Background: Pulmonary arterial hypertension (PAH) is a condition with severe morbidity and mortality. It is associated with an increase in incidence of all forms of arrhythmias which further increase morbidity and mortality. The monocrotaline (MCT) model of pulmonary hypertension (PH) in the rat is analogous to PAH in humans and was used to study how PH causes arrhythmias. Methods: A single injection of MCT or a volume matched saline injection (control) was given to the rats on day 0 of the protocol. The hearts of both groups of rats were studied in vivo with echocardiography (echo) and electrocardiogram (ECG). The rat’s condition was monitored and they were electively sacrificed when they showed symptoms or on day 28. Live cardiac tissue was studied using the Langendorff preparation and a right atrial preparation that incorporated the sinoatrial (SA) and atrioventricular (AV) nodes. Molecular biology techniques including reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry were used identify changes in the heart caused by PH. The effects of macitentan, an endothelin receptor antagonist used in the treatment of PAH, on the MCT injected rats was assessed using echo and ECGResults: Echo demonstrated that the MCT treated rats developed severe pulmonary hypertension with a decreased pulmonary artery acceleration time (P<0.005) and an increased pulmonary artery deceleration (P<0.005). The MCT treated rats also developed right ventricular hypertrophy (P<0.05) and dilation (P<0.005). The in vivo ECG demonstrated QT prolongation (P<0.005). Ex vivo functional experiments demonstrated QT prolongation (P<0.005) and prolonged ventricular effective refractory period (P<0.005). AV node dysfunction was also seen in the ex vivo experiments with an increased AV effective refractory period (P<0.05), AV functional refractory period (P<0.05) and incidence of complete heart block (P<0.05). RT-qPCR demonstrated significant changes in the mRNA expression of several ion channels and exchanges, Ca2+ handling proteins and autonomic receptors including a downregulation of HCN4 and CaV1.2 in the AV nodal tissues (P<0.05). Treatment of established pulmonary hypertension led to a reduction in the prolongation of the QT interval caused by MCT administration at day 21 (P<0.05).Conclusions: PH causes arrhythmogenic changes including prolonged repolarisation in the working myocardium and AV node dysfunction. These changes may be caused by dysregulation of ion channels and Ca2+ handling proteins. These ion channels and Ca2+handling proteins may play a key role in both physiological and pathological processes within the AV node.

No / Lymphocytes have many ion channels. These ion channels contribute to T cell-mediated autoimmune and/or inflammatory responses and therefore are targets for pharmacological immune modulation [1]. Lymphocytes are also suitable surrogate cells for cancer [2] and other diseases states [3] where inflammation is associated with increasing disease incidence. Non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, have been associated with anti-tumour effects in cancers [4]. We recently compared DNA damage caused by the nanoparticle forms (NPs) of the NSAIDs, aspirin and ibuprofen and their bulk forms in peripheral blood lymphocytes of patients with respiratory diseases and healthy individuals in the Comet and micronucleus assays [5]. In this present study, we investigate electrophysiological changes from lymphocytes after NP exposure and compare these results with their DNA damage. 10 ml peripheral blood was collected from patients and healthy control individuals. Ethical permission was obtained from the Bradford Ethics Committee REC ref no: 09/H1313/37, ReDA no: 1202, and the University of Bradford ref no: 0405/8. Ibuprofen USP was purchased from Albermarle Europe sprl (Belgium). Pharmcoat 606 (HPMC) was kindly donated by Shinetsu (Japan). Aspirin and sodium lauryl sulphate were purchased from Sigma. Kollidon 30 (PVP K-30) was purchased from BASF (UK). Bulk and nano compound suspensions of aspirin and ibuprofen (IBU) were kindly prepared by Lena Nanoceutics (Bradford, UK). Whole blood collected from healthy individuals and cancer patients were treated for 30 mins with 500µg/ml of IBU bulk and nano forms separately. Whole-cell currents were recorded with normal patch clamping technique. The extracellular solution contained (in mM) the following: NaCl 125; KCl 5; MgCl2 1; CaCl2 2.5; HEPES 10; pH 7.4. The electrode internal solution contained (in mM) the following: KF 120; MgCl2 2; HEPES 10; EGTA 10; and CaCl2 1, pH 7.4. All experiments were carried out at room temperature. Compared with untreated cells, lymphocytes treated with IBU in NP form had lower whole-cell currents and the activities of ion channels were inhibited by 20% compared to those in bulk form. This result is mirrored by the DNA damage which occurred in lymphocytes after exposure to nanoparticles [5]. Although the intracellular biochemical mechanisms and ion channels involved in our nanoparticle toxicity remain to be determined, this study provides direct evidence that 500 μg/ml IBU in nano form can cause membrane damage to lymphocytes after a relatively short exposure. Such cytotoxicity of nanoparticles in lymphocytes may be associated with early membrane damage. Further detailed investigation is needed to explain the changes of lymphocytes in response to different concentrations of NPs in real time. / Poster communications

Phosphatidylinositol bisphosphate (PIP2) directly regulates electrophysiological activity in a diverse family of ion channels whether the effect is stimulatory or inhibitory. Much has been unveiled about the apparent affinity and modulatory function of PIP2 using a chemically modified dioctanoyl PIP2 (diC8), a membrane delimited cytosolic co-factor in inside-out macropatch experiments. Yet, the scarcity of molecular tools that permit fine external control in whole-cell systems has precluded future studies from probing the physiological role of PIP2 in cells in the presence of a fully intact cytoplasm. Here we introduce light as an external control for PIP2 through photocaging of diC8, and test its activation of Kir2.3 (IRK3), an inwardly rectifying ion channel that has previously shown to possess moderate binding affinity to PIP2, in excised, inside-out macropatches. Our experiments revealed that photocaged-diC8 and irradiated photocaged-diC8 have significantly different activation kinetics than the fully active diC8. Surprisingly, the activation of caged-diC8 by UV irradiation attenuated Kir2.3 activity, while the inactivated diC8 (caged-diC8) resulted in similar magnitude of channel activity compared to the currents elicited by unmodified diC8. Interestingly, we also show that application of both activated (irradiated) and inactive (caged) diC8 in macropatches generated highly fluctuating ion channel activity.

ABSTRACT The Chloride Intracellular Channel (CLIC) proteins are a family of amphitropic proteins that can convert from soluble to integral membrane forms. CLIC1 is a member of this family that functions as a chloride channel in the plasma and nuclear membranes of cells. Although high-resolution structural data exists for the soluble form of monomeric CLIC1, not much is known about the integral membrane forms’ structure. The exact mechanism and signals involved in the conversion of the soluble form to membrane-inserted form are also not clear. Studies were undertaken in the absence and presence of membrane models. Analysis of the structure and stability of CLIC1 in the absence of membrane investigated the effect of possible signals or triggers that may play a crucial role in the conversion of the soluble form to integral membrane form. Exposing CLIC1 to oxidizing conditions results in the formation of a dimeric form. The CLIC1 dimer was found to be less stable than the monomeric form based on unfolding kinetic studies. The stability of the dimer was also less influenced by salt concentration, compared with the monomer. The effect of pH on the structure of CLIC1 is of physiological relevance since the movement of soluble CLIC1 in the cytoplasm or nucleoplasm toward the membrane will involve the protein being exposed to a lower pH micro-environment. Hydrogen exchange mass spectrometry was used to study the structural dynamics of CLIC1 at pH 7.0 and pH 5.5. At neutral pH, domain II is more stable than the more flexible thioredoxin domain I. The thioredoxin-fold therefore is more likely to unfold and rearrange to insert into membranes. Because of the high stability of domain II this region is probably where the folding nucleus of the protein is. At pH 5.5 it was found that the a1, a3 and a6 helices, which are spatially adjacent to one another across the domain interface, were destabilized. This destabilization may be the trigger for CLIC1 to unfold and rearrange into a membrane insertion-competent form. The role of the primary sequence and unique three-dimensional structure of CLIC1 in membrane insertion was investigated in a bioinformatics-based study that looked at conserved residue features such as hydropathy and charge. Hidden helical propensities and Ncapping motifs in the a1-b2 region were found, which may have important implications for locating putative transmembrane regions. Analysis of the structure and thermodynamics of CLIC1 interacting with membranes investigated changes in secondary structure, tertiary structure, hydrodynamic volume and thermodynamics when CLIC1 is exposed to membrane-mimicking models. The effect of a variety of conditions such as pH and redox, cysteine-modifiying agents (NEM), ligands (GSH), and inhibitors (IAA) on CLIC1 membrane interaction were studied. It was found that CLIC1 interacted with membranes more favourably at lower pH and that NEM completely inhibited CLIC1 interaction with micelles.

Abstract In dim light, reliable coding of visual information becomes compromised, unless the sensitivity of the visual system to light is improved by structural and functional adaptations. Thus far, many adaptations for night vision in the compound eyes of nocturnal insects have been described, but little is known about the mechanisms underlying the electrochemical signalling in their photoreceptors. In this thesis, whole-cell patch-clamp and mathematical modelling are utilised to study basic electrical properties and ionic currents in photoreceptors of two nocturnal insects, the American cockroach Periplaneta americana and the field cricket Gryllus bimaculatus. Photoreceptors in both species showed large input resistance, membrane capacitance and phototransduction gain (large single photon responses) compared with most studied diurnal insects, providing improved sensitivity to light. The photoreceptors also expressed two voltage-sensitive outward currents: a transient current and a sustained current. The cricket photoreceptor expressed a dominating transient current, which is a typical characteristic for insects adapted for slow vision in dim light. By contrast, in the majority of cockroach photoreceptors the sustained current dominated, which is more common among fast diurnal species. Model simulations indicated that the sustained current is necessary for improved photoreceptor dynamics. Examination of light-induced currents suggested that the functional variability in cockroach photoreceptors is in part derived from variations in the total area of the photosensitive membrane. Recordings of light-induced currents also revealed that the cockroach light-gated channels are only moderately Ca2+-selective and that the polarisation-sensitive photoreceptors of the cricket may utilise phototransduction machinery in some details different from that in regular photoreceptors. Furthermore, the dynamics and information transfer rates of polarisation-sensitive photoreceptors in the cricket were clearly inferior to their regular counterparts, suggesting that they are not necessary for image formation.

Introduction: Factors that influence atrioventricular (AV) nodal conduction are complex and not well understood. Multiple studies have been performed to explain the mechanisms responsible for AV nodal conduction but the AV node (AVN) remains a "riddle". With ageing there is an increase in the incidence of AV nodal dysfunction leading to AV block. Methodology: I have performed electrophysiological (EP) and immunohistochemistry experiments on male Wistar-Hanover rats aged 3 months (equivalent to 20 year old humans; n=24) and 2 years (equivalent to 70 year old humans; n=15). AH interval, Wenkebach cycle length (WCL) and AV node effective refractory period (AVNERP) were measured. I used cesium (Cs+ = 2 mM) to block HCN channels responsible for the funny current "If " (and therefore the membrane clock), and ryanodine (2 μM) to block RyR2 channels responsible for Ca2+ release from the sarcoplasmic reticulum (and therefore the Ca2+ clock) in the two age groups. Protein expression in each group (from n=9 young and n=8 old rats) from different regions of the AV conduction axis: inferior nodal extension (INE), compact node (CN), proximal penetrating bundle (PPB) and distal penetrating or His bundle (His) were studied using immunofluorescence and confocal microscopy. The expression of the gap junction channels Cx43 and Cx40 and ion channel’s including HCN4 (responsible for If current), Nav1.5 (major cardiac Na+ channel responsible for INa) and Cav1.3 (L-type Ca2+ channel), and calcium handling proteins, RyR2 and SERCA 2a (involved in Ca2+ release and reuptake from cardiac sarcoplasmic reticulum, SR) were studied. Semi-quantitative signal intensity of these channels was measured using Volocity software. Structural characteristics of the tissue were studied using histology (Masson’s trichome stain and picrosirius red stain for collagen). Statistical analysis was performed with Prism 6.0. Electrophysiological measurements were performed using Spike2.Results: Without drugs to block the If current and Ca2+ release from the SR, there was a significant prolongation of the AH interval (P<0.005), WCL (P<0.005) and AVNERP (P<0.001) with ageing. In young rats (but not old rats), Cs+ prolonged the AH interval (P<0.001), WCL (P<0.01) and AVNERP (P<0.01). Ryanodine prolonged the AH interval (P<0.01) and WCL (P<0.01) in young and old rats. Immunofluorescence revealed that with ageing: Cx43 is downregulated in the PPB and His (P<0.05); Cx40 is upregulated in the INE and CN (P<0.05); HCN4 is downregulated in the His bundle (P=0.05); Nav1.5 is downregulated in the CN and PB (P<0.05); RyR2 is downregulated in the CN and PPB (P<0.05); SERCA2a and Cav1.3 is upregulated in the PPB (P<0.05). Histology confirmed that with ageing that the cells of CN, PPB and His are more loosely packed and irregularly arranged. There is cellular hypertrophy, decrease in the number of nuclei and increase in the collagen content with ageing. The clinical study has shown that elderly patients with syncope and cardiac conduction system disease are at risk of high mortality and recurrent transient loss of consciousness. Conclusion: For the first time, we have shown that both HCN and RyR2 channels play an important role in AV nodal conduction. With ageing the expression of HCN4 and the role of If in AV nodal conduction decreases, whereas the role of Ca2+ clock in AV nodal conduction was unchanged, although the expression of RyR2 and SERCA2a changes. The clinical study suggests that AV nodal disease is associated with significant morbidity and higher mortality among elderly patients who present with transient loss of consciousness.

Opioids are gold-standard analgesics for pain relief in chronic pain conditions. Paradoxically, chronic opioid use causes an enhanced pain sensitivity termed ‘Opioid-induced hyperalgesia’ (OIH). OIH is a clinically relevant problem associated with the use of opioids. In addition to decreasing quality of life, increased pain from OIH necessitates increasing dosages of analgesics to effectively control the pain, resulting in an increased risk of opioid epidemics, addiction, and overdose. To prevent this clinically important effect, it is necessary to understand how chronic opioid use causes hyperalgesia. Our preliminary studies revealed that synaptic plasticity in the spinal dorsal horn (SDH) is dependent on neuron type in the OIH model and occurs concurrently with hyperalgesia, suggesting central sensitization as a mechanism of OIH. We found that astrocyte ablation blocked mechanical hyperalgesia and neuron type-dependent synaptic plasticity, indicating that astrocytes are critically involved in OIH. Additionally, morphine treatment upregulated IL-1β expression in the SDH in our preliminary experiments. Inhibition of IL-1β prevented OIH and blocked the repeated morphine-induced synaptic plasticity in the SDH, suggesting IL-1β is a key player in the pathogenesis of OIH. Astrocytes and other glial cells are critical in the development and maintenance of neuroinflammatory conditions, such as OIH, through the release of proinflammatory cytokines (PICs), including IL-1β. The mechanosensitive ion channel, Piezo1, was recently found to be upregulated in astrocytes and microglia under LPS-induced inflammatory conditions, and activation of Piezo1 was found to reduce IL-1β expression in LPS-inflamed primary mouse astrocytes. The goal of this study was to investigate the function of Piezo1 as a potential treatment for neuroinflammatory diseases of the CNS in a model of LPS-induced inflammation. In this study, we created a culture cell model of LPS-induced astrocytic neuroinflammation using the C8-S type II astrocyte culture cell line. We used a multi-disciplinary approach of electrophysiology and imaging to assess changes in calcium flux induced by the selective Piezo1 agonist, Yoda1, and mechanosensitive ion channel activity in the LPS-stimulated C8-S culture astrocytes. We found that calcium flux is increased in LPS stimulation and augmented by additional Yoda1 treatment. We also found that LPS stimulation increases mechanosensitive ion currents and stiffens cell membranes using patch-clamp electrophysiology techniques. These results indicate that Piezo1 is likely upregulated in the LPS model of cultured astrocytes, thus mechanosensitive responses are increased. Results from these experiments reveal key information about the mechanical properties of Piezo1 and poise Piezo1 as a promising therapeutic for OIH and other neuroinflammatory diseases caused by astrocytic IL-1β release.

A number of studies have demonstrated the ability of free fatty acids to activate taste cells and elicit behavioral responses consistent with there being a taste of fat. Here I show for the first time that long chain unsaturated free fatty acid, linoleic acid, depolarizes taste cells and elicits a robust intracellular calcium rise via the activation of transient receptor potential channel type M5. The linoleic acid-induced responses depend on G protein-phospholipase C pathway indicative of the involvement of G protein-coupled receptors in the transduction of fatty acids. Mice lacking transient receptor potential channel type M5 exhibit no preference for and show reduced sensitivity to linoleic acid. Together, these studies show that transient receptor potential channel type M5 plays an essential role in fatty acid transduction and suggest that fat may reflect a bona fide sixth primary taste. Studies to identify the types of taste cells that respond to fatty acids show that both type II and type III taste cells express fatty acid-activated receptors. Fatty acids elicit robust intracellular calcium rise primarily in type II taste cells and a subset of type III taste cells. However, a significant subset of type II taste cells respond to high potassium chloride, which has been broadly used as the indicator for type III taste cells as well, suggesting the expression of voltage-gated calcium channels in these cells. This finding conflicts with previous studies that type II taste cells lack voltage-gated calcium channels. To explore if voltage-gated calcium channels are expressed in subsets of type II taste cells, transgenic mice with type II or III taste cells marked by green fluorescent proteins are used. Results show that a subset of type II taste cells exhibit voltage-gated calcium currents, verifying the expression of voltage-gated calcium channels in these cells. These results question the utility of being able to use high potassium chloride solution to identify unequivocally type III taste cells within the taste buds. A model for the transduction of fatty acids in taste cells consistent with these findings and our previous data is presented.

At the neuromuscular junction, nicotinic acetylcholine receptors (AChRs) convert chemical stimuli into electrical signals. They are heteropentameric membrane protein complexes assembled from four evolutionary related subunits (two α subunits, and one each of the β-, δ-, and ε-subunits), arranged around a central ion-conducting pore, which is regulated by the neurotransmitter acetylcholine. Understanding how the binding of acetylcholine leads to channel opening is of fundamental importance. While it is known that channel opening results from a global conformational change involving the cooperative action of all five subunits, how the subunits achieve this cooperativity is unclear. Our hypothesis is that this subunit cooperation is maintained through coevolution of the subunits, and thus studies of subunit coevolution can provide insight into subunit cooperativity. Using an ancestral reconstruction approach, combined with single-molecule patch clamp electrophysiology, we have begun dissecting the mechanistic consequences of preventing coevolution of the acetylcholine receptor β-subunit. This approach has allowed us to identify new amino acid determinants of acetylcholine receptor function.

No / channels that are critical for their development and function. Many ion channels contribute to T cell-mediated autoimmune and/or inflammatory responses, so they are attractive targets for pharmacological immune modulations. In this study, we conduct patch clamp experiments to exam the whole cell currents from lymphocytes after nanoparticles exposure with the aim to test if nanoparticles exposure brings any electrophysiological changes for lymphocytes, and to compare the electrophysiological responses of lymphocytes to drugs in nanoparticles forms. Our result suggests a potential inhibition of effects of IBU N on lymphocytes. Such cytotoxicity of nanoparticles in Lymphocytes may be mainly associated with the early membrane damage. These results are also mirrored by the DNA damages occurred on lymphocytes after exposure of nanoparticles. Further detailed investigation is needed to explain the changes of Lymphocytes in response to NPs in real time and dose differences. This would provide useful information in the evaluation of toxicology of nanoparticles and in understanding the underlying mechanism of their effects on ion channels in health and diseases.