Glucose and hypotonicity induced cell swelling stimulate insulin release from pancreatic β-cells but the mechanisms are poorly understood. Recently, Piezo1 was identified as a mechanically-activated nonselective Ca 2+ permeable cationic channel in a range of mammalian cells. As cell swelling induced insulin release could be through stimulation of Ca 2+ permeable stretch activated channels, we hypothesised a role for Piezo1 in cell swelling induced insulin release. Two rat β-cell lines (INS-1 and BRIN-BD11) and freshly-isolated mouse pancreatic islets were studied. Intracellular Ca 2+ measurements were performed using the fura-2 Ca 2+ indicator dye and ionic current was recorded by whole cell patch-clamp. Piezo1 agonist Yoda1, a competitive antagonist of Yoda1 (Dooku1) and an inactive analogue of Yoda1 (2e) were used as chemical probes. Piezo1 mRNA and insulin secretion were measured by RT-PCR and ELISA respectively. Piezo1 mRNA was detected in both β-cell lines and mouse islets. Yoda1 evoked Ca 2+ entry was inhibited by Yoda1 antagonist Dooku1 as well as other Piezo1 inhibitors gadolinium and ruthenium red, and not mimicked by 2e. Yoda1, but not 2e, stimulated Dooku1-sensitive insulin release from β-cells and pancreatic islets. Hypotonicity and high glucose increased intracellular Ca 2+ and enhanced Yoda1 Ca 2+ influx responses. Yoda1 and hypotonicity induced insulin release were significantly inhibited by Piezo1 specific siRNA. Pancreatic islets from mice with haploinsufficiency of Piezo1 released less insulin upon exposure to Yoda1. The data show that Piezo1 channel agonist induces insulin release from β-cell lines and mouse pancreatic islets suggesting a role for Piezo1 in cell swelling induced insulin release. Hence Piezo1 agonists have the potential to be used as enhancers of insulin release.

The anti-cancer receptor tyrosine kinase inhibitors include known cardiotoxins: a component of this toxicity may be mediated by effects on cardiac fibroblasts (CFs). We hypothesised that imatinib mesylate (imatinib) and sunitinib malate (sunitinib) cause significant dysfunction in adult CFs. Following in vitro treatments with imatinib or sunitinib, adult rat CF viability was assessed by fluorescein diacetate assay, proliferation measured by bromodeoxyuridine nuclear incorporation and changes to the expression of CF secretome components determined by real time quantitative RT-PCR. Imatinib and sunitinib significantly reduced cell viability over 48 h, with EC50 values of 11.0 μM (imatinib) and 4.5 μM (sunitinib) respectively. Imatinib reduced CF proliferation from 35.5 ± 3.2% in control to 23.0 ± 5.5% (3 μM; p < 0.001) and to 9.4 ± 2.5% (10 μM; p < 0.001), whereas sunitinib reduced proliferation to 22.9 ± 3.1% (1 μM; p < 0.001) and to 15 ± 1.0% (3 μM; p < 0.001). Further, 10 μM imatinib increased mRNA expression of TGFB1 7-fold, (p < 0.01), IL6 6-fold (p < 0.01), and IL1B 7-fold (p < 0.05) and reduced PDGFD 15-fold (p < 0.01); whereas sunitinib specifically reduced IL1B mRNA expression 17-fold (p < 0.01). Overall, these findings show tyrosine kinase inhibitors cause significant dysfunction in CFs. These data point to an important role for the PDGF pathway in governing CF functions, including survival and proliferation.

Rise in plasma free fatty acids (FFAs) represents a major risk factor for obesity-induced type 2 diabetes. Saturated FFAs cause a progressive decline in insulin secretion by promoting pancreatic β-cell death through increased production of reactive oxygen species (ROS). Recent studies have demonstrated that palmitate (a C16-FFA)-induced rise in ROS causes β-cell death by triggering mitochondrial fragmentation, but the underlying mechanisms are unclear. Using the INS1-832/13 β-cell line, here we demonstrate that palmitate generates the ROS required for mitochondrial fission by activating NOX (NADPH oxidase)-2. More importantly, we show that chemical inhibition, RNAi-mediated silencing and knockout of ROS-sensitive TRPM (transient receptor potential melastatin)-2 channels prevent palmitate-induced mitochondrial fission. Although TRPM2 activation affects the intracellular dynamics of Ca2+ and Zn2+, chelation of Zn2+ alone was sufficient to prevent mitochondrial fission. Consistent with the role of Zn2+, palmitate caused a rise in mitochondrial Zn2+, leading to Zn2+-dependent mitochondrial recruitment of Drp-1 (a protein that catalyses mitochondrial fission) and loss of mitochondrial membrane potential. In agreement with the previous reports, Ca2+ caused Drp-1 recruitment, but it failed to induce mitochondrial fission in the absence of Zn2+. These results indicate a novel role for Zn2+ in mitochondrial dynamics. Inhibition or knockout of TRPM2 channels in mouse islets and RNAi-mediated silencing of TRPM2 expression in human islets prevented FFA/cytokine-induced β-cell death, findings that are consistent with the role of abnormal mitochondrial fission in cell death. To conclude, our results reveal a novel, potentially druggable signalling pathway for FFA-induced β-cell death. The cascade involves NOX-2-dependent production of ROS, activation of TRPM2 channels, rise in mitochondrial Zn2+, Drp-1 recruitment and abnormal mitochondrial fission.

Diabetic stress increases the production of reactive oxygen species (ROS), leading to mitochondrial fragmentation and dysfunction. Wehypothesized that ROS-sensitive TRPM2 channels mediated diabetic stress-induced mitochondrial fragmentation. We found that chemical inhibitors, RNAi silencing, and genetic knockout of TRPM2 channels abolished the ability of high glucose to causemitochondrial fission in endothelial cells, a cell type that is particularly vulnerable to diabetic stress. Similar to high glucose, increasing ROS in endothelial cells by applying H2O2 induced mitochondrial fission. Ca2+ that entered through TRPM2 induced lysosomal membrane permeabilization, which led to the release of lysosomal Zn2+ and a subsequent increase in mitochondrial Zn2+. Zn2+ promoted the recruitment of the fission factor Drp-1 to mitochondria to trigger their fission. This signaling pathwaymay operate in aging-associated illnesses inwhich excessive mitochondrial fragmentation plays a central role.

Metformin is the main drug of choice for treating type 2 diabetes, yet the therapeutic regimens and side effects of the compound are all undesirable and can lead to reduced compliance. The aim of this study was to elucidate the mechanism of action of two novel compounds which improved glucose handling and weight gain in mice on a high-fat diet. Wildtype C57Bl/6 male mice were fed on a high-fat diet and treated with novel, anti-diabetic compounds. Both compounds restored the glucose handling ability of these mice. At a cellular level, these compounds achieve this by inhibiting complex I activity in mitochondria, leading to AMP-activated protein kinase activation and subsequent increased glucose uptake by the cells, as measured in the mouse C2C12 muscle cell line. Based on the inhibition of NADH dehydrogenase (IC50 27µmolL(-1)), one of these compounds is close to a thousand fold more potent than metformin. There are no indications of off target effects. The compounds have the potential to have a greater anti-diabetic effect at a lower dose than metformin and may represent a new anti-diabetic compound class. The mechanism of action appears not to be as an insulin sensitizer but rather as an insulin substitute.

Reactive oxygen species (ROS) can cause pancreatic β-cell death by activating transient receptor potential (melastatin) 2 (TRPM2) channels. Cell death has been attributed to the ability of these channels to raise cytosolic Ca2+. Recent studies however revealed that TRPM2 channels can also conduct Zn2+, but the physiological relevance of this property is enigmatic. Given that Zn2+ is cytotoxic, we asked whether TRPM2 channels can permeate sufficient Zn2+ to affect cell viability. To address this, we used the insulin secreting (INS1) β-cell line, human embryonic kidney (HEK)-293 cells transfected with TRPM2 and pancreatic islets. H2O2 activation of TRPM2 channels increases the cytosolic levels of both Ca2+ and Zn2+ and causes apoptotic cell death. Interestingly, chelation of Zn2+ alone was sufficient to prevent β-cell death. The source of the cytotoxic Zn2+ is intracellular, found largely sequestered in lysosomes. Lysosomes express TRPM2 channels, providing a potential route for Zn2+ release. Zn2+ release is potentiated by extracellular Ca2+ entry, indicating that Ca2+-induced Zn2+ release leads to apoptosis. Knockout of TRPM2 channels protects mice from β-cell death and hyperglycaemia induced by multiple low-dose streptozotocin (STZ; MLDS) administration. These results argue that TRPM2-mediated, Ca2+-potentiated Zn2+ release underlies ROS-induced β-cell death and Zn2+, rather than Ca2+, plays a primary role in apoptosis.

Members of the six-transmembrane segment family of ion channels share a common structural design. However, there are sequence differences between the members that confer distinct biophysical properties on individual channels. Currently, we do not have 3D structures for all members of the family to help explain the molecular basis for the differences in their biophysical properties and pharmacology. This is due to low-level expression of many members in native or heterologous systems. One exception is rat Kv1.2 which has been overexpressed in Pichia pastoris and crystallised. Here, we tested chimaeras of rat Kv1.2 with the hERG channel for function in Xenopus oocytes and for overexpression in Pichia . Chimaera containing the S1–S6 transmembrane region of HERG showed functional and pharmacological properties similar to hERG and could be overexpressed and purified from Pichia . Our results demonstrate that rat Kv1.2 could serve as a surrogate to express difficult-to-overexpress members of the six-transmembrane segment channel family.

Voltage-gated ion (K+, Na+, Ca2+) channels contain a pore domain (PD) surrounded by four voltage sensing domains (VSD). Each VSD is made up of four transmembrane helices, S1-S4. S4 contains 6-7 positively charged residues (arginine/lysine) separated two hydrophobic residues, whereas S1-S3 contribute to two negatively charged clusters. These structures are conserved among all members of the voltage-gated ion channel family and play essential roles in voltage gating. The role of S4 charged residues in voltage gating is well established: During depolarization, they move out of the membrane electric field, exerting a mechanical force on channel gates, causing them to open. However, the role of the intervening hydrophobic residues in voltage sensing is unclear. Here we studied the role of these residues in the prototypical Shaker potassium channel. We have altered the physicochemical properties of both charged and hydrophobic positions of S4 and examined the effect of these modifications on the gating properties of the channel. For this, we have introduced cysteines at each of these positions, expressed the mutants in Xenopus oocytes, and examined the effect of in situ addition of charge, via Cd2+, on channel gating by two-electrode voltage clamp. Our results reveal a face of the S4 helix (comprising residues L358, L361, R365 and R368) where introduction of charge at hydrophobic positions destabilises the closed state and removal of charges from charged positions has an opposite effect. We propose that hydrophobic residues play a crucial role in limiting gating to a physiological voltage range.

The zebrafish, Danio rerio, is emerging as an important model organism for the pathophysiological study of some human kidney diseases, but the sites of expression and physiological roles of a number of protein orthologues in the zebrafish nephron remain mostly undefined. Here we show that a zebrafish potassium channel is orthologous to the mammalian kidney potassium channel, ROMK. The cDNA (kcnj1) encodes a protein (Kcnj1) that when expressed in Xenopus laevis oocytes displayed pH- and Ba2+-sensitive K+-selective currents, but unlike the mammalian channel, was completely insensitive to the peptide inhibitor tertiapin-Q. In the pronephros, kcnj1 transcript expression was restricted to a distal region and overlapped with that of sodium–chloride cotransporter Nkcc, chloride channel ClC-Ka, and ClC-Ka/b accessory subunit Barttin, indicating the location of the diluting segment. In a subpopulation of surface cells, kcnj1 was coexpressed with the a1a.4 isoform of the Na+/K+-ATPase, identifying these cells as potential K+ secretory cells in this epithelium. At later stages of development, kcnj1 appeared in cells of the developing gill that also expressed the a1a.4 subunit.Morpholino antisense-mediated knockdown of kcnj1 was accompanied by transient tachycardia followed by bradycardia, effects consistent with alterations in extracellular K+ concentration in the embryo.Our findings indicate that Kcnj1 is expressed in cells associated with osmoregulation and acts as a K+ efflux pathway that is important in maintaining extracellular levels of K+ in the developing embryo.

Abstract The hERG potassium channel is a member of the voltage gated potassium (Kv) channel family, comprising a pore domain and four voltage sensing domains (VSDs). Like other Kv channels, the VSD senses changes in membrane voltage and transmits the signal to gates located in the pore domain; the gates open at positive potentials (activation) and close at negative potentials, thereby controlling the ion flux. hERG, however, differs from other Kv channels in that it is activated slowly but inactivated rapidly – a property that is crucial for the role it plays in the repolarization of the cardiac action potential. Voltage-gating requires movement of gating charges across the membrane electric field, which is accomplished by the transmembrane movement of the fourth transmembrane segment, S4, of the VSD containing the positively charged arginine or lysine residues. Here we ask if the functional differences between hERG and other Kv channels could arise from differences in the transmembrane movement of S4. To address this, we have introduced single cysteine residues into the S4 region of the VSD, expressed the mutant channels in Xenopus oocytes and examined the effect of membrane impermeable para-chloromercuribenzene sulphonate on function by the two-electrode voltage clamp technique. Our results show that depolarization results in the accessibility of seven consecutive S4 residues, including the first two charged residues, K525 and R528, to extracellularly applied reagent. These data indicate that the extent of S4 movement in hERG is similar to other Kv channels, including the archabacterial KvAP and the Shaker channel of Drosophila.

The ATP-sensitive potassium (K(ATP)) channel controls insulin secretion by coupling glucose metabolism to excitability of the pancreatic beta-cell membrane. The channel comprises four subunits each of Kir6.2 and the sulphonylurea receptor (SUR1), encoded by KCNJ11 and ABCC8, respectively. Mutations in these genes that result in reduced activity or expression of K(ATP) channels lead to enhanced beta-cell excitability, insulin hypersecretion and hypoglycaemia, and in humans lead to the clinical condition congenital hyperinsulinism (CHI). Here we have investigated the molecular basis of the focal form of CHI caused by one such mutation in Kir6.2, E282K. The study led to the discovery that Kir6.2 contains a di-acidic ER exit signal, (280)DLE(282), which promotes concentration of the channel into COPII-enriched ER exit sites prior to ER export via a process that requires Sar1-GTPase. The E282K mutation abrogates the exit signal, and thereby prevents the ER export and surface expression of the channel. When co-expressed, the mutant subunit was able to associate with the wild-type Kir6.2 and form functional channels. Thus unlike most mutations, the E282K mutation does not cause protein mis-folding. Since in focal CHI, maternal chromosome containing the K(ATP) channel genes is lost, beta-cells of the patient would lack wild-type Kir6.2 to rescue the mutant Kir6.2 subunit expressed from the paternal chromosome. The resultant absence of functional K(ATP) channels leads to insulin hypersecretion. Taken together, we conclude that surface expression of K(ATP) channels is critically dependent on the Sar1-GTPase-dependent ER exit mechanism and abrogation of the di-acidic ER exit signal leads to CHI.

Voltage-gated potassium channels are six-transmembrane (S1-S6) proteins that form a central pore domain (4 x S5-S6) surrounded by four voltage sensor domains (S1-S4), which detect changes in membrane voltage and control pore opening. Upon depolarization, the S4 segments move outward carrying charged residues across the membrane field, thereby leading to the opening of the pore. The mechanism of S4 motion is controversial. We have investigated how S4 moves relative to the pore domain in the prototypical Shaker potassium channel. We introduced pairs of cysteines, one in S4 and the other in S5, and examined proximity changes between each pair of cysteines during activation, using Cd2+ and copper-phenanthroline, which crosslink the cysteines with metal and disulphide bridges, respectively. Modelling of the results suggests a novel mechanism: in the resting state, the top of the S3b-S4 voltage sensor paddle lies close to the top of S5 of the adjacent subunit, but moves towards the top of S5 of its own subunit during depolarization - this motion is accompanied by a reorientation of S4 charges to the extracellular phase.

To test the hypothesis that the Kch gene of Escherichia coli encodes a potassium channel, we have transformed E. coli with an expression vector containing the Kch sequence and observed the effect of over-expression of Kch on E. coli. We found that: (i) over-expression of Kch is toxic to E. coli, but the toxicity could be prevented by supplementing the growth medium with K(+), Rb(+), and NH(4)(+), but not Na(+), consistent with the properties of a potassium selective pore; (ii) Cs(+), a blocker of potassium channels, rescues the growth of Kch over-expressing cells; and (iii) when the putative pore-forming region of Kch, containing the signature sequence, was replaced with the corresponding region of the eukaryotic Shaker potassium channel, and the resultant construct expressed in E. coli, the cells became critically dependent on K(+) supply for survival. These data are consistent with the proposed function of Kch, i.e., K(+) conduction.

Voltage-gated potassium (K(v)) channels are integral membrane proteins, composed of four subunits, each comprising six (S1-S6) transmembrane segments. S1-S4 comprise the voltage-sensing domain, and S5-S6 with the linker P-loop forms the ion conducting pore domain. During activation, S4 undergoes structural rearrangements that lead to the opening of the channel pore and ion conduction. To obtain details of these structural changes we have used the engineered disulfide bridge approach. For this we have introduced the L361C mutation at the extracellular end of S4 of the Shaker K channel and expressed the mutant channel in Xenopus oocytes. When exposed to mild oxidizing conditions (ambient oxygen or copper phenanthroline), Cys-361 formed an intersubunit disulfide bridge as revealed by the appearance of a dimeric band on Western blotting. As a consequence, the mutant channel suffered a significant loss in conductance (measured by two-electrode voltage clamp). Removal of native cysteines failed to prevent the disulfide formation, indicating that Cys-361 forms a disulfide with its counterpart in the neighboring subunit. The effect was voltage-dependent and occurred during channel activation after Cys-361 has been exposed to the extracellular phase. Although the disulfide bridge reduced the maximal conductance, it caused a hyperpolarizing shift in the conductance-voltage relationship and reduced the deactivation kinetics of the channel. The latter two effects suggest stabilization of the open state of the channel. In conclusion, we report that during activation the intersubunit distance between the N-terminal ends of the S4 segments of the L361C mutant Shaker K channel is reduced.

The location of adenosine A(1) receptors in the rat kidney was investigated using immunolabelling with antibodies raised to a 15-amino-acid sequence near the C-terminus of the receptor (antibody I) and to a 14-amino-acid sequence in the second extracellular loop (antibody II). In the cortex, antibody I bound to adenosine A(1) receptors in mesangial cells and afferent arterioles, whilst antibody II bound to receptors in proximal convoluted tubules. In the medulla, both antibodies bound to receptors in collecting ducts and the papillary surface epithelium. These observations provide support for the diverse functional roles previously proposed for the adenosine A(1) receptor in the kidney. The labelling of distinct but different structures in the cortex by antibodies raised to different amino acid sequences on the A(1) receptor protein suggests that differing forms of the receptor are present in this region of the kidney.

In the rat, treatment with the alkylxanthine 8-cyclopentyl-1,3-dipropylxanthine (CPX) at a dose of 0.1 mg kg-1 antagonizes adenosine-induced falls in renal blood flow and reduces the severity of glycerol-induced acute renal failure. Treatment of glycerol-injected rats with 0.03 mg kg-1 of CPX resulted in no significant improvements in a range of indices of renal function. However, treatment with 0.1 or 0.3 mg kg-1 doses of CPX did significantly ameliorate acute renal failure although there were no significant differences in the degree of protection of renal function afforded by these two doses. In glycerol-injected rats, 0.1 or 0.3 mg kg-1 CPX administered either as a single dose or repeated doses every 12 h for two days did not inhibit renal phosphodiesterase. Thus the beneficial effects of CPX can be produced by doses that have no effect on renal phosphodiesterase activity whereas 0.1 mg kg-1 of CPX has been shown previously to antagonize the actions of adenosine. The findings provide further evidence that the beneficial effect of CPX in glycerol-induced acute renal failure is a consequence of adenosine antagonism and not phosphodiesterase inhibition.