The Role of Chloride Channels in the Multidrug Resistance

Nowadays, one of medicine’s main and most challenging aims is finding effective ways to treat cancer. Unfortunately, although there are numerous anti-cancerous drugs, such as cisplatin, more and more cancerous cells create drug resistance. Thus, it is equally important to find new medicines and research the drug resistance phenomenon and possibilities to avoid this mechanism. Ion channels, including chloride channels, play an important role in the drug resistance phenomenon. Our article focuses on the chloride channels, especially the volume-regulated channels (VRAC) and CLC chloride channels family. VRAC induces multidrug resistance (MDR) by causing apoptosis connected with apoptotic volume decrease (AVD) and VRAC are responsible for the transport of anti-cancerous drugs such as cisplatin. VRACs are a group of heterogenic complexes made from leucine-rich repetition with 8A (LRRC8A) and a subunit LRRC8B-E responsible for the properties. There are probably other subunits, which can create those channels, for example, TTYH1 and TTYH2. It is also known that the ClC family is involved in creating MDR in mainly two mechanisms—by changing the cell metabolism or acidification of the cell. The most researched chloride channel from this family is the CLC-3 channel. However, other channels are playing an important role in inducing MDR as well. In this paper, we review the role of chloride channels in MDR and establish the role of the channels in the MDR phenomenon.

Consensus channelome of dinoflagellates revealed by transcriptomic analysis sheds light on their physiology

Ion channels are membrane protein complexes mediating passive ion flux across the cell membranes. Every organism has a certain set of ion channels that define its physiology. Dinoflagellates are ecologically important microorganisms characterized by effective physiological adaptability, which backs up their massive proliferations that often result in harmful blooms (red tides). In this study, we used a bioinformatics approach to identify homologs of known ion channels that belong to 36 ion channel families. We demonstrated that the versatility of the dinoflagellate physiology is underpinned by a high diversity of ion channels including homologs of animal and plant proteins, as well as channels unique to protists. The analysis of 27 transcriptomes allowed reconstructing a consensus ion channel repertoire (channelome) of dinoflagellates including the members of 31 ion channel families: inwardly-rectifying potassium channels, two-pore domain potassium channels, voltage-gated potassium channels (Kv), tandem Kv, cyclic nucleotide-binding domain-containing channels (CNBD), tandem CNBD, eukaryotic ionotropic glutamate receptors, large-conductance calcium-activated potassium channels, intermediate/small-conductance calcium-activated potassium channels, eukaryotic single-domain voltage-gated cation channels, transient receptor potential channels, two-pore domain calcium channels, four-domain voltage-gated cation channels, cation and anion Cys-loop receptors, small-conductivity mechanosensitive channels, large-conductivity mechanosensitive channels, voltage-gated proton channels, inositole-1,4,5- trisphosphate receptors, slow anion channels, aluminum-activated malate transporters and quick anion channels, mitochondrial calcium uniporters, voltage-dependent anion channels, vesicular chloride channels, ionotropic purinergic receptors, animal volage-insensitive cation channels, channelrhodopsins, bestrophins, voltage-gated chloride channels H+/Cl- exchangers, plant calcium-permeable mechanosensitive channels, and trimeric intracellular cation channels. Overall, dinoflagellates represent cells able to respond to physical and chemical stimuli utilizing a wide range of Gprotein coupled receptors- and Ca2+-dependent signaling pathways. The applied approach not only shed light on the ion channel set in dinoflagellates, but also provided the information on possible molecular mechanisms underlying vital cellular processes dependent on the ion transport.

Recombinantly Expressed MeICT, a new Toxin from Mesobuthus Eupeus Scorpion, Inhibits Glioma Cell Proliferation and Downregulates Annexin A2 and FOXM1 Genes

Gliomas are highly invasive and lethal malignancy that do not respond to current therapeutic approaches. Novel therapeutic agents are required to target molecular mechanisms involved in glioma progression. MeICT is a new short-chain toxin isolated from Mesobuthus eupeus scorpion venom. This toxin contained 34 amino acid residues and belongs to chloride channels toxins. In this study, the coding sequence of MeICT was cloned into the pET32Rh vector and a high yield of soluble recombinant MeICT was expressed and purified. Recombinant MeICT-His significantly inhibited the proliferation and migration of glioma cells at low concentration. In vivo studies showed that MeICT was not toxic when administrated to mice at high doses. We also determined the effect of MeICT on the mRNA expression of MMP-2, Annexin A2 and FOXM-2 that are key molecules in the progression and invasion of glioma. Expression of Annexin A2 and FOXM1 mRNA was significantly down-regulated following treatment with MeICT. However, no significant decrease in the expression of MMP-2 gene was identified. In this study a short toxin with four disulfide bonds was successfully produced and its anti-cancer effects was detected. Our findings suggest that recombinant MeICT can be considered as a new potent agent for glioma targeting.

On the molecular mechanism of excitation–transcription coupling in skeletal muscle

An important question in neuromuscular biology is how skeletal muscle cells decipher the stimulation pattern coming from motoneurons to define their phenotype-activating transcriptional changes in a process named excitation–transcription coupling. We have shown in adult muscle fibers that 20 Hz electrical stimulation (ES) activates a signaling cascade that starts with Cav1.1 activation, ATP release trough pannexin-1 channel, activation of purinergic receptors, and IP3-dependent Ca2+ signals inducing transcriptional changes related to muscle plasticity from fast to slow phenotype. Extracellular addition of 30 µM ATP mimics transcriptional changes induced by ES at 20 Hz. ATP release occurs in two peaks, the first around 15 s after ES and a second around 300 s after ES. In the present work, we used apyrase to hydrolyze ATP 60 s after ES, maintaining the first peak and eliminating the second peak. In this condition, transcriptional changes were abolished, indicating that the second peak is the one crucial to activate transcription. Additionally, we observed a small depolarization of fibers after ES. The addition of 30 to 100 µM external ATP also induced depolarization of muscle fibers. This depolarization was unable to activate contraction but was able to induce transcriptional changes induced by 20 Hz ES. These changes were completely inhibited by the IP3R blocker xestospongin B, suggesting that IP3-dependent events are triggered at these membrane depolarization values. Moreover, transcriptional changes induced by addition of 30 µM extracellular ATP was blocked by incubation of fibers with 25 µM Nifedipine. These results suggest that the second ATP peak observed after 20 Hz ES is responsible for transcriptional activation by inducing small depolarizations of fiber membranes that are also sensed by Cav1.1. Finally, we show evidence that downstream of purinergic receptors, PKC is activated, likely causing phosphorylation of ClC-1 chloride channels, possibly responsible for depolarization after 20 Hz.

Cranial Nerves I and II

Cranial nerves I (olfactory nerve) and II (optic nerve) are supratentorial, paired cranial nerves. This chapter provides an overview of their anatomy. Cranial nerve I is a special visceral afferent nerve carrying sensory information about odors. Olfactory receptors lie in the nasal cavity. Odorants activate receptors within the cilia of olfactory sensory neurons and trigger the opening of a cyclic nucleotide–gated channel. This channel allows a calcium influx and the opening of calcium-activated chloride channels. Depolarization then occurs.

Zn2+ triggered two-step mechanism of CLIC1 membrane insertion and activation into chloride channels

The CLIC protein family displays the unique feature of altering its structure from a soluble form to a membrane-bound chloride channel. CLIC1, a member of this family, is found in the cytoplasm or in internal and the plasma membranes, with membrane relocalisation linked to endothelial disfunction, tumour proliferation and metastasis. The molecular switch promoting CLIC1 activation remains unclear. Here, cellular chloride efflux assays and immunofluorescence microscopy studies have identified Zn2+ intracellular release as the trigger for CLIC1 activation and membrane relocalisation. Biophysical assays confirmed specific binding to Zn2+, inducing membrane association and enhancing chloride efflux in a pH dependent manner. Together, our results identify a two-step mechanism with Zn2+ binding as the molecular switch promoting CLIC1 membrane insertion, followed by pH activation of chloride efflux.

Calcium-Activated Chloride Channels in Myometrial and Vascular Smooth Muscle

In smooth muscle tissues, calcium-activated chloride channels (CaCC) provide the major anionic channel. Opening of these channels leads to chloride efflux and depolarization of the myocyte membrane. In this way, activation of the channels by a rise of intracellular [Ca2+], from a variety of sources, produces increased excitability and can initiate action potentials and contraction or increased tone. We now have a good mechanistic understanding of how the channels are activated and regulated, due to identification of TMEM16A (ANO1) as the molecular entity of the channel, but key questions remain. In reviewing these channels and comparing two distinct smooth muscles, myometrial and vascular, we expose the differences that occur in their activation mechanisms, properties, and control. We find that the myometrium only expresses “classical,” Ca2+-activated, and voltage sensitive channels, whereas both tonic and phasic blood vessels express classical, and non-classical, cGMP-regulated CaCC, which are voltage insensitive. This translates to more complex activation and regulation in vascular smooth muscles, irrespective of whether they are tonic or phasic. We therefore tentatively conclude that although these channels are expressed and functionally important in all smooth muscles, they are probably not part of the mechanisms governing phasic activity. Recent knockdown studies have produced unexpected functional results, e.g. no effects on labour and delivery, and tone increasing in some but decreasing in other vascular beds, strongly suggesting that there is still much to be explored concerning CaCC in smooth muscle.

Chloride transport modulators as drug candidates

Chloride transport across cell membranes is broadly involved in epithelial fluid transport, cell volume and pH regulation, muscle contraction, membrane excitability, and organellar acidification. The human genome encodes at least 53 chloride transporting proteins with expression in cell plasma or intracellular membranes, which include chloride channels, exchangers and cotransporters, some having broad anion specificity. Loss of function mutations in chloride transporters cause a wide variety of human diseases, including cystic fibrosis, secretory diarrhea, kidney stones, salt wasting nephropathy, myotonia, osteopetrosis, hearing loss and goiter. While impactful advances have been made in the past decade in drug treatment of cystic fibrosis using small molecule modulators of the defective cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, other chloride channels and solute carrier proteins (SLCs) represent relatively underexplored target classes for drug discovery. New opportunities have emerged for development of chloride transport modulators as potential therapeutics for secretory diarrheas, constipation, dry eye disorders, kidney stones, polycystic kidney disease, hypertension and osteoporosis. Approaches to chloride transport-targeted drug discovery are reviewed herein, with focus on chloride channel and exchanger classes in which recent preclinical advances have been made in the identification of small molecule modulators and in proof of concept testing in experimental animal models.