Modulation Of Membrane Potential Research Articles

Two Neonatal Diabetes Mutations on Transmembrane Helix 15 of SUR1 Increase Affinity for ATP and ADP at Nucleotide Binding Domain 2

K(ATP) channels, (SUR1/Kir6.2)(4) (sulfonylurea receptor type 1/potassium inward rectifier type 6.2) respond to the metabolic state of pancreatic β-cells, modulating membrane potential and insulin exocytosis. Mutations in both subunits cause neonatal diabetes by overactivating the pore. Hyperactive channels fail to close appropriately with increased glucose metabolism; thus, β-cell hyperpolarization limits insulin release. K(ATP) channels are inhibited by ATP binding to the Kir6.2 pore and stimulated, via an uncertain mechanism, by magnesium nucleotides at SUR1. Glibenclamide (GBC), a sulfonylurea, was used as a conformational probe to compare nucleotide action on wild type versus Q1178R and R1182Q SUR1 mutants. GBC binds with high affinity to aporeceptors, presumably in the inward facing ATP-binding cassette configuration; MgATP reduces binding affinity via a shift to the outward facing conformation. To determine nucleotide affinities under equilibrium, non-hydrolytic conditions, Mg(2+) was eliminated. A four-state equilibrium model describes the allosteric linkage. The K(D) for ATP(4-) is ~1 versus 12 mM, Q1178R versus wild type, respectively. The linkage constant is ~10, implying that outward facing conformations bind GBC with a lower affinity, 9-10 nM for Q1178R. Thus, nucleotides cannot completely inhibit GBC binding. Binding of channel openers is reported to require ATP hydrolysis, but diazoxide, a SUR1-selective agonist, concentration-dependently augments ATP(4-) action. An eight-state model describes linkage between diazoxide and ATP(4-) binding; diazoxide markedly increases the affinity of Q1178R for ATP(4-) and ATP(4-) augments diazoxide binding. NBD2, but not NBD1, has a higher affinity for ATP (and ADP) in mutant versus wild type (with or without Mg(2+)). Thus, the mutants spend more time in nucleotide-bound conformations, with reduced affinity for GBC, that activate the pore.

Graphene Interfaced with Biological Cells: Opportunities and Challenges.

By interfacing the quantum mechanical properties of nanomaterials with the complex processes in biology, several bio/nano systems have evolved with applications in biosensors, cellular devices, drug delivery, and biophotoluminescence. One recent breakthrough has been the application of graphene, a two-dimensional (2-D) sheet of sp(2) hybridized carbon atoms arranged in a honeycomb lattice, as a sensitive platform for interfacing with biological cells to detect intra- and extracellular phenomena, including cellular excretion and cell membrane's potential modulation. In this Perspective, we discuss the recent results on graphene/cell interfacial devices and the principles defining the modulation of charge-carrier properties in graphene and its derivatives via interaction with cellular membranes. Graphene's high sensitivity in these applications evolves from the π-carrier cloud confined within an atom-thick layer, quantum-capacitance-induced doping enhancement, closely spaced electronic bands, and a large surface area. We discuss the effect of the electronegativity of the cell wall and the dynamic changes in its chemical potential on doping specific carriers into graphene. Finally, we discuss the challenges and opportunities of graphene-interfaced biocellular systems.

Indole prevents Escherichia coli cell division by modulating membrane potential

Indole is a bacterial signalling molecule that blocks E. coli cell division at concentrations of 3–5mM. We have shown that indole is a proton ionophore and that this activity is key to the inhibition of division. By reducing the electrochemical potential across the cytoplasmic membrane of E. coli, indole deactivates MinCD oscillation and prevents formation of the FtsZ ring that is a prerequisite for division. This is the first example of a natural ionophore regulating a key biological process. Our findings have implications for our understanding of membrane biology, bacterial cell cycle control and potentially for the design of antibiotics that target the cell membrane.

Escherichia Coli Regulates Cell Division by Modulating Membrane Potential

Indole is a very important biological molecule being produced by more than 85 Gram positive and Gram negative bacterial species, including Escherichia coli. Among its several physiological functions indole is shown to stop bacterial cell division when secreted in large quantities.Here we study the effect of indole on the electrical properties of the lipid membranes. Using electrophysiology, we show that the indole molecules are able to short-circuit lipid membranes. We also show that the rate of mitochondrial oxygen consumption measured in the presence of succinate increases when the indole concentration is titrated up. Therefore indole is capable of lowering the energetic barrier for ion permeation across lipid membranes. Since the bacterial cell division is strongly influenced by the ability of the cell to maintain a membrane potential(1) this is the first example of a bacterium using an ionophore to regulate the bacterial cell division. These findings have implications for our understanding of membrane biology, bacterial cell cycle control and potentially for the design of antibiotics that target the cell membrane.1. Strahl, H. & Hamoen, L.W. Membrane potential is important for bacterial cell division. Proceedings of the National Academy of Sciences 107, 12281-12286 (2010).

Down-Regulation of the Large-Conductance Ca<sup>2+</sup>-Activated K<sup>+</sup> Channel, K<sub>Ca</sub>1.1 in the Prostatic Stromal Cells of Benign Prostate Hyperplasia

Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel encoded by K(Ca)1.1 plays an important role in the control of smooth muscle tone by modulating membrane potential and intracellular Ca(2+) mobilization. BK(Ca) channel is functionally expressed in prostatic smooth muscle cells, and is activated by α(1)-adrenoceptor agonists. The main objective of this study was to elucidate the pathophysiological significance of changes in prostatic K(Ca)1.1 expressions in benign prostatic hyperplasia (BPH). Our previous study has shown that K(Ca)3.1 encoding intermediate-conductance K(Ca) (IK(Ca)) channel is up-regulated in stromal cells of implanted urogenital sinuses (UGSs) of stromal hyperplasia BPH model rats and in those of prostatic tissues from BPH patients. In the present study, the results from real-time polymerase chain reaction (PCR), Western blot, and immunohistochemical analyses showed significant down-regulation of K(Ca)1.1 transcripts and proteins and negative correlation between K(Ca)1.1 and K(Ca)3.1 transcript expressions in prostatic stromal cells of both BPH model rats and BPH patients. Corresponding to down-regulation of K(Ca)1.1 expression in stromal cells of implanted UGSs, membrane depolarization by application of the BK(Ca) channel blocker was disappeared. Down-regulation of K(Ca)1.1 may be involved in the phenotype switch from contractile profile to proliferative one in prostatic stromal cells of BPH patients.

Calcium Signaling in Smooth Muscle

Changes in intracellular Ca(2+) are central to the function of smooth muscle, which lines the walls of all hollow organs. These changes take a variety of forms, from sustained, cell-wide increases to temporally varying, localized changes. The nature of the Ca(2+) signal is a reflection of the source of Ca(2+) (extracellular or intracellular) and the molecular entity responsible for generating it. Depending on the specific channel involved and the detection technology employed, extracellular Ca(2+) entry may be detected optically as graded elevations in intracellular Ca(2+), junctional Ca(2+) transients, Ca(2+) flashes, or Ca(2+) sparklets, whereas release of Ca(2+) from intracellular stores may manifest as Ca(2+) sparks, Ca(2+) puffs, or Ca(2+) waves. These diverse Ca(2+) signals collectively regulate a variety of functions. Some functions, such as contractility, are unique to smooth muscle; others are common to other excitable cells (e.g., modulation of membrane potential) and nonexcitable cells (e.g., regulation of gene expression).

An Inducible Expression System of the Calcium-Activated Potassium Channel 4 to Study the Differential Impact on Embryonic Stem Cells

Rationale. The family of calcium-activated potassium channels consists of four members with varying biological functions and conductances. Besides membrane potential modulation, SK channels have been found to be involved in cardiac pacemaker cell development from ES cells and morphological shaping of neural stem cells. Objective. Distinct SK channel subtype expression in ES cells might elucidate their precise impact during cardiac development. We chose SK channel subtype 4 as a potential candidate influencing embryonic stem cell differentiation. Methods. We generated a doxycycline inducible mouse ES cell line via targeted homologous recombination of a cassette expressing a bicistronic construct encoding SK4 and a fluorophore from the murine HPRT locus. Conclusion. We characterized the mouse ES cell line iSK4-AcGFP. The cassette is readily expressed under the control of doxycycline, and the overexpression of SK4 led to an increase in cardiac and pacemaker cell differentiation thereby serving as a unique tool to characterize the cell biological variances due to specific SK channel overexpression.

Functional Changes in Neocortical Activity in Huntington's Disease Model Mice: An in vivo Intracellular Study

Studies of animal models of Huntington's disease (HD) have revealed that neocortical and neostriatal neurons of these animals in vitro exhibit a number of morphological and physiological changes, including increased input resistance and changes in neocortical synaptic inputs. We measured the functional effects of polyglutamate accumulation in neocortical neurons in R6/2 mice (8–14 weeks of age) and their age-matched non-transgenic littermates using in vivo intracellular recordings. All neurons showed spontaneous membrane potential fluctuations. The current/voltage and the firing properties of the HD neocortical neurons were significantly altered, especially in the physiologically relevant current range around and below threshold. As a result, membrane potential transitions from the Down state to Up state were evoked with smaller currents in HD neocortical neurons than in controls. The excitation-to-frequency curves of the HD mice were significantly steeper than those of controls, indicating a smaller input–output dynamic range for these neurons. Increased likelihood of Down to Up state transitions could cause pathological recruitment of corticostriatal assemblies by increasing correlated neuronal activity. We measured coherence of the in vivo intracellular recordings with simultaneously recorded electrocorticograms. We found that the peak of the coherence at <5 Hz was significantly smaller in the HD animals, indicating that the amount of coherence in the state transitions of single neurons is less correlated with global activity than non-transgenic controls. We propose that decreased correlation of neocortical inputs may be a major physiological cause underlying the errors in sensorimotor pattern generation in HD.

Support for a synaptic chain model of neuronal sequence generation

SummaryIn songbirds, the remarkable temporal precision of song is generated by a sparse sequence of bursts in the premotor nucleus HVC (proper name). To distinguish between two possible classes of models of neural sequence generation, we carried out intracellular recordings of HVC neurons in singing birds. We found that the subthreshold membrane potential is characterized by a large rapid depolarization 5–10 ms prior to burst onset, consistent with a synaptically-connected chain of neurons in HVC. We found no evidence for the slow membrane potential modulation predicted by models in which burst timing is controlled by subthreshold dynamics. Furthermore, bursts ride on an underlying depolarization of ~10ms duration, likely the result of a regenerative calcium spike within HVC neurons that could facilitate the propagation of activity through a chain network with high temporal precision. Our results shed light on the fundamental mechanisms by which neural circuits can generate complex sequential behaviours.

Membrane potential modulation of ionomycin-stimulated Ca2+ entry via Ca2+/H+ exchange and SOC in rat submandibular acinar cells

Ionomycin (IM) at 5 microM mediates the Ca(2+)/H(+) exchange, while IM at 1 microM activates the store-operated Ca(2+) entry channels (SOCs). In this study, the effects of depolarization on both pathways were examined in rat submandibular acinar cells by increasing extracellular K(+) concentration ([K(+)](o)). IM (5 microM, the Ca(2+)/H(+) exchange) increased the intracellular Ca(2+) concentration ([Ca(2+)](i)) to an extremely high value at 151 mM [K(+)](o). However, with increasing [K(+)](o), the rates of Ca(2+) entry decreased in a linear relationship. The reversal potential (E (rev)) for the Ca(2+)/H(+) exchange was +93 mV, suggesting that IM (5 microM) exchanges 1 Ca(2+) for 1 H(+). Thus, depolarization decreases the Ca(2+) influx via the Ca(2+)/H(+) exchange because of its electrogenicity (1 Ca(2+) for 1 H(+)). On the other hand, IM (1 microM, the SOCs) abolished an increase in [Ca(2+)](i) at 151 mM [K(+)](o). With increasing [K(+)](o), the rate of Ca(2+) entry immediately decreased linearly. The E (rev) for the SOC was +3.7 mV, suggesting that the SOCs are nonselective cation channels and less selective for Ca(2+) over Na(+) (P (Ca)/P (Na) = 8.2). Moreover, an increase in extracellular Ca(2+) concentration (20 mM) enhanced the Ca(2+) entry via the SOCs at 151 mM [K(+)](o), suggesting depolarization does not inhibit the SOCs and decreases the driving force for the Ca(2+) entry. This suggests that membrane potential changes induced by a secretory stimulation finely regulate the [Ca(2+)](i) via the SOCs in rat submandibular acinar cells. In conclusion, IM increases [Ca(2+)](i) via two pathways depending on its concentration, the exchange of 1 Ca(2+) for 1 H(+) at 5 muM and the SOCs at 1 microM.

Effect of nanoparticle surface charge at the plasma membrane and beyond.

Herein, we demonstrate that the surface charge of gold nanoparticles (AuNPs) plays a critical role in modulating membrane potential of different malignant and nonmalignant cell types and subsequent downstream intracellular events. The findings presented here describe a novel mechanism for cell-nanoparticle interactions and AuNP uptake: modulation of membrane potential and its effect on intracellular events. These studies will help understand the biology of cell-nanoparticle interactions and facilitate the engineering of nanoparticles for specific intracellular targets.

Cellular prostheses: functional abiotic nanosystems to probe, manipulate, and endow function in live cells

A class of nanoscale (∼1–10 nm) structures designed to probe, manipulate, or endow function by direct interfacing with live cells is considered. Such a concept of cellular-level prostheses is illustrated via the example of light-activated nanoscale photodiodes capable of creating local electric fields that modulate existing voltage-gated ion channels in excitable cells. The dynamics of the membrane potential modulation by such photovoltaic functional abiotic nanosystems (PV-FANs) is modeled through an appropriate equivalent circuit. The feasibility of exceeding the typical ∼10 mV depolarization threshold for activating the action potentials is examined. In view of the continuing advances in the ability to design, synthesize, and characterize abiotic nanoscale systems that can provide desired function, several approaches to the implementation of PV-FANs are discussed. The FANs as “cellular prostheses” can provide a variety of functions in response to different stimuli and represent a paradigm-changing opportunity at the frontiers of nanomedicine. From the Clinical Editor A class of nanoscale (~1-10nm) structures designed to probe, manipulate, or endow live cell functions is demonstrated in this work. More specifically, light-activated nanoscale photodiodes were found capable of creating local electric fields that modulate existing voltage gated ion channels in excitable cells, thus allowing the generation of action potentials in excitable cells via external light stimulus in a controlled fashion.

Cationic amphiphilic polyproline helix P11LRR targets intracellular mitochondria

We demonstrate that P11LRR, a recently developed amphiphilic polyproline, cell penetrating agent, is able to locate inside the mitochondria of various cell lines when administrated at high concentrations. Mitochondrial targeting was verified by confocal fluorescence co-localization of P11LRR-fluorescein with Mitotracker Red. Elimination of mitochondrial membrane potential dramatically inhibits the localization of P11LRR to mitochondria. Concentration-dependency experiments suggest that cellular internalization of P11LRR occurs via two different pathways: endocytosis and direct transport. Results indicate that the latter pathway predominates at high concentrations of P11LRR, resulting in localization of the agent to the mitochondria. The membrane translocation pathway was further confirmed by two endocytosis inhibitors, cytochalasin D and phenylarsine oxide, and by modulation of plasma membrane potential. The potential of using P11LRR as a mitochondrial drug delivery vector was demonstrated through the delivery of a covalently linked small antioxidant, dimethyltyrosine (Dmt), which allowed for the reduction of chemically induced reactive oxygen species within the mitochondria.

Allosteric Control of Gating Mechanisms Revisited: The Large Conductance Ca2+-Activated K+ Channel

Large-conductance Ca2+-activated K+ channels (BK) play a fundamental role in modulating membrane potential in many cell types. The gating of BK channels and its modulation by Ca2+ and voltage has been the subject of intensive research over almost three decades, yielding several of the most complicated kinetic mechanisms ever proposed. A large number of open and closed states disposed, respectively, in two planes, named tiers, characterize these mechanisms. Transitions between states in the same plane are cooperative and modulated by Ca2+. Transitions across planes are highly concerted and voltage-dependent. Here we reexamine the validity of the two-tiered hypothesis by restricting attention to the modulation by Ca2+. Large single channel data sets at five Ca2+ concentrations were simultaneously analyzed from a Bayesian perspective by using hidden Markov models and Markov-chain Monte Carlo stochastic integration techniques. Our results support a dramatic reduction in model complexity, favoring a simple mechanism derived from the Monod-Wyman-Changeux allosteric model for homotetramers, able to explain the Ca2+ modulation of the gating process. This model differs from the standard Monod-Wyman-Changeux scheme in that one distinguishes when two Ca2+ ions are bound to adjacent or diagonal subunits of the tetramer.

Burst Spiking of a Single Cortical Neuron Modifies Global Brain State

Different global patterns of brain activity are associated with distinct arousal and behavioral states of an animal, but how the brain rapidly switches between different states remains unclear. We here report that repetitive high-frequency burst spiking of a single rat cortical neuron could trigger a switch between the cortical states resembling slow-wave and rapid-eye-movement sleep. This is reflected in the switching of the membrane potential of the stimulated neuron from slow UP/DOWN oscillations to a persistent-UP state or vice versa, with concurrent changes in the temporal pattern of cortical local field potential (LFP) recorded several millimeters away. These results point to the power of single cortical neurons in modulating the behavioral state of an animal.

Apoptosis induction in human lung adenocarcinoma cells by oil‐soluble allyl sulfides: Triggers, pathways, and modulators

DAS (diallyl sulfide), DADS (diallyl disulfide), and DATS (diallyl trisulfide) are major oil-soluble allyl sulfides (OAS) that represent major garlic constituents. The anticarcinogenic and antimutagenic effects of these substances have been extensively studied during the last decades. Previous reports suggest that induction of apoptosis by OASs might contribute to their chemopreventive effects. In this study, we report that OASs DADS and DATS induce significant apoptosis in human lung adenocarcinoma A549 cells, whereas DAS does not. Differential modulation of reactive oxygen intermediates (ROI) and mitochondria membrane potential (MMP) may account for the apoptotic effects of DADS and DATS. The underlying molecular mechanisms of apoptosis induction by both compounds include activation of C-Jun N-terminal kinase (JNK), up-regulation of p53, and down-regulation of bcl-2 expression. In our test series, up-regulation of extracellular signal-regulated protein kinase (ERK) was dispensable for apoptosis induction; DAS, DADS, or DATS did not modify expression of MAPK p38, bax, and bcl-xL. Further investigation revealed that the specific JNK inhibitor SP600125 and the antioxidant NAC blocked DADS and DATS-induced apoptosis, whereas ERK inhibitors did not. Additionally, our data provide the first evidence that Fas-mediated cell death pathway is partly involved in DADS but not DATS-mediated cell death. Taken together, our work has elucidated the triggers, important modulators, and signal transduction pathways in DADS and DATS-mediated apoptosis.

Potassium currents induced by hydrostatic pressure modulate membrane potential and transmitter release in vestibular type II hair cells

Vestibular type II hair cells respond to increases in the hydrostatic pressure with pressure-dependent K(+) currents. We examined whether such currents may modulate transmitter release (assessed as membrane capacitance increments) by altering membrane potentials and voltage-gated Ca(2+) currents. Capacitance increments were dependent on voltage-gated Ca(2+) influx. Stimulating currents (0.7 nA) in current clamp induced depolarisations that were more negative by 8.7 +/- 2.1 mV when the bath height was elevated from 0.2 to 0.5 cm. In voltage clamp, protocols were used that simulated the time course of the membrane potential in current clamp at either low (control) or high hydrostatic pressure (high bath). The low bath protocol induced significantly larger Ca(2+) currents and increases in capacitance than the high bath protocol. We conclude that pressure-dependent K(+) currents may alter the voltage response of vestibular hair cells to an extent critical for Ca(2+) currents and transmitter release. This mechanism may contribute to vestibular dysfunction in Meniere's disease.

Dopamine D2 and Adenosine A2A Receptors Regulate NMDA-Mediated Excitation in Accumbens Neurons Through A2A–D2 Receptor Heteromerization

Bursting activity of striatal medium spiny neurons results from membrane potential oscillations between a down- and an upstate that could be regulated by G-protein-coupled receptors. Among these, dopamine D(2) and adenosine A(2A) receptors are highly enriched in striatal neurons and exhibit strong interactions whose physiological significance and molecular mechanisms remain partially unclear. More particularly, respective involvements of common intracellular signaling cascades and A(2A)-D(2) receptor heteromerization remain unknown. Here we show, by performing perforated-patch-clamp recordings on brain slices and loading competitive peptides, that D(2) and A(2A) receptors regulate the induction by N-methyl-D-aspartate of a depolarized membrane potential plateau through mechanisms relying upon specific protein-protein interactions. Indeed, D(2) receptor activation abolished transitions between a hyperpolarized resting potential and a depolarized plateau potential by regulating the Ca(V)1.3a calcium channel activity through interactions with scaffold proteins Shank1/3. Noticeably, A(2A) receptor activation had no effect per se but fully reversed the effects of D(2) receptor activation through a mechanism in which A(2A)-D(2) receptors heteromerization is strictly mandatory, demonstrating therefore a first direct physiological relevance of these heteromers. Our results show that membrane potential transitions and firing patterns in striatal neurons are tightly controlled by D(2) and A(2A) receptors through specific protein-protein interactions including A(2A)-D(2) receptors heteromerization.

Structure of spontaneous UP and DOWN transitions self-organizing in a cortical network model.

Synaptic plasticity is considered to play a crucial role in the experience-dependent self-organization of local cortical networks. In the absence of sensory stimuli, cerebral cortex exhibits spontaneous membrane potential transitions between an UP and a DOWN state. To reveal how cortical networks develop spontaneous activity, or conversely, how spontaneous activity structures cortical networks, we analyze the self-organization of a recurrent network model of excitatory and inhibitory neurons, which is realistic enough to replicate UP–DOWN states, with spike-timing-dependent plasticity (STDP). The individual neurons in the self-organized network exhibit a variety of temporal patterns in the two-state transitions. In addition, the model develops a feed-forward network-like structure that produces a diverse repertoire of precise sequences of the UP state. Our model shows that the self-organized activity well resembles the spontaneous activity of cortical networks if STDP is accompanied by the pruning of weak synapses. These results suggest that the two-state membrane potential transitions play an active role in structuring local cortical circuits.

Latrunculin A depolarizes starfish oocytes

Depolymerization of the actin cytoskeleton may liberate Ca 2+ from InsP 3-sensitive stores in some cell types, including starfish oocytes, while inhibiting Ca 2+ influx in others. However, no information is available on the modulation of membrane potential ( V m) by actin. The present study was aimed to ascertain whether the widely employed actin depolymerizing drug, latrunculin A (Lat A), affects V m in mature oocytes of the starfish Astropecten aranciacus. Lat A induced a membrane depolarization which was mimicked by cytochalasin D, another popular actin disruptor, and prevented by jasplakinolide, a stabilizer of the actin network. Lat A-elicited depolarization consisted in a positive shift in V m which reached the threshold of activation of voltage-gated Ca 2+ channels (VGCC), thus triggering an action potential. Lat A-promoted depolarization lacked the action potential in Ca 2+-free sea water, while it was abolished upon removal of external Na +. Moreover, membrane depolarization was prevented by pre-injection of BAPTA and heparin, but not ryanodine. These data indicate that Lat A induces a membrane depolarization by releasing Ca 2+ from InsP 3Rs. The Ca 2+ signal in turn activates a Ca 2+-dependent Na + entry, which causes the positive shift in V m and stimulates the VGCC.