore, the role of high-affinity potassium transporter, the salt overly sensitive pathway, the most well-defined Na⁺ exclusion pathway that exports Na⁺ from cell into xylem and tonoplast localized cation transporters that compartmentalizes Na⁺ in vacuoles need to be studied in detail and applied to make the plant adaptable to saline soil. Knowledge on the regulation of expression of these transporters by the hormones, microRNAs and other non-coding RNAs can be utilized to manipulate the ion transport. Here, we reviewed paradigm of the ion transporters in salt stress signalling pathways from the recent and past studies aiding transformation of basic knowledge into biotechnological applications to generate engineered salt stress tolerant crops.

     

The plant vacuole: emitter and receiver of calcium signals

This review portrays the plant vacuole as both a source and a target of Ca²⁺ signals. In plants, the vacuole represents a Ca²⁺ store of enormous size and capacity. Total and free Ca²⁺ concentrations in the vacuole vary with plant species, cell type, and environment, which is likely to have an impact on vacuolar function and the release of vacuolar Ca²⁺. It is known that cytosolic Ca²⁺ signals are often generated by release of the ion from internal stores, but in very few cases has a role of the vacuole been directly demonstrated. Biochemical and electrophysical studies have provided evidence for the operation of ligand- and voltage-gated Ca²⁺-permeable channels in the vacuolar membrane. The underlying molecular mechanisms are largely unknown with one exception: the slow vacuolar channel, encoded by TPC1, is the only vacuolar Ca²⁺-permeable channel cloned to date. However, due to its complex regulation and its low selectivity amongst cations, the role of this channel in Ca²⁺ signalling is still debated. Many transport proteins at the vacuolar membrane are also targets of Ca²⁺ signals, both by direct binding of Ca²⁺ and by Ca²⁺-dependent phosphorylation. This enables the operation of feedback mechanisms and integrates vacuolar transport systems in the wider signalling network of the plant cell.     

Annexins — Modulators of EGF receptor signalling and trafficking

At the cell surface, activation of the epidermal growth factor (EGF) receptor triggers a complex network of signalling events that regulate a variety of cellular processes. For signal termination, the activated EGF receptor is internalised and targeted to lysosomes for degradation. Microdomain localization at the plasma membrane and endocytic transport of the EGFR is important for the formation of compartment-specific signalling complexes and is regulated by scaffolding and targeting proteins. This includes Ca²⁺-effector proteins, such as calmodulin and annexins (Anx), in particular AnxA1, AnxA2, AnxA6 and as shown recently, AnxA8. Given that these annexins show differences in their expression patterns, subcellular localization and mode of action, they are likely to differentially contribute and cooperate in the fine-tuning of EGFR activity. In support of this hypothesis, current literature suggests these annexins to be involved in different steps that control the endocytic transport and signalling of the EGF receptor. This review summarizes how the coordinated activity of AnxA1, AnxA2, AnxA6 and AnxA8 can contribute to regulate EGF receptor localization and activity.     

Organohalide Perovskites are Fast Ionic Conductors

Fast ionic conductors constitute a family of materials exhibiting high values of the ionic conductivity while their crystal structure remains rather rigid. Perovskite‐like compounds are known to be good ionic conductors with applications as solid electrolytes. In hybrid halide perovskites both intrinsic (native) and extrinsic defect migration are regarded to occur. Ion diffusivity analysis is inherently ambiguous in all‐solid‐state configurations because of the multicomponent environment. Here, a liquid electrolyte in contact to the perovskite material forms a reservoir of Li⁺ that is forced to intercalate and migrate within the perovskite electrode. This approach decouples different contributions to transport in such a way that ion diffusion kinetics is easily accessible by means of impedance methods. The room‐temperature chemical diffusion coefficient of lithium ion within the perovskite lattice exhibits values as high as D _μ ≈ 10^?7 cm² s^?1, which implies conductivities within the range of 10^?3 Ω^?1 cm^?1 for highly lithiated electrodes. This confirms the superionic intrinsic property of organohalide perovskites from a direct and unambiguous measurement that does not rely upon simulation tools.     

Cell marking in Arabidopsis thaliana and its application to patch–clamp studies

Ion transport processes at the plasma membrane of plant cells are frequently studied by applying membrane-patch voltage-clamp (patch–clamp) electrophysiological techniques to isolated protoplasts. As plants are composed of many tissues and cell types, and each tissue and cell type may be specialized to a particular function and possess a unique complement of transport proteins, it is important to certify the anatomical origin of the protoplasts used for patch–clamp studies. This paper describes a general molecular genetic approach to marking specific cell types for subsequent patch–clamp studies and presents a specific example: a comparison of the K⁺ currents in protoplasts from cortical and stelar cells of Arabidopsis roots. Transgenic Arabidopsis were generated in which the expression of green fluorescent protein (GFP) from Aequoria victoria was driven by the CaMV 35S promoter (line mGFP3). In roots of the transgenic mGFP3 line, visible fluorescence was restricted to the stele. Protoplasts were generated from roots of the mGFP3 line and K⁺ currents in non-fluorescent (cortical/epidermal) and fluorescent (stelar) protoplasts were assayed using patch–clamp techniques. It was found that both the frequency of observing inward rectifying K⁺ channel (IRC) activity and the relative occurrence of IRC compared to outward rectifying K⁺ channels were significantly lower in protoplasts from cortical/epidermal cells compared to cells of the stele. The presence of GFP did not affect the occurrence or biophysical properties of K⁺ channels. It is concluded that the generation of transgenic Arabidopsis expressing GFP in a cell-specific fashion is a convenient and reliable way to mark protoplasts derived from contrasting cell types for subsequent patch–clamp studies.     

Gangliosides play important roles in the nervous system by regulating ion concentrations

Gangliosides are important components of the neuronal cell membrane and play a vital role in the development of neurons and the brain. They participate in neurotransmission and are considered as the structural basis of learning and memory. Gangliosides participate in several and important physiological processes, such as cell differentiation, cell signaling, neuroprotection, nerve regeneration and apoptosis. The stability of ion concentration in excitable cells is particularly important in the maintenance of a steady state of cells and in the regulation of physiological functions. Ion concentration has been found to be related to the ganglioside’s regulation in many neurological diseases, and several studies have found that they can stabilize intracellular ion concentration by regulating ion channels, which highlights their important regulatory role in neuronal excitability and synaptic transmission. Gangliosides can influence some forms of ion transport, by directly binding to ion transporters or through indirect binding and activation of transport proteins via appropriate signaling pathways. Therefore, the important and special role of gangliosides in the homeostasis of ion concentration is becoming a hot topic in the field and a theoretical basis in promoting help gangliosides use as key drugs for the treatment of nervous system diseases.

     

CAX‐ing a wide net: Cation/H+ transporters in metal remediation and abiotic stress signalling

Cation/proton exchangers (CAXs) are a class of secondary energised ion transporter that are being implicated in an increasing range of cellular and physiological functions. CAXs are primarily Ca²⁺ efflux transporters that mediate the sequestration of Ca²⁺ from the cytosol, usually into the vacuole. Some CAX isoforms have broad substrate specificity, providing the ability to transport trace metal ions such as Mn²⁺ and Cd²⁺, as well as Ca²⁺. In recent years, genomic analyses have begun to uncover the expansion of CAXs within the green lineage and their presence within non‐plant species. Although there appears to be significant conservation in tertiary structure of CAX proteins, there is diversity in function of CAXs between species and individual isoforms. For example, in halophytic plants, CAXs have been recruited to play a role in salt tolerance, while in metal hyperaccumulator plants CAXs are implicated in cadmium transport and tolerance. CAX proteins are involved in various abiotic stress response pathways, in some cases as a modulator of cytosolic Ca²⁺ signalling, but in some situations there is evidence of CAXs acting as a pH regulator. The metal transport and abiotic stress tolerance functions of CAXs make them attractive targets for biotechnology, whether to provide mineral nutrient biofortification or toxic metal bioremediation. The study of non‐plant CAXs may also provide insight into both conserved and novel transport mechanisms and functions.     

Direct control of store-operated calcium channels by ultrafast laser

Ca²⁺ channels are essential to cell birth, life, and death. They can be externally activated by optogenetic tools, but this requires robust introduction of exogenous optogenetic genes for expression of photosensitive proteins in biological systems. Here we present femtoSOC, a method for direct control of Ca²⁺ channels solely by ultrafast laser without the need for optogenetic tools or any other exogenous reagents. Specifically, by focusing and scanning wavelength-tuned low-power femtosecond laser pulses on the plasma membrane for multiphoton excitation, we directly induced Ca²⁺ influx in cultured cells. Mechanistic study reveals that photoexcited flavins covalently bind cysteine residues in Orai1 via thioether bonds, which facilitates Orai1 polymerization to form store-operated calcium channels (SOCs) independently of STIM1, a protein generally participating in SOC formation, enabling all-optical activation of Ca²⁺ influx and downstream signaling pathways. Moreover, we used femtoSOC to demonstrate direct neural activation both in brain slices in vitro and in intact brains of living mice in vivo in a spatiotemporal-specific manner, indicating potential utility of femtoSOC.Subject terms: Biological techniques, Ion channel signalling, Calcium signalling     

Ion channel–transporter interactions

All living cells require membrane proteins that act as conduits for the regulated transport of ions, solutes and other small molecules across the cell membrane. Ion channels provide a pore that permits often rapid, highly selective and tightly regulated movement of ions down their electrochemical gradient. In contrast, active transporters can move moieties up their electrochemical gradient. The secondary active transporters (such as SLC superfamily solute transporters) achieve this by coupling uphill movement of the substrate to downhill movement of another ion, such as sodium. The primary active transporters (including H⁺/K⁺-ATPases and Na⁺/K⁺-ATPases) utilize ATP hydrolysis as an energy source to power uphill transport. It is well known that proteins in each of these classes work in concert with members of the other classes to ensure, for example, ion homeostasis, ion secretion and restoration of ion balance following action potentials. More recently, evidence is emerging of direct physical interaction between true ion channels, and some primary or secondary active transporters. Here, we review the first known members of this new class of macromolecular complexes that we term “chansporters”, explore their biological roles and discuss the pathophysiological consequences of their disruption. We compare functional and/or physical interactions between the ubiquitous KCNQ1 potassium channel and various active transporters, and examine other newly discovered chansporter complexes that suggest we may be seeing the tip of the iceberg in a newly emerging signaling modality.     

Singlet oxygen-mediated and EXECUTER-dependent signalling and acclimation of Arabidopsis thaliana exposed to light stress

Plants respond to environmental changes by acclimation that activates defence mechanisms and enhances the plant''s resistance against a subsequent more severe stress. Chloroplasts play an important role as a sensor of environmental stress factors that interfere with the photosynthetic electron transport and enhance the production of reactive oxygen species (ROS). One of these ROS, singlet oxygen (¹O₂), activates a signalling pathway within chloroplasts that depends on the two plastid-localized proteins EXECUTER 1 and 2. Moderate light stress induces acclimation protecting photosynthetic membranes against a subsequent more severe high light stress and at the same time activates ¹O₂-mediated and EXECUTER-dependent signalling. Pre-treatment of Arabidopsis seedlings with moderate light stress confers cross-protection against a virulent Pseudomonas syringae strain. While non-pre-acclimated seedlings are highly susceptible to the pathogen regardless of whether ¹O₂- and EXECUTER-dependent signalling is active or not, pre-stressed acclimated seedlings without this signalling pathway lose part of their pathogen resistance. These results implicate ¹O₂- and EXECUTER-dependent signalling in inducing acclimation but suggest also a contribution by other yet unknown signalling pathways during this response of plants to light stress.