Activation Of Cation Channels Research Articles

Trpm4 gene invalidation leads to cardiac hypertrophy and electrophysiological alterations.

RationaleTRPM4 is a non-selective Ca2+-activated cation channel expressed in the heart, particularly in the atria or conduction tissue. Mutations in the Trpm4 gene were recently associated with several human conduction disorders such as Brugada syndrome. TRPM4 channel has also been implicated at the ventricular level, in inotropism or in arrhythmia genesis due to stresses such as ß-adrenergic stimulation, ischemia-reperfusion, and hypoxia re-oxygenation. However, the physiological role of the TRPM4 channel in the healthy heart remains unclear.ObjectivesWe aimed to investigate the role of the TRPM4 channel on whole cardiac function with a Trpm4 gene knock-out mouse (Trpm4 -/-) model.Methods and ResultsMorpho-functional analysis revealed left ventricular (LV) eccentric hypertrophy in Trpm4 -/- mice, with an increase in both wall thickness and chamber size in the adult mouse (aged 32 weeks) when compared to Trpm4+/+ littermate controls. Immunofluorescence on frozen heart cryosections and qPCR analysis showed no fibrosis or cellular hypertrophy. Instead, cardiomyocytes in Trpm4-/- mice were smaller than Trpm4+/+with a higher density. Immunofluorescent labeling for phospho-histone H3, a mitosis marker, showed that the number of mitotic myocytes was increased 3-fold in the Trpm4-/-neonatal stage, suggesting hyperplasia. Adult Trpm4 -/- mice presented multilevel conduction blocks, as attested by PR and QRS lengthening in surface ECGs and confirmed by intracardiac exploration. Trpm4-/-mice also exhibited Luciani-Wenckebach atrioventricular blocks, which were reduced following atropine infusion, suggesting paroxysmal parasympathetic overdrive. In addition, Trpm4 -/- mice exhibited shorter action potentials in atrial cells. This shortening was unrelated to modifications of the voltage-gated Ca2+ or K+ currents involved in the repolarizing phase.ConclusionsTRPM4 has pleiotropic roles in the heart, including the regulation of conduction and cellular electrical activity which impact heart development.

Piezo2 is the major transducer of mechanical forces for touch sensation in mice.

The sense of touch provides critical information about our physical environment by transforming mechanical energy into electrical signals. It is postulated that mechanically activated cation channels initiate touch sensation, but the identity of these molecules in mammals has been elusive. Piezo2 is a rapidly adapting, mechanically activated ion channel expressed in a subset of sensory neurons of the dorsal root ganglion and in cutaneous mechanoreceptors known as Merkel-cell-neurite complexes. It has been demonstrated that Merkel cells have a role in vertebrate mechanosensation using Piezo2, particularly in shaping the type of current sent by the innervating sensory neuron; however, major aspects of touch sensation remain intact without Merkel cell activity. Here we show that mice lacking Piezo2 in both adult sensory neurons and Merkel cells exhibit a profound loss of touch sensation. We precisely localize Piezo2 to the peripheral endings of a broad range of low-threshold mechanoreceptors that innervate both hairy and glabrous skin. Most rapidly adapting, mechanically activated currents in dorsal root ganglion neuronal cultures are absent in Piezo2 conditional knockout mice, and ex vivo skin nerve preparation studies show that the mechanosensitivity of low-threshold mechanoreceptors strongly depends on Piezo2. This cellular phenotype correlates with an unprecedented behavioural phenotype: an almost complete deficit in light-touch sensation in multiple behavioural assays, without affecting other somatosensory functions. Our results highlight that a single ion channel that displays rapidly adapting, mechanically activated currents in vitro is responsible for the mechanosensitivity of most low-threshold mechanoreceptor subtypes involved in innocuous touch sensation. Notably, we find that touch and pain sensation are separable, suggesting that as-yet-unknown mechanically activated ion channel(s) must account for noxious (painful) mechanosensation.

Role of early signalling events in plant-insect interactions.

The response of plants to the stress caused by herbivores involves several different defence mechanisms. These responses begin at the plant cell plasma membrane, where insect herbivores interact physically by causing mechanical damage and chemically by introducing elicitors or by triggering plant-derived signalling molecules. The earliest plant responses to herbivore contact are represented by ion flux unbalances generated in the plant cell plasma membrane at the damaged site. Differences in the charge distribution generate plasma transmembrane potential (V m) variation, the first event, which eventually leads to the initiation of signal transduction pathways and gene expression. Calcium signalling and the generation of reactive oxygen and nitrogen species are early events closely related to V m variations. This review provides an update on recent developments and advances in plant early signalling in response to herbivory, with particular emphasis on the electrophysiological variations of the plasma membrane potential, calcium signalling, cation channel activity, production of reactive oxygen and nitrogen species, and formation of a systemically moving signal from wounded tissues. The roles of calcium-dependent protein kinases and calcineurin signalling are also discussed.

Cation channels are involved in brassinosteroid signalling in higher plants

Brassinosteroids (BRs) are an important class of plant hormones with a multitude of functions. They have been intensively investigated for their biosynthesis, distribution and physiological functions. The aim of this study was to examine possible effects of BRs on the plant plasma membrane cation conductances and Ca2+ signalling. The wheat root protoplasts (tested by patch-clamping) and excised arabidopsis roots (analysed by Ca2+-aequorin chemiluminometry), were used. In the whole-cell plasma membrane patches, 24-epibrassinolide, 28-homobrassionolide or 24-epicastasterone (1μM) were applied exogenously. 24-Epicastasterone increased the activity of the K+ efflux conductance in 50% of tested protoplasts while 24-epibrassonolide and 28-homobrassionolide did not modify the plasma membrane currents. Addition of 24-epicastasterone at the cytosolic side (to the pipette solution) resulted in dramatic stimulation of a time-dependent K+ efflux current (in 30% of protoplasts) and an activation of Ca2+ influx currents (in 30% of protoplasts). Gadolinium ions, which are blockers of cation channels, inhibited the 24-epicastasterone-induced cation channel activities. In Arabidopsis thaliana plants constitutively expressing aequorin, exogenous 24-epibrassonolide, 28-homobrassionolide and 24-epicastasterone induced a transient elevation of the cytosolic free Ca2+, which was inhibited by Gd3+ and mediated by Ca2+ influx from the bathing solution. In Ca2+-aequorin tests, 10μM of exogenous BRs was the minimal concentration at which statistically significant changes of the cytosolic Ca2+ were observed. In conclusion, the obtained results suggest that the plasma membrane of root cells contains the brassinosteroid-activated cation-permeable channels, which can probably be involved in rapid regulation of the K+ homeostasis and Ca2+ signalling.

Mitochondrial fission augments capsaicin-induced axonal degeneration.

Capsaicin, an agonist of transient receptor potential vanilloid receptor 1, induces axonal degeneration of peripheral sensory nerves and is commonly used to treat painful sensory neuropathies. In this study, we investigated the role of mitochondrial dynamics in capsaicin-induced axonal degeneration. In capsaicin-treated rodent sensory axons, axonal swellings, decreased mitochondrial stationary site length and reduced mitochondrial transport preceded axonal degeneration. Increased axoplasmic Ca2+ mediated the alterations in mitochondrial length and transport. While sustaining mitochondrial transport did not reduce axonal swellings in capsaicin-treated axons, preventing mitochondrial fission by overexpression of mutant dynamin-related protein 1 increased mitochondrial length, retained mitochondrial membrane potentials and reduced axonal loss upon capsaicin treatment. These results establish that mitochondrial stationary site size significantly affects axonal integrity and suggest that inhibition of Ca2+-dependent mitochondrial fission facilitates mitochondrial function and axonal survival following activation of axonal cationic channels.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-014-1354-3) contains supplementary material, which is available to authorized users.

Light-induced termination of spiral wave arrhythmias by optogenetic engineering of atrial cardiomyocytes.

Atrial fibrillation (AF) is the most common cardiac arrhythmia and often involves reentrant electrical activation (e.g. spiral waves). Drug therapy for AF can have serious side effects including proarrhythmia, while electrical shock therapy is associated with discomfort and tissue damage. Hypothetically, forced expression and subsequent activation of light-gated cation channels in cardiomyocytes might deliver a depolarizing force sufficient for defibrillation, thereby circumventing the aforementioned drawbacks. We therefore investigated the feasibility of light-induced spiral wave termination through cardiac optogenetics. Neonatal rat atrial cardiomyocyte monolayers were transduced with lentiviral vectors encoding light-activated Ca(2+)-translocating channelrhodopsin (CatCh; LV.CatCh∼eYFP↑) or eYFP (LV.eYFP↑) as control, and burst-paced to induce spiral waves rotating around functional cores. Effects of CatCh activation on reentry were investigated by optical and multi-electrode array (MEA) mapping. Western blot analyses and immunocytology confirmed transgene expression. Brief blue light pulses (10 ms/470 nm) triggered action potentials only in LV.CatCh∼eYFP↑-transduced cultures, confirming functional CatCh-mediated current. Prolonged light pulses (500 ms) resulted in reentry termination in 100% of LV.CatCh∼eYFP↑-transduced cultures (n = 31) vs. 0% of LV.eYFP↑-transduced cultures (n = 11). Here, CatCh activation caused uniform depolarization, thereby decreasing overall excitability (MEA peak-to-peak amplitude decreased 251.3 ± 217.1 vs. 9.2 ± 9.5 μV in controls). Consequently, functional coresize increased and phase singularities (PSs) drifted, leading to reentry termination by PS-PS or PS-boundary collisions. This study shows that spiral waves in atrial cardiomyocyte monolayers can be terminated effectively by a light-induced depolarizing current, produced by the arrhythmogenic substrate itself, upon optogenetic engineering. These results provide proof-of-concept for shockless defibrillation.

Serotonergic neurons of the Drosophila air-puff-stimulated flight circuit.

Monoaminergic modulation of insect flight is well documented. Recently, we demonstrated that synaptic activity is required in serotonergic neurons for Drosophila flight. This requirement is during early pupal development, when the flight circuit is formed, as well as in adults. Using a Ca2+-activity-based GFP reporter, here we show that serotonergic neurons in both prothoracic and mesothoracic segments are activated upon air-puff-stimulated flight. Moreover ectopic activation of the entire serotonergic system by TrpA1, a heat activated cation channel, induces flight, even in the absence of an air-puff stimulus.

Ion channels of the nuclear membrane of hippocampal neurons

Rise in Ca(2+) concentration in the nucleus affects gene transcription and has been implicated in neuroprotection, transcription-dependent neuronal plasticity, and pain modulation, but the mechanism of regulation of nuclear Ca(2+) remains poorly understood. The nuclear envelope is a part of the endoplasmic reticulum and may be one of the sources of nuclear Ca(2+) . Here, we studied ion channels in the nuclear membrane of hippocampal neurons using the patch-clamp technique. We have found that the nuclear membrane of CA1 pyramidal and dentate gyrus granule (DG), but not CA3 pyramidal neurons, was enriched in functional inositol 1,4,5-trisphosphate receptors/Ca(2+) -release channels (IP3 Rs) localized mainly in the inner nuclear membrane. A single nuclear ryanodine receptor (RyR) has been detected only in DG granule neurons. Nuclei of the hippocampal neurons also expressed a variety of spontaneously active cation and anion channels specific for each type of neuron. In particular, large-conductance ion channels selective for monovalent cations (LCC) were coexpressed with IP3 Rs. These data suggest that: (1) the nuclear membranes of hippocampal neurons contain distinct sets of ion channels, which are specific for each type of neuron; (2) IP3 Rs, but not RyRs are targeted to the inner nuclear membrane of CA1 pyramidal and DG granule, but they were not found in the nuclear membranes of CA3 pyramidal neurons; (3) the nuclear envelope of these neurons is specialized to release Ca(2+) into the nucleoplasm which may amplify Ca(2+) signals entering the nucleus from the cytoplasm or generate Ca(2+) transients on its own; (4) LCC channels are an integral part the of Ca(2+) -releasing machinery providing a route for counterflow of К(+) and thereby facilitating Ca(2+) movement in and out of the Ca(2+) store.

Piezo2 is required for Merkel-cell mechanotransduction

SummaryHow we sense touch remains fundamentally unknown1,2. The Merkel cell-neurite complex is a gentle touch receptor in the skin that mediates slowly-adapting (SA) responses of Aβ sensory fibers to encode fine details of objects3-6. This mechanoreceptor complex was recognized to play an essential role in sensing gentle touch nearly 50 years ago3,4. However, whether Merkel cells or afferent fibers themselves sense mechanical force is still debated, and the molecular mechanism of mechanotransduction is unknown1,2,7-12. Interestingly, synapse-like junctions are observed between Merkel cells and associated afferents6,13-15, and yet it is unclear if Merkel cells are inherently mechanosensitive or whether they can rapidly transmit such information to the neighboring nerve1,2,16,17. Here we show for the first time that Merkel cells produce touch-sensitive currents in vitro. Piezo2, a mechanically-activated (MA) cation channel, is expressed in Merkel cells. We engineered mice deficient in Piezo2 in the skin, but not in sensory neurons, and show that Merkel cell mechanosensitivity completely depends on Piezo2. In these mice, Merkel cell-neurite complex-mediated SA responses in vivo show reduced static firing rates, and moreover, they display moderately decreased behavioral responses to gentle touch. Our results indicate that Piezo2 is the Merkel cell mechanotransduction channel and provide the first line of evidence that Piezos play a physiological role in mechanosensation in mammals. Furthermore, our data present evidence for a two-receptor site model, where both Merkel cells and innervating afferents act in concert as mechanosensors. The two-receptor system could provide this mechanoreceptor complex with a tuning mechanism to achieve highly sophisticated responses to a given mechanical stimulus15,18,19.

Mechanisms and physiological roles of K+ efflux from root cells

Potassium is the most abundant macronutrient, which is involved in a multitude of physiological processes. Potassium uptake in roots is crucial for plants; however, K(+) efflux can also occur and has important functions. Potassium efflux from roots is mainly induced by stresses, such as pathogens, salinity, freezing, oxidants and heavy metals. Reactive oxygen species (ROS) and exogenous purines also cause this reaction. The depolarisation and activation of cation channels are required for K(+) efflux from plant roots. Potassium channels and nonselective cation channels (NSCCs) are involved in this process. Some of them are 'constitutive', while the others require a chemical agent for activation. In Arabidopsis, there are 77 genes that can potentially encode K(+)-permeable channels. Potassium-selective channel genes include 9 Shaker and 6 Tandem-Pore K(+) channels. Genes of NSCCs are more abundant and present by 20 cyclic nucleotide gated channels, 20 ionotropic glutamate receptors, 1 two-pore channel, 10 mechanosensitive-like channels, 2 mechanosensitive 'Mid1-Complementing Activity' channels, 1 mechanosensitive Piezo channel, and 8 annexins. Two Shakers (SKOR and GORK) and several NSCCs are expressed in root cell plasma membranes. SKOR mediates K(+) efflux from xylem parenchyma cells to xylem vessels while GORK is expressed in the epidermis and functions in K(+) release. Both these channels are activated by ROS. The GORK channel activity is stimulated by hydroxyl radicals that are generated in a Ca(2+)-dependent manner in stress conditions, such as salinity or pathogen attack, resulting in dramatic K(+) efflux from root cells. Potassium loss simulates cytosolic proteases and endonucleases, leading to programmed cell death. Other physiological functions of K(+) efflux channels include repolarisation of the plasma membrane during action potentials and the 'hypothetical' function of a metabolic switch, which provides inhibition of energy-consuming biosyntheses and releasing energy for defence and reparation needs.

Two-Back Makes Step Forward in Brain Imaging Genomics

The combination of brain imaging and genetics promises to identify neural mechanisms underlying complex traits such as cognition and psychiatric disorder. The study by Heck etal. (2014) in this issue of Neuron highlights voltage-gated cation channel activity as a likely link and culprit.

Functional Swapping between Transmembrane Proteins TMEM16A and TMEM16F

The transmembrane proteins TMEM16A and -16F each carry eight transmembrane regions with cytoplasmic N and C termini. TMEM16A carries out Ca(2+)-dependent Cl(-) ion transport, and TMEM16F is responsible for Ca(2+)-dependent phospholipid scrambling. Here we established assay systems for the Ca(2+)-dependent Cl(-) channel activity using 293T cells and for the phospholipid scramblase activity using TMEM16F(-/-) immortalized fetal thymocytes. Chemical cross-linking analysis showed that TMEM16A and -16F form homodimers in both 293T cells and immortalized fetal thymocytes. Successive deletion from the N or C terminus of both proteins and the swapping of regions between TMEM16A and -16F indicated that their cytoplasmic N-terminal (147 amino acids for TMEM16A and 95 for 16F) and C-terminal (88 amino acids for TMEM16A and 68 for 16F) regions were essential for their localization at plasma membranes and protein stability, respectively, and could be exchanged. Analyses of TMEM16A and -16F mutants with point mutations in the pore region (located between the fifth and sixth transmembrane regions) indicated that the pore region is essential for both the Cl(-) channel activity of TMEM16A and the phospholipid scramblase activity of TMEM16F. Some chemicals such as epigallocatechin-3-gallate and digallic acid inhibited the Cl(-) channel activity of TMEM16A and the scramblase activity of TMEM16F with an opposite preference. These results indicate that TMEM16A and -16F use a similar mechanism for sorting to plasma membrane and protein stabilization, but their functional domains significantly differ.

Transduction for Pheromones in the Main Olfactory Epithelium Is Mediated by the Ca2+-Activated Channel TRPM5

Growing evidence suggests that the main olfactory epithelium contains a subset of olfactory sensory neurons (OSNs) responding to pheromones. One candidate subpopulation expresses the calcium activated cation channel TRPM5 (transient receptor potential channel M5). Using GFP driven by the TRPM5 promoter in mice, we show that this subpopulation responds to putative pheromones, urine, and major histocompatibility complex peptides, but not to regular odors or a pheromone detected by other species. In addition, this subpopulation of TRPM5-GFP+ OSNs uses novel transduction. In regular OSNs, odorants elicit activation of the cyclic nucleotide-gated (CNG) channel, leading to Ca2+ gating of Cl- channels; in TRPM5-GFP+ OSNs, the Ca2+ -activated Cl- ANO2 (anoctamin 2) channel is not expressed, and pheromones elicit activation of the CNG channel leading to Ca2+ gating of TRPM5. In conclusion, we show that OSNs expressing TRPM5 respond to pheromones, but not to regular odors through the opening of CNG channels leading to Ca2+ gating of TRPM5.

Converging genetic and functional brain imaging evidence links neuronal excitability to working memory, psychiatric disease, and brain activity.

Working memory, the capacity of actively maintaining task-relevant information during a cognitive task, is a heritable trait. Working memory deficits are characteristic for many psychiatric disorders. We performed genome-wide gene set enrichment analyses in multiple independent data sets of young and aged cognitively healthy subjects (n= 2,824) and in a large schizophrenia case-control sample (n= 32,143). The voltage-gated cation channel activity gene set, consisting of genes related to neuronal excitability, was robustly linked to performance in working memory-related tasks across ages and toschizophrenia. Functional brain imaging in 707 healthy participants linked this gene setalso to working memory-related activity in the parietal cortex and the cerebellum. Gene set analyses may help to dissect the molecular underpinnings of cognitive dimensions, brain activity, and psychopathology.

Common Mechanisms Underlying Refractive Error Identified in Functional Analysis of Gene Lists From Genome-Wide Association Study Results in 2 European British Cohorts

To date, relatively few genes responsible for a fraction of heritability have been identified by means of large genetic association studies of refractive error. To explore the genetic mechanisms that lead to refractive error in the general population. Genome-wide association studies were carried out in 2 British population-based independent cohorts (N = 5928 participants) to identify genes moderately associated with refractive error. Enrichment analyses were used to identify sets of genes overrepresented in both cohorts. Enriched groups of genes were compared between both participating cohorts as a further measure against random noise. Groups of genes enriched at highly significant statistical levels were remarkably consistent in both cohorts. In particular, these results indicated that plasma membrane (P = 7.64 × 10⁻³⁰), cell-cell adhesion (P = 2.42 × 10⁻¹⁸), synaptic transmission (P = 2.70 × 10⁻¹⁴), calcium ion binding (P = 3.55 × 10⁻¹⁵), and cation channel activity (P = 2.77 × 10⁻¹⁴) were significantly overrepresented in relation to refractive error. These findings provide evidence that development of refractive error in the general population is related to the intensity of photosignal transduced from the retina, which may have implications for future interventions to minimize this disorder. Pathways connected to the procession of the nerve impulse are major mechanisms involved in the development of refractive error in populations of European origin.

TRPs as chemosensors (ROS, RNS, RCS, gasotransmitters).

The transient receptor potential (trp) gene superfamily encodes TRP proteins that act as multimodal sensor cation channels for a wide variety of stimuli from outside and inside the cell. Upon chemical or physical stimulation of cells, TRP channels transduce electrical and/or Ca(2+) signals via their cation channel activities. These functional features of TRP channels allow the body to react and adapt to different forms of environmental changes. Indeed, members of one class of TRP channels have emerged as sensors of reactive oxygen species (ROS), reactive nitrogen species (RNS), reactive carbonyl species (RCS), and gaseous messenger molecules including molecular oxygen (O2), hydrogen sulfide (H2S), and carbon dioxide (CO2). Hydrogen peroxide (H2O2), an ROS, triggers the production of ADP-ribose, which binds and activates TRPM2. In addition to TRPM2, TRPC5, TRPV1, and TRPA1 are also activated by H2O2 via modification of cysteine (Cys) free sulfhydryl groups. Nitric oxide (NO), a vasoactive gaseous molecule, regulates TRP channels directly via Cys S-nitrosylation or indirectly via cyclic GMP (cGMP)/protein kinase G (PKG)-dependent phosphorylation. Anoxia induced by O2-glucose deprivation and severe hypoxia activates TRPM7 and TRPC6, respectively, whereas TRPA1 serves as a sensor of mild hypoxia and hyperoxia in vagal and sensory neurons. TRPA1 also detects other gaseous molecules, such as hydrogen sulfide (H2S) and carbon dioxide (CO2). In this review, we highlight our current knowledge of TRP channels as chemosensors for ROS, RNS, RCS, and gaseous molecules and discuss their functional impacts on physiological and pathological events.

TRPM5.

TRPM5 is a Ca(2+)-activated cation channel that mediates signaling in taste and other chemosensory cells. Within taste cells, TRPM5 is the final element in a signaling cascade that starts with the activation of G protein-coupled receptors by bitter, sweet, or umami taste molecules and that requires the enzyme PLCβ2. PLCβ2 breaks down PIP2 into DAG and IP3, and the ensuing release of Ca(2+) from intracellular stores activates TRPM5. Since its initial discovery in the taste system, TRPM5 has been found to be distributed in sparse chemosensory cells located throughout the digestive track, in the respiratory system, and in the olfactory system. It is also found in pancreatic islets, where it contributes to insulin secretion. This review highlights recent work on the mechanisms of the activation of the TRPM5 channel and its regulation by voltage, phosphoinositides, temperature, and pH. The distribution of the channel in the body and its functional contribution to various sensory and nonsensory processes are discussed.

Calcium dynamics underlying the myogenic response of the renal afferent arteriole

The renal afferent arteriole reacts to an elevation in blood pressure with an increase in muscle tone and a decrease in luminal diameter. This effect, known as the myogenic response, is believed to stabilize glomerular filtration and to protect the glomerulus from systolic blood pressure increases, especially in hypertension. To study the mechanisms underlying the myogenic response, we developed a mathematical model of intracellular Ca2+ signaling in an afferent arteriole smooth muscle cell. The model represents detailed transmembrane ionic transport, intracellular Ca2+ dynamics, the kinetics of myosin light chain phosphorylation, and the mechanical behavior of the cell. It assumes that the myogenic response is initiated by pressure-induced changes in the activity of nonselective cation channels. Our model predicts spontaneous vasomotion at physiological luminal pressures and KCl- and diltiazem-induced diameter changes comparable to experimental findings. The time-periodic oscillations stem from the dynamic exchange of Ca2+ between the cytosol and the sarcoplasmic reticulum, coupled to the stimulation of Ca2+-activated potassium (KCa) and chloride (ClCa) channels, and the modulation of voltage-activated L-type channels; blocking sarco/endoplasmic reticulum Ca2+ pumps, ryanodine receptors (RyR), KCa, ClCa, or L-type channels abolishes these oscillations. Our results indicate that the profile of the myogenic response is also strongly dependent on the conductance of ClCa and L-type channels, as well as the activity of plasmalemmal Ca2+ pumps. Furthermore, inhibition of KCa is not necessary to induce myogenic contraction. Lastly, our model suggests that the kinetic behavior of L-type channels results in myogenic kinetics that are substantially faster during constriction than during dilation, consistent with in vitro observations (Loutzenhiser R, Bidani A, Chilton L. Circ. Res. 90: 1316–1324, 2002).

Optogenetic Excitation of the Pancreatic β-Cell

The β-cell is a subunit of the Islet of Langerhans, a micro-organ of the pancreas which maintains glucose homeostasis. This endocrine cell senses changes in blood glucose levels and releases the hormone insulin. An electrophysiological process governing insulin release occurs through initial uptake of blood glucose and generation of ATP which binds the K-ATP channel causing inhibition and subsequent membrane depolarization. Influx of calcium from voltage-gated channels then interact with insulin secretory vesicles to promote exocystosis into the blood stream. In order to directly influence calcium dynamics, insulin release, and to study the electrophysical properties of the β-cell we used Channelrhodopsin-2 (ChR2), a light activated cation channel capable of creating membrane depolarization with light stimulation. Transient transfection of the MIN6 β-cell line using ChR2-YFP was carried out followed by calcium imaging using Fura2-AM. Following ChR2 stimulation at 490nm we observed local depolarization events in positively transfected cells. We demonstrated the creation of new oscillatory behavior at high glucose levels that can be set at specified frequencies and demonstrate that ChR2 can be used to overcome disruption in the K-ATP channel. Next, we isolated intact islets from a mouse model with β-cell specific expression of ChR2-YFP. We demonstrated global depolarization events upon ChR2 stimulation and performed insulin secretion assays to demonstrate enhanced insulin secretion. In addition, a computational multi-cellular islet model including ChR2 was used for comparison between in-vitro and in-silico experiments. This work demonstrates the ability of cell-specific electrophysiological control in the islet of Langerhans which will give further insight into β-cell behavior. Future work will include analyzing local depolarization on global islet behavior, examining the effect of ChR2 on islet-islet coupling, and developing an in-vivo model; which will include testing the feasibility for reversing diabetes.

Calcium homeostasis in red blood cells of dialysis patients in dependence of erythropoietin treatment

Calcium homeostasis in red blood cells of dialysis patients in dependence of erythropoietin treatment