Changes In Membrane Potential Of Cells Research Articles

Do Prestin Variants Persist within the Cochlea to Affect Charge Movement In Vitro?

Introduction. Prestin-associated voltage sensors exhibit charge movement, q, in response to changes in cell-membrane potential. Summed over all participating atoms, q is the product of each atom's charge and the relative distance it moves within the dielectric thickness of the membrane. The quotient of q, κB (Boltzmann's constant) and T (temperature) is the voltage sensitivity, α of the sensor. It is determined experimentally for a large number (>104) of sensors by measuring the voltage-dependent capacitance of an outer hair cell (OHC) or prestin-transfected cell.Observations. OHCs isolated from pigmented guinea pigs exhibit a higher α when they originate from the basal region of the cochlea compared to the apical region. In addition, the maximum charge measured for each OHC is inversely proportional (not directly proportional) to the measured α, suggesting the sensors do not act independently.Explanation. Prestin is the fifth member of solute carrier family 26, where SLC26A5a of Homo sapiens, also known as isoform X1 of Cavia porcellus, is the dominant variant. Three less common isoforms were reported in humans with two different variants predicted to exist in the domestic guinea pig. We hypothesize that isoforms of prestin coexist and their arrangement within an oligomer vary to account for the gradients in α, where each component of an oligomer translates a different fraction of q and the measured voltage sensitivity, represents their ensemble average.Result We find evidence forsplice variants in the cochlea of pigmented guinea pigs upon conducting reverse transcription of mRNA with PCR. The findings will be discussed with respect to experimental measurements of charge movement.

Aluminium alleviates manganese toxicity to rice by decreasing root symplastic Mn uptake and reducing availability to shoots of Mn stored in roots.

Manganese (Mn) and aluminium (Al) phytotoxicities occur mainly in acid soils. In some plant species, Al alleviates Mn toxicity, but the mechanisms underlying this effect are obscure. Rice (Oryza sativa) seedlings (11 d old) were grown in nutrient solution containing different concentrations of Mn(2+) and Al(3+) in short-term (24 h) and long-term (3 weeks) treatments. Measurements were taken of root symplastic sap, root Mn plaques, cell membrane electrical surface potential and Mn activity, root morphology and plant growth. In the 3-week treatment, addition of Al resulted in increased root and shoot dry weight for plants under toxic levels of Mn. This was associated with decreased Mn concentration in the shoots and increased Mn concentration in the roots. In the 24-h treatment, addition of Al resulted in decreased Mn accumulation in the root symplasts and in the shoots. This was attributed to higher cell membrane surface electrical potential and lower Mn(2+) activity at the cell membrane surface. The increased Mn accumulation in roots from the 3-week treatment was attributed to the formation of Mn plaques, which were probably related to the Al-induced increase in root aerenchyma. The results show that Al alleviated Mn toxicity in rice, and this could be attributed to decreased shoot Mn accumulation resulting from an Al-induced decrease in root symplastic Mn uptake. The decrease in root symplastic Mn uptake resulted from an Al-induced change in cell membrane potential. In addition, Al increased Mn plaques in the roots and changed the binding properties of the cell wall, resulting in accumulation of non-available Mn in roots.

Chloride and its Clinical Implications in Today’s Clinical Practice: Not an Orphan Electrolyte

Though chloride is the predominant extracellular anion, it is mostly seen just as an anion accompanying sodium and hardly receives attention in textbooks. But independent evaluation of serum chloride may unearth several clinical and acid-base disorders. It is used in formulas to estimate serum anion gap, urine anion gap, and strong ion difference (Stewart method). Several critical functions of a cell such as maintenance of cell volume, neutralization of H+ in lysosomal vesicles, epithelial fluid transport, change in cell membrane potential and ligand-gated transmission in the post-synaptic membrane utilize chloride channels. In addition, chloride forms an integral part of anion exchanger proteins coded by SLC26A gene family. Chloride is an essential component of intravenous fluids used in day-to-day clinical practice. The role and contribution of chloride rich fluids and resulting acidosis in causing inferior outcomes in sepsis, renal vasoconstriction, and acute kidney injury has been debated. Numerous genetic diseases are known to be related with chloride channels and proteins abnormalities. In the following review, I would like to bring much needed attention towards importance of chloride in human physiology. The following hypothetical clinical case will be just a spark for fiery chloride.

Small Aβ1-42 oligomer-induced membrane depolarization of neuronal and microglial cells: role of N-methyl-D-aspartate receptors.

Although it is well documented that soluble beta amyloid (Aβ) oligomers are critical factors in the pathogenesis of Alzheimer's disease (AD) by causing synaptic dysfunction and neuronal death, the primary mechanisms by which Aβ oligomers trigger neurodegeneration are not entirely understood. We sought to investigate whether toxic small Aβ(1-42) oligomers induce changes in plasma membrane potential of cultured neurons and glial cells in rat cerebellar granule cell cultures leading to neuronal death and whether these effects are sensitive to the N-methyl-D-aspartate receptor (NMDA-R) antagonist MK801. We found that small Aβ(1-42) oligomers induced rapid, protracted membrane depolarization of both neurons and microglia, whereas there was no change in membrane potential of astrocytes. MK801 did not modulate Aβ-induced neuronal depolarization. In contrast, Aβ1(-42) oligomer-induced decrease in plasma membrane potential of microglia was prevented by MK801. Small Aβ(1-42) oligomers significantly elevated extracellular glutamate and caused neuronal necrosis, and both were prevented by MK801. Also, small Aβ(1-42) oligomers decreased resistance of isolated brain mitochondria to calcium-induced opening of mitochondrial permeability transition pore. In conclusion, the results suggest that the primary effect of toxic small Aβ oligomers on neurons is rapid, NMDA-R-independent plasma membrane depolarization, which leads to neuronal death. Aβ oligomers-induced depolarization of microglial cells is NMDA-R dependent.

Characterization of cadmium plasma membrane transport in gills of a mangrove crab Ucides cordatus

Membrane pathway for intracellular cadmium (Cd2+) accumulation is not fully elucidated in many organisms and has not been studied in crab gill cells. To characterize membrane Cd2+ transport of anterior and posterior gill cells of Ucides cordatus, a hypo-hyper-regulating crab, a change in intracellular Cd2+ concentration under various experimental conditions was examined by using FluoZin, a fluorescent probe. The membrane Cd2+ transport was estimated by the augmentation of FluoZin fluorescence induced by extracellular application of CdCl2 and different inhibitors. Addition of extracellular calcium (Ca2+) to the cells affected little the fluorescence of FluoZin, confirming that Cd2+ was the main ion increasing intracellular fluorescence. Ca2+ channels blockers (nimodipine and verapamil) decreased Cd2+ influx as well as vanadate, a Ca2+-ATPase blocker. Chelating intracellular Ca2+ (BAPTA) decreased Cd2+ influx in gill cells, while increasing intracellular Ca2+ (caffeine) augmented Cd influx. Cd2+ and ATP added at different temporal conditions were not effective at increasing intracellular Cd2+ accumulation. Ouabain (Na+/K+-ATPase inhibitor) increased Cd2+ influx probably through a change in intracellular Na and/or a change in cell membrane potential. Routes of Cd2+ influx, a non-essential metal, through the gill cell plasma membrane of crabs are suggested.

Membrane current series monitoring: essential reduction of data points to finite number of stable parameters.

In traditional studies of changes in cell membrane potential or trans-membrane currents a large part of the recorded data presents “a pure noise.” This noise results mainly from the random openings of membrane ionic channels. Different types of stationary or non-stationary noise analysis have been used in electrophysiological experiments for identification of channels kinetic states. But these methods have a limited power and often cannot answer to the main question of the experimental study: do external factors induce a significant change of channels kinetics? A new method suggested in the current study is based on the scaling properties of the beta-distribution function that allows reducing the series containing 200,000 and more data points to analysis of only 10–20 stable parameters. The following clusterization using the generalized Pearson correlation function allows taking into account the influence of an external factor and combine/separate different parameters of interest into a statistical cluster considering the influential parameter. This method which we call BRC (Beta distribution-Reduction-Clusterization) opens new possibilities in creation of a largely reduced database while extracting specific fingerprints of the long-term series. The BRC method was validated using patch clamp current recordings containing 250,000 data points obtained from the living cells and from open tip electrode. The numerical distinction between these two series in terms of the reduced parameters was obtained.

Antioxidative dietary compounds modulate gene expression associated with apoptosis, DNA repair, inhibition of cell proliferation and migration.

Many dietary compounds are known to have health benefits owing to their antioxidative and anti-inflammatory properties. To determine the molecular mechanism of these food-derived compounds, we analyzed their effect on various genes related to cell apoptosis, DNA damage and repair, oxidation and inflammation using in vitro cell culture assays. This review further tests the hypothesis proposed previously that downstream products of COX-2 (cyclooxygenase-2) called electrophilic oxo-derivatives induce antioxidant responsive elements (ARE), which leads to cell proliferation under antioxidative conditions. Our findings support this hypothesis and show that cell proliferation was inhibited when COX-2 was down-regulated by polyphenols and polysaccharides. Flattened macrophage morphology was also observed following the induction of cytokine production by polysaccharides extracted from viili, a traditional Nordic fermented dairy product. Coix lacryma-jobi (coix) polysaccharides were found to reduce mitochondrial membrane potential and induce caspase-3- and 9-mediated apoptosis. In contrast, polyphenols from blueberries were involved in the ultraviolet-activated p53/Gadd45/MDM2 DNA repair system by restoring the cell membrane potential. Inhibition of hypoxia-inducible factor-1 by saponin extracts of ginsenoside (Ginsen) and Gynostemma and inhibition of S100A4 by coix polysaccharides inhibited cancer cell migration and invasion. These observations suggest that antioxidants and changes in cell membrane potential are the major driving forces that transfer signals through the cell membrane into the cytosol and nucleus, triggering gene expression, changes in cell proliferation and the induction of apoptosis or DNA repair.

Multiparametric One-Color Assays for Functional Assessment of Cardiomyocytes

Heart-related health problems remain a leading cause of mortality in developed countries. To efficiently fight these problems, cardiology needs an information-rich screening for new drug candidates. Recent achievements in stem cell research provide a long-awaited solution by producing human stem-cell derived cardiomyocytes and, thus, providing various disease-in-a-dish models of cardiac disorders.Two biologically distinct parameters are required to efficiently characterize the cardiomyocytes function: changes in cell membrane potential and distribution of intracellular calcium ions. Unfortunately, due to experimental limitations and lack of appropriate fluorescent indicators, the traditional approach to perform such an assessment is to record one signal at a time.Here we present novel approach to perform multi-parametric assessment of cardiomyocytes while using two functionally different fluorescent indicators excited by the same wavelength of light. Specifically, using the IC200 HCS screening system (Vala Sciences), we performed a functional assessment of stem cell-derived cardiomyocytes by parallel optical recording of two physiologically distinct signals in real time: dynamic changes in cell membrane voltage as well as redistribution of calcium ions in cells. To do that, in addition to a calcium indicator Fluo4, we used a novel highly efficient voltage-sensitive fluorescent dye (VF 2.1) to detect voltage changes in cardiomyocytes.To perform data analysis, we have developed a dedicated software program that by creating spatially distinct subsets of masks is separating calcium and voltage signals. Using our multiparametric assay, we have performed several screening campaigns using benchmark compounds such as modulators of ion channels involved in generation of cardiac action potential (hERG channels, sodium and calcium voltage-gated ion channels), as well as drugs that affect the intracellular calcium handling. In summary, we have developed a multi-parametric approach for comprehensive screening of a human stem cell-derived cardiomyocytes for drug discovery and cardiotoxicity purposes.

Real-time monitoring of changes in plasma membrane potential via imaging of fluorescence resonance energy transfer at individual cell resolution in suspension

A method for monitoring heterogeneity in changes of plasma membrane potential (PMP) at an individual cell resolution while in suspension, utilizing a simple and low-cost wide-field illumination arrangement, is presented. The method is modeled via HEK-293 cell line in suspension, double stained with coumarin and oxonol (donor and acceptor), which were loaded into an array of nanoliter wells, each designed to preserve the individuality of the nontethered cell it holds during vigorous biomanipulation. Depolarization of PMP was induced by high K(+) solution, reducing the proximity between the membrane fluorophores and subsequently reducing the efficiency (E%) of resonance energy transfer between them. Spatial plots of E% were produced from both images of fluorescence intensity and polarization. The spatial resolution of E% plots seem to be higher, and their contrast greater, when calculated from the polarization, rather than from the intensity of the fluorescence.

Early changes in membrane potential of Saccharomyces cerevisiae induced by varying extracellular K+, Na+ or H+ concentrations

Recently we introduced a fluorescent probe technique that makes possible to convert changes of equilibrium fluorescence spectra of 3,3'-dipropylthiadicarbocyanine, diS-C3(3), measured in yeast cell suspensions under defined conditions into underlying membrane potential differences, scaled in millivolts (Plasek et al. in J Bioenerg Biomembr 44: 559-569, 2012). The results presented in this paper disclose measurements of real early changes of plasma membrane potential induced by the increase of extracellular K(+), Na(+) and H(+) concentration in S. cerevisiae with and without added glucose as energy source. Whereas the wild type and the ∆tok1 mutant cells exhibited similar depolarization curves, mutant cells lacking the two Trk1,2 potassium transporters revealed a significantly decreased membrane depolarization by K(+), particularly at lower extracellular potassium concentration [K(+)]out. In the absence of external energy source plasma membrane depolarization by K(+) was almost linear. In the presence of glucose the depolarization curves exhibited an exponential character with increasing [K(+)]out. The plasma membrane depolarization by Na(+) was independent from the presence of Trk1,2 transporters. Contrary to K(+), Na(+) depolarized the plasma membrane stronger in the presence of glucose than in its absence. The pH induced depolarization exhibited a fairly linear relationship between the membrane potential and the pHo of cell suspensions, both in the wild type and the Δtrk1,2 mutant strains, when cells were energized by glucose. In the absence of glucose the depolarization curves showed a biphasic character with enhanced depolarization at lower pHo values.

Piracetam induces plasma membrane depolarization in rat brain synaptosomes

Piracetam is a cyclic derivative of γ-aminobutyric acid (GABA). It was the first nootropic drug approved for clinical use. However, mechanism of its action is still not clear. In present paper, I investigated effects of piracetam on neurotransmitter release, plasma membrane potential monitored by fluorescent dye DiSC3(5) and chloride transport monitored by fluorescent dye SPQ in rat brain synaptosomes. It was shown that piracetam (1mM) induces slow weak plasma membrane depolarization. This effect was decreased on 43% and 58% by both AMPA/kainate receptor blockers NBQX (10μM) and CNQX (100μM), respectively, on 84% by GABA ionotropic receptor blocker picrotoxin (50μM) and on 91% upon withdrawal of HCO3− ions from incubation medium. GABA (1mM) and kainate (100μM) were found not to produce changes of plasma membrane potential. Also, it was found that piracetam induces chloride efflux which seems to be the reason of depolarization. Thereby, piracetam induces depolarization of plasma membrane of isolated neuronal presynaptic endings by picrotoxin-sensitive way.

Heating during infrared neural stimulation

Infrared neural stimulation (INS) has recently evoked interest as an alternative to electrical stimulation. The mechanism of activation is the heating of water, which induces changes in cell membrane potential but may also trigger heat sensitive receptors. To further elucidate the mechanism, which may be dependent on cell type, a detailed description of the temperature distribution is necessary. A good control of the resulting temperature during INS is also necessary to avoid excessive heating that may damage the cells. Here we present a detailed model for the heating during INS and apply it for INS of in vitro neural networks of rat cerebral cortex neurons. A model of the heating during INS of a cell culture in a non-turbid media was prepared using multiphysics software. Experimental parameters such as initial temperature, beam distribution, pulse length, pulse duration, frequency and laser-cell distance were used. To verify the model, local temperature measurements using open pipette resistance were conducted. Furthermore, cortical neurons in culture were stimulated by a 500 mW pulsed diode laser (wavelength 1,550 nm) launched into a 200 µm multimodal optical fiber positioned 300 µm from the glass surface. The radiant exposure was 5.2 J/cm(2) . The model gave detailed information about the spatial and temporal temperature distribution in the heated volume during INS. Temperature measurements using open pipette resistance verified the model. The peak temperature experienced by the cells was 48°C. Cortical neurons were successfully stimulated using the 1,550 nm laser and single cell activation as well as neural network inhibition were observed. The model shows the spatial and temporal temperature distribution in the heated volume and could serve as a useful tool for future studies of the heating during INS.

Examining the Role of Mitochondria in Ca2+ Signaling in Native Vascular Smooth Muscle

Mitochondrial Ca2+ uptake contributes important feedback controls to limit the time course of Ca2+signals. Mitochondria regulate cytosolic [Ca2+] over an exceptional breath of concentrations (∼200 nM to >10 μM) to provide a wide dynamic range in the control of Ca2+ signals. Ca2+ uptake is achieved by passing the ion down the electrochemical gradient, across the inner mitochondria membrane, which itself arises from the export of protons. The proton export process is efficient and on average there are less than three protons free within the mitochondrial matrix. To study mitochondrial function, the most common approaches are to alter the proton gradient and to measure the electrochemical gradient. However, drugs which alter the mitochondrial proton gradient may have substantial off target effects that necessitate careful consideration when interpreting their effect on Ca2+ signals. Measurement of the mitochondrial electrochemical gradient is most often performed using membrane potential sensitive fluorophores. However, the signals arising from these fluorophores have a complex relationship with the electrochemical gradient and are altered by changes in plasma membrane potential. Care is again needed in interpreting results. This review provides a brief description of some of the methods commonly used to alter and measure mitochondrial contribution to Ca2+ signaling in native smooth muscle.

Membrane "potential-omics": toward voltage imaging at the cell population level in roots of living plants.

Genetically encoded voltage-sensitive fluorescent proteins (VSFPs) are being used in neurobiology as non-invasive tools to study synchronous electrical activities in specific groups of nerve cells. Here we discuss our efforts to adapt this “light-based electrophysiology” for use in plant systems. We describe the production of transgenic plants engineered to express different versions of VSFPs that are targeted to the plasma membrane and internal membranes of root cells. The aim is to optically record concurrent changes in plasma membrane potential in populations of cells and at multiple membrane systems within single cells in response to various stimuli in living plants. Such coordinated electrical changes may globally orchestrate cell behavior to elicit successful reactions of the root as a whole to varying and unpredictable environments. Findings from membrane “potential-omics” can eventually be fused with data sets from other “omics” approaches to forge the integrated and comprehensive understanding that underpins the concept of systems biology.

Monitoring of real changes of plasma membrane potential by diS-C3(3) fluorescence in yeast cell suspensions

The fluorescent dye 3,3'-dipropylthiadicarbocyanine, diS-C(3)(3), is a suitable probe to monitor real changes of plasma membrane potential in yeast cells which are too small for direct membrane potential measurements with microelectrodes. A method presented in this paper makes it possible to convert changes of equilibrium diS-C(3)(3) fluorescence spectra, measured in yeast cell suspensions under certain defined conditions, into underlying membrane potential differences, scaled in the units of millivolts. Spectral analysis of synchronously scanned diS-C(3)(3) fluorescence allows to assess the amount of dye accumulated in cells without otherwise necessary sample taking and following separation of cells from the medium. Moreover, membrane potential changes can be quantified without demanding calibration protocols. The applicability of this approach was demonstrated on the depolarization of Rhodotorula glutinis yeast cells upon acidification of cell suspensions and/or by increasing extracellular K(+) concentration.

Real‐time flow cytometry for the kinetic analysis of oncosis

The standard method of distinguishing apoptotic and oncotic cells has been by microscopic analysis of nuclei and cell membrane morphology. Thus a rapid test for analyzing large numbers of cells in the study of cell necrobiology has not been possible until the recent advent of the Amnis Image-stream and real-time Lab-on-a-Chip technologies. An interesting difference between apoptosis and oncosis is that they are ATP dependent and independent processes, respectively. Here we describe an assay measuring real-time kinetic changes in the potential differences of the inner mitochondrial membrane (mmp) and the plasma membrane (pmp) in cells immediately before and after the addition of the inducing agent. Live cells were loaded with carbocyanine dye DiIC(1) (5) and bis-oxonol (DiBAC(4) (5)) to measure mmp and pmp in conjunction with annexin V-FITC and DAPI labeling for gating out annexin V binding cells and dead cells respectively. Live cells gave specific membrane signatures in response to apoptotic or oncotic reagents in real-time. Apoptosis showed little change in mmp and pmp signals over the course of 25 min, the mitochondria only showed a slight hyperpolarization. In contrast chemical treatment with oxidative phosphorylation blocker, sodium azide (SA) caused an immediate hyperpolarization spike followed by a complete abrogation of mmp over a 25 min time course. Treatment with SA (1%) also caused plasma membrane depolarization. Likewise detergent (0.01% Triton X-100) treatments also caused abrogation of mmp and depolarization of pmp. Whereas heat shock (42°C) treatment showed only a slight mitochondrial membrane potential depolarization. These flow cytometric observations were confirmed by confocal microscopy. This novel real-time kinetic assay measuring mitochondrial and plasma membrane potential changes has important implications in the field of cell necrobiology in that it allows the researcher to differentiate apoptotic and oncotic processes in an immediate manner for the first time.

Further Characterization of the Electrogenicity and pH Sensitivity of the Human Organic Anion-Transporting Polypeptides OATP1B1 and OATP1B3

Organic anion-transporting polypeptides (OATPs) are involved in the liver uptake of many endogenous and xenobiotic compounds, such as bile acids and drugs, respectively. Using Xenopus laevis oocytes and Chinese hamster ovary (CHO) cells expressing rat Oatp1a1, human OATP1B1, or OATP1B3, the sensitivity of these transporters to extracellular/intracellular pH (pHo/pHi) and changes in plasma membrane potential (ΔΨ) was investigated. In X. laevis oocytes, nonspecific plasma membrane permeability increased only at pHo below 4.5. Above this value, both using oocytes and CHO cells, extracellular acidification affected differently the specific transport of taurocholic acid (TCA) and estradiol 17β-d-glucuronide (E(2)17βG) by Oatp1a1 (stimulation), OATP1B1 (inhibition), and OATP1B3 (stimulation). Changes in substrate uptake in the presence of valinomycin (K(+)-ionophore), carbonyl cyanide 3-chlorophenylhydrazone and nigericin (protonophores), and amiloride (Na(+)/H(+)-inhibitor) and cation replacement in the medium were studied with fluorescent probes for measuring substrate uptake (cholylglycyl amidofluorescein) and changes in pHi (SNARF-4F) and ΔΨ [DilC(1)(5)]. The results suggest that activity of these three carriers is sodium/potassium-independent and affected differently by changes in pHo and ΔΨ: Oatp1a1 was confirmed to be an electroneutral anion exchanger, whereas the function of both OATP1B1 and OATP1B3 was markedly affected by the magnitude of ΔΨ. Moreover, electrophysiological measurements revealed the existence of a net anion influx associated to OATP1B1/OATP1B3-mediated transport of TCA, E(2)17βG, and estrone-3-sulfate. Furthermore, a leakage of Na(+) through OATP1B1 and OATP1B3, which is not coupled to substrate transport, was found. In conclusion, these results suggest that OATP1B1 and OATP1B3 are electrogenic transporters whose activity may be strongly affected under circumstances of displacement of local pH.

Palmitoylation of the S0-S1 Linker Regulates Cell Surface Expression of Voltage- and Calcium-activated Potassium (BK) Channels

S-Palmitoylation is rapidly emerging as an important post-translational mechanism to regulate ion channels. We have previously demonstrated that large conductance calcium- and voltage-activated potassium (BK) channels are palmitoylated within an alternatively spliced (STREX) insert. However, these studies also revealed that additional site(s) for palmitoylation must exist outside of the STREX insert, although the identity or the functional significance of these palmitoylated cysteine residues are unknown. Here, we demonstrate that BK channels are palmitoylated at a cluster of evolutionary conserved cysteine residues (Cys-53, Cys-54, and Cys-56) within the intracellular linker between the S0 and S1 transmembrane domains. Mutation of Cys-53, Cys-54, and Cys-56 completely abolished palmitoylation of BK channels lacking the STREX insert (ZERO variant). Palmitoylation allows the S0-S1 linker to associate with the plasma membrane but has no effect on single channel conductance or the calcium/voltage sensitivity. Rather, S0-S1 linker palmitoylation is a critical determinant of cell surface expression of BK channels, as steady state surface expression levels are reduced by ∼55% in the C53:54:56A mutant. STREX variant channels that could not be palmitoylated in the S0-S1 linker also displayed significantly reduced cell surface expression even though STREX insert palmitoylation was unaffected. Thus our work reveals the functional independence of two distinct palmitoylation-dependent membrane interaction domains within the same channel protein and demonstrates the critical role of S0-S1 linker palmitoylation in the control of BK channel cell surface expression.

Direct voltage control of endogenous lysophosphatidic acid G-protein-coupled receptors inXenopusoocytes

Lysophosphatidic acid (LPA) G-protein-coupled receptors (GPCRs) play important roles in a variety of physiological and pathophysiological processes, including cell proliferation, angiogenesis, central nervous system development and carcinogenesis. Whilst many ion channels and transporters are recognized to be controlled by a change in cell membrane potential, little is known about the voltage dependence of other proteins involved in cell signalling. Here, we show that the InsP(3)-mediated Ca(2+) response stimulated by the endogenous LPA GPCR in Xenopus oocytes is potentiated by membrane depolarization. Depolarization was able to repetitively stimulate transient [Ca(2+)](i) increases after the initial agonist-evoked response. In addition, the initial rate and amplitude of the LPA-dependent Ca(2+) response were significantly modulated by the steady holding potential over the physiological range, such that the response to LPA was potentiated at depolarized potentials and inhibited at hyperpolarized potentials. Enhancement of LPA receptor-evoked Ca(2+) mobilization by membrane depolarization was observed over a wide range of agonist concentrations. Importantly, the amplitude of the depolarization-evoked intracellular Ca(2+) increase displayed an inverse relationship with agonist concentration such that the greatest effect of voltage was observed at near-threshold levels of agonist. Voltage-dependent Ca(2+) release was not induced by direct elevation of InsP(3) or by activation of heterotrimeric G-proteins in the absence of agonist, indicating that the LPA GPCR itself represents the primary site of action of membrane voltage. This novel modulation of LPA signalling by membrane potential may have important consequences for control of Ca(2+) signals both in excitable and non-excitable tissues.

Impact of low intensity millimetre waves on cell functions

Investigations on the biological impact of low levels of millimetre-wave energy date back to the first experiments on the generation and detection of these high-frequency signals by Sir Jagadis Chunder Bose at the end of the 19th century. Slightly more than a hundred years later, millimetre-wave transmission has become a ubiquitous commercial reality. Despite the widespread use of millimetre-wave transmitters for communications, radar and even non-lethal weapons systems, only a handful of researchers have funded programmes focusing on millimetre-wave interactions with biological systems. As such, there is a growing need for a better understanding of the mechanisms of these interactions and their possible adverse and therapeutic implications. Independent of the health impact of long-term exposure to high doses of millimetre-wave energy on whole organisms, there exists the potential for subtle effects on specific tissues or organs which can best be quantified in studies which examine real-time changes in cellular function as energy is applied. In this Letter, a series of experiments are presented which show changes in cell membrane potential and the action potential firing rate of cortical neurons under short (1 min) exposures to continuous-wave 60 GHz radiation at µW/cm2 power levels, more than 1000 times below the US government maximum permissible exposure. The findings have implications for non-contact stimulation and control of neurologic function, and might prove useful in a variety of health applications from suppression of peripheral neuropathic pain to the treatment of central neurological disorders.