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The ATP-gated P2X7 receptors (P2X7Rs) play a key role in the release of pro-inflammatory cytokines in response to immunological challenges. Pannexin 1 (Panx1), conventionally described as a hemichannel forming protein, was suggested to be involved in the formation of the P2X7 large pore, which provides a conduit for large molecules such as fluorescent dyes. Firstly, this thesis demonstrated that the P2X7R-mediated dye uptake, a phenomenon attributed to the activation of Panx1, was suppressed by acidic pH and this inhibition was abolished in a P2X7 mutant (aspartic acid 197 to alanine) that was insensitive to extracellular pH. Then, the functional properties of human or mouse Panx1 in HEK293 cells were analysed in the absence of P2X7. The Panx1 currents were not affected by extracellular/intracellular calcium, but were reversibly inhibited by adenosine triphosphate (ATP) and non-specific anion channel blockers. Ion substitution experiments showed that Panx1 was permeable only to monovalent anions and single channel studies revealed a medium sized unitary conductance of Panx1 (~65 pS). Based on the evidence, this thesis concluded that Panx1 is an anion channel but not a hemichannel as originally proposed.

<p>Chloride ion channels are macromolecular pores providing for passage of chloride ions (and certain other inorganic and organic anions) through the cell membrane, down their electrochemical gradients. Chloride channels are differentially expressed in various cells, to best suit specific cellular activities. They are present in practically all living cells, and regardless of cell specialization they play an important role in vital housekeeping functions of cell-volume and pH regulation and in membrane potential stabilization. Regulation of cell volume underlies the structural integrity and constancy of the intracellular milieu. A variety of metabolic pathways have been shown to be sensitive to cell volume, and alterations of cell volume and osmoregulation processes can influence various intracellular signaling and organizing factors.</p><p>Volume-regulated anion channels (VRACs) are believed to play a pivotal role in cell-volume regulating processes. In this report I present data from macroscopic patch-clamp studies of VRACs performed in a fibroblast cell line and from single channel studies of chloride channels (tentatively related to VRACs) in mouse brown adipocytes in primary culture.</p><p>One of the characteristic features of the VRACs is their dependence on the presence of cytoplasmic ATP. In whole-cell experiments, removal of ATP from the pipette solution almost completely prevented activation of VRACs, whereas substitution of ATP with the nonhydrolyzable analog ATPγS did not alter the activation of VRACs. The inhibitors of protein tyrosine kinases (PTK) tyrphostin A25 and B46 depressed VRAC currents in both cases (ATP and ATPγS), but a PTK ineffective analog (tyrphostin A1) did not affect VRAC currents. We infer that in the cell preparation we used, ATP has a dual role in VRAC regulation: it is required for channel-protein phosphorylation and it can influence channel activity through non-hydrolytic binding in a ligand-receptor manner. It can additionally be suggested that tyrosine-specific protein kinases can be involved in the regulation of VRACs, independently of the effects of ATP. We also studied cell cycle-related changes in activation of VRACs by osmotic swelling of cells chemically arrested at different phases of the cell cycle. We found no significant changes during most of the cell cycle, except short periods before and after mitosis and in the quiescent G0 state.</p><p>The single Cl^-channels of brown adipocytes resemble in their electrophysiological phenotype outwardly rectifying Cl^- channels (ORCCs). We investigated the sensitivity of these channels to intracellular Ca²⁺. It appeared that the commonly used Ca²⁺-chelators EGTA and BAPTA could influence the ORCCs currents by themselves, independently of their calcium chelating effects. In some channels, these chelators induced classical flickery-type block of activity, whereas in others there was quasi-blockage, i.e. a peculiar combination of flickery blockage and overall channel activation. The chloride channel blocking agents DIDS and SITS mimicked the true/quasi blockage of EGTA and BAPTA. These phenomena add to the structure-function characteristics of the ORCC molecule. Moderate inhibitory effect of Ca²⁺ within a physiological range of intracellular concentrations (sub-µM) was also detected; however, the biological relevance of this observation, as well as of these Cl^- channels in general, remains to be clarified.</p>

Chloride ion channels are macromolecular pores providing for passage of chloride ions (and certain other inorganic and organic anions) through the cell membrane, down their electrochemical gradients. Chloride channels are differentially expressed in various cells, to best suit specific cellular activities. They are present in practically all living cells, and regardless of cell specialization they play an important role in vital housekeeping functions of cell-volume and pH regulation and in membrane potential stabilization. Regulation of cell volume underlies the structural integrity and constancy of the intracellular milieu. A variety of metabolic pathways have been shown to be sensitive to cell volume, and alterations of cell volume and osmoregulation processes can influence various intracellular signaling and organizing factors. Volume-regulated anion channels (VRACs) are believed to play a pivotal role in cell-volume regulating processes. In this report I present data from macroscopic patch-clamp studies of VRACs performed in a fibroblast cell line and from single channel studies of chloride channels (tentatively related to VRACs) in mouse brown adipocytes in primary culture. One of the characteristic features of the VRACs is their dependence on the presence of cytoplasmic ATP. In whole-cell experiments, removal of ATP from the pipette solution almost completely prevented activation of VRACs, whereas substitution of ATP with the nonhydrolyzable analog ATPγS did not alter the activation of VRACs. The inhibitors of protein tyrosine kinases (PTK) tyrphostin A25 and B46 depressed VRAC currents in both cases (ATP and ATPγS), but a PTK ineffective analog (tyrphostin A1) did not affect VRAC currents. We infer that in the cell preparation we used, ATP has a dual role in VRAC regulation: it is required for channel-protein phosphorylation and it can influence channel activity through non-hydrolytic binding in a ligand-receptor manner. It can additionally be suggested that tyrosine-specific protein kinases can be involved in the regulation of VRACs, independently of the effects of ATP. We also studied cell cycle-related changes in activation of VRACs by osmotic swelling of cells chemically arrested at different phases of the cell cycle. We found no significant changes during most of the cell cycle, except short periods before and after mitosis and in the quiescent G0 state. The single Cl- channels of brown adipocytes resemble in their electrophysiological phenotype outwardly rectifying Cl- channels (ORCCs). We investigated the sensitivity of these channels to intracellular Ca2+. It appeared that the commonly used Ca2+-chelators EGTA and BAPTA could influence the ORCCs currents by themselves, independently of their calcium chelating effects. In some channels, these chelators induced classical flickery-type block of activity, whereas in others there was quasi-blockage, i.e. a peculiar combination of flickery blockage and overall channel activation. The chloride channel blocking agents DIDS and SITS mimicked the true/quasi blockage of EGTA and BAPTA. These phenomena add to the structure-function characteristics of the ORCC molecule. Moderate inhibitory effect of Ca2+ within a physiological range of intracellular concentrations (sub-µM) was also detected; however, the biological relevance of this observation, as well as of these Cl- channels in general, remains to be clarified.

Cardiac ischemic preconditioning (IPC) is defined as brief periods of ischemia and reperfusion that protect the heart against longer ischemia and reperfusion. IPC triggers Cl- efflux and protein kinase C epsilon (PKCe) translocation to the particulate fraction. Chloride channel 2 (ClC-2) is volume regulated and is a potential end effector of IPC. The goal of my study was to investigate the involvement of PKCε and ClC-2 protein module in IPC of isolated adult rabbit ventricular myocytes. Co-immunoprecipitation (co-IP) assays on HEK 293 cells, transfected with ClC-2-Flag, confirmed that ClC-2 interacts with PKCe. Subcellular fractionation showed that PKCe/ClC-2 protein module is localized to the sarcolemma of cardiomyocytes. Lastly, ischemia/reperfusion injury was simulated in cardiomyocytes with 45min simulated ischemia (SI)/60min simulated reperfusion (SR) and IPC was induced by pre-treatment with 10min SI/20min SR. Co-IP after each time interval showed that IPC transiently enhanced PKCe/ClC-2 interaction. PKC inhibitor, GF109203X, abrogated the enhanced interaction.

Cardiac ischemic preconditioning (IPC) is defined as brief periods of ischemia and reperfusion that protect the heart against longer ischemia and reperfusion. IPC triggers Cl- efflux and protein kinase C epsilon (PKCe) translocation to the particulate fraction. Chloride channel 2 (ClC-2) is volume regulated and is a potential end effector of IPC. The goal of my study was to investigate the involvement of PKCε and ClC-2 protein module in IPC of isolated adult rabbit ventricular myocytes. Co-immunoprecipitation (co-IP) assays on HEK 293 cells, transfected with ClC-2-Flag, confirmed that ClC-2 interacts with PKCe. Subcellular fractionation showed that PKCe/ClC-2 protein module is localized to the sarcolemma of cardiomyocytes. Lastly, ischemia/reperfusion injury was simulated in cardiomyocytes with 45min simulated ischemia (SI)/60min simulated reperfusion (SR) and IPC was induced by pre-treatment with 10min SI/20min SR. Co-IP after each time interval showed that IPC transiently enhanced PKCe/ClC-2 interaction. PKC inhibitor, GF109203X, abrogated the enhanced interaction.

Calcium-activated chloride channels (CaCCs) are ubiquitously expressed in a plethora of cell types and, consequently, are involved in numerous cellular processes as diverse as epithelial secretion, regulation of cardiac excitability and smooth muscle contraction. Current pharmacology of CaCCs is limited to compounds with low potency and poor selectivity. The lack of knowledge surrounding the molecular identity of the CaCC has greatly hindered the development of more specific drugs and has impaired our understanding of the channel physiology and biophysics. The recent discovery that the TMEM16A gene codes for CaCCs has offered hope for new developments in these areas. CaCCs have been suggested as possible targets to treat a variety of conditions including asthma as well as pulmonary and systemic hypertension. Due to the ubiquitous expression of CaCCs and the ability of the channel to interact with a number of pharmacological compounds with diverse chemical structures however, it was hypothesised that TMEM16A could be a possible source for off-target drug effects and may represent a concern for safety pharmacology. The principal aim of this thesis was to assess the functional significance of TMEM16A in the cardiovascular system, as this is one of the major systems of concern for safety pharmacology and accounts for the largest number of post-market drug withdrawals. The main findings of this study can be summarised as follows: 1) RT-PCR analysis revealed a ubiquitous expression of TMEM16A in tissues of the rat and human cardiovascular systems, including systemic and pulmonary arteries as well as cardiac tissue. Analysis also revealed the presence of multiple TMEM16A splice variants in all rat tissues examined, in addition to a number of other TMEM16x family members. 2) Myography experiments using the “classical” CaCC blocker niflumic acid and newly identified TMEM16A blockers confirmed a functional role for TMEM16A in phenylephrine-induced vascular smooth muscle contraction. 3) The suitability of currently available Cl- channel blockers for use as pharmacological tools for TMEM16A research was assessed using conventional whole-cell patch clamp and high-throughput electrophysiology techniques to respectively compare their potencies and selectivity over other cardiovascular ion channels. Of the compounds tested, DIDS and T16Ainh-A01 appeared the most suitable blockers; however all compounds had a degree of non-selectivity, raising concerns for their use in functional studies. In conclusion, these findings provide evidence for the ubiquitous expression and functional significance of TMEM16A within the cardiovascular system and support the hypothesis that TMEM16A is a concern for safety pharmacology and should be included into future pre-clinical safety assays. The inadequacy of current inhibitors however highlights the urgency for the development of novel potent and selective channel modulators for future TMEM16A research.

Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society. A volume-regulated anion channel (VRAC) has been electrophysiologically characterized in innumerable mammalian cell types. VRAC is activated by cell swelling and mediates the volume regulatory efflux of Cl− and small organic solutes from cells. Two groups recently identified the mammalian leucine-rich repeat containing protein LRRC8A as an essential VRAC component. LRRC8A must be coexpressed with at least one of the other four members of this gene family, LRRC8B-E, to reconstitute VRAC activity in LRRC8−/− cells. LRRC8 genes likely arose with the origin of chordates. We identified LRRC8A and LRRC8C-E orthologs in the zebrafish genome and demonstrate that zebrafish embryo cells and differentiated adult cell types express a swelling-activated Cl− current indistinguishable from mammalian VRAC currents. Embryo cell VRAC currents are virtually eliminated by morpholino knockdown of the zebrafish LRRC8A ortholog lrrc8aa. VRAC activity is fully reconstituted in LRRC8−/− human cells by coexpression of zebrafish lrrc8aa and human LRRC8C cDNAs. lrrc8aa expression varies during zebrafish embryogenesis and lrrc8aa knockdown causes pericardial edema and defects in trunk elongation and somatogenesis. Our studies provide confirmation of the importance of LRRC8A in VRAC activity and establish the zebrafish as a model system for characterizing the molecular regulation and physiological roles of VRAC and LRRC8 proteins.

Yes / Ion channels regulate many aspects of cell physiology, including cell proliferation, motility, and migration, and aberrant expression and activity of ion channels is associated with various stages of tumor development, with K+ and Cl- channels now being considered the most active during tumorigenesis. Accordingly, emerging in vitro and preclinical studies have revealed that pharmacological manipulation of ion channel activity offers protection against several cancers. Merkel cell polyomavirus (MCPyV) is a major cause of Merkel cell carcinoma (MCC), primarily due to the expression of two early regulatory proteins termed small and large tumour antigens (ST and LT, respectively). Several molecular mechanisms have been attributed to MCPyVmediated cancer formation but thus far, no studies have investigated any potential link to cellular ion channels. Here we demonstrate that Cl- channel modulation can reduce MCPyV STinduced cell motility and invasiveness. Proteomic analysis revealed that MCPyV ST upregulates two Cl- channels; CLIC1 and CLIC4, which when silenced, inhibit MCPyV STinduced motility and invasiveness, implicating their function as critical to MCPyV-induced metastatic processes. Consistent with these data, we confirmed that CLIC1 and CLIC4 are upregulated in primary MCPyV-positive MCC patient samples. We therefore, for the first time, implicate cellular ion channels as a key host cell factor contributing to virus-mediated cellular transformation. Given the intense interest in ion channel modulating drugs for human disease, this highlights CLIC1 and CLIC4 activity as potential targets for MCPyV-induced MCC. / BBSRC DTP studentship (BB/J014443/1) and Royal Society University Research Fellowship to JM (UF100419)