An in-situ surface electrochemistry approach toward whole-cell studies: Charge transfer between Geobacter sulfurreducens and electrified metal/electrolyte interfaces through linker molecules

Electrochemical reactivity and structure properties of electrogenic bacteria, Geobacter sulfurreducens (Gs) were studied to explore the heterogeneous electron transfer at the bacteria/electrode interface using electrochemical and in-situ spectroscopic techniques. The redox behavior of Gs adsorbed on a gold electrode, which is modified with a ω-functionalized self-assembled monolayer (SAM) of alkanethiols, depends strongly on the terminal group. The latter interacts directly with outermost cytochromes embedded into the outer membrane of the Gs cells. The redox potential of bacterial cells bound electrostatically to a carboxyl-terminated SAM is close to that observed for bacteria attached to a bare gold electrode, revealing a high electronic coupling at the cell/SAM interface. The redox potentials of bacterial cells adsorbed on amino- and pyridyl-terminated SAMs are significantly different suggesting that the outermost cytochromes changes their conformation upon adsorption on these SAMs. No redox activity of Gs was found with CH3-, N(CH3)3+- and OH-terminated SAMs. Complementary in-situ spectroscopic studies on bacteria/SAMs/Au electrode assemblies were carried out to monitor structure changes of the bacterial cells upon polarization. Spectro-electrochemical techniques revealed the electrochemical turnover of the oxidized and reduced states of outer membrane cytochromes (OMCs) in Gs, providing evidence that the OMCs are responsible for the direct electron transfer to metal electrodes, such as gold or silver, during the electricity production. Furthermore, we observed spectroscopic signatures of the native structure of the OMCs and no conformational change during the oxidation/reduction process of the microorganisms. These findings indicate that the carboxyl-anchoring group provides biocompatible conditions for the outermost cytochromes of the Gs, which facilitate the heterogeneous electron transfer at the microorganism/electrode interface.

Biochemical, single-channel, whole-cell patch clamp, and pharmacological analyses of endogenous TRPM4 channels in HEK293 cells

Human embryonic kidney cells 293 (HEK293) are widely used as cellular heterologous expression systems to study transfected ion channels. This work characterizes the endogenous expression of TRPM4 channels in HEK293 cells. TRPM4 is an intracellular Ca(2+)-activated non-selective cationic channel expressed in many cell types. Western blot analyses have revealed the endogenous expression of TRPM4. Single channel 22pS conductance with a linear current-voltage relationship was observed using the inside-out patch clamp configuration in the presence of intracellular Ca(2+). The channels were permeable to the monovalent cations Na(+) and K(+), but not to Ca(2+). The open probability was voltage-dependent, being higher at positive potentials. Using the whole-cell patch clamp "ruptured patch" configuration, the amplitude of the intracellular Ca(2+)-activated macroscopic current was dependent on time after patch rupture. Initial transient activation followed by a steady-increase reaching a plateau phase was observed. Biophysical analyses of the macroscopic current showed common properties with those from HEK293 cells stably transfected with human TRPM4b, with the exception of current time course and Ca(2+) sensitivity. The endogenous macroscopic current reached the plateau faster and required 61.9±3.5μM Ca(2+) to be half-maximally activated versus 84.2±1.5μM for the transfected current. The pharmacological properties, however, were similar in both conditions. One hundred μM of flufenamic acid and 9-phenanthrol strongly inhibited the endogenous current. Altogether, the data demonstrate the expression of endogenous TRMP4 channels in HEK293 cells. This observation should be taken into account when using this cell line to study TRPM4 or other types of Ca(2+)-activated channels.

An in situ surface electrochemistry approach towards whole-cell studies: the structure and reactivity of a Geobacter sulfurreducens submonolayer on electrified metal/electrolyte interfaces

A direct electron transfer process between bacterial cells of electrogenic species Geobacter sulfurreducens (Gs) and electrified electrode surfaces was studied to exploit the reactivity of Gs submonolayers on gold and silver surfaces. A submonolayer of Gs was prepared and studied to explore specifically the heterogeneous electron transfer properties at the bacteria/electrode interface. In situ microscopic techniques characterised the morphology of the Gs submonolayers under the operating conditions. In addition, complementary in situ spectroscopic techniques that allowed us to access in situ molecular information of the Gs with high surface selectivity and sensitivity were employed. The results provided clear evidence that the outermost cytochrome C in Gs is responsible for the heterogeneous electron transfer, which is in direct contact with the metal electrode. Feasibility of single cell in situ studies under operating conditions was demonstrated where the combination of surface-electrochemical tools at the nano- and micro-scale with microbiological approaches can offer unique opportunities for the emerging field of electro-microbiology to explore processes and interactions between microorganisms and electrical devices.

Pitfalls when examining gap junction hemichannels: interference from volume-regulated anion channels

Human HeLa cells transfected with mouse connexin45 were used to explore the experimental conditions suitable to measure currents carried by gap junction hemichannels. Experiments were performed with a voltage-clamp technique and whole-cell recording. Lowering [Ca(2+)](o) from 2 mM to 20 nM evoked an extra current, I (m), putatively carried by Cx45 hemichannels. However, the variability of I (m) (size, voltage sensitivity, kinetics) suggested the involvement of other channels. The finding that growth medium in the incubator increased the osmolarity with time implied that volume-regulated anion channels (VRAC) may participate. This assumption was reinforced by the following observations. On the one hand, keeping [Ca(2+)](o) normal while the osmolarity of the extracellular solution was reduced from 310 to 290 mOsm yielded a current characteristic of VRAC; I (VRAC) activated/deactivated at negative/positive voltage, giving rise to the conductance functions g (VRAC,inst)=f(V (m)) (inst: instantaneous; V (m): membrane potential) and g (VRAC,ss)=f(V (m)) (ss: steady state). Moreover, it was reversibly inhibited by mibefradil, a Cl(-)channel blocker (binding constant K (d)=38 microM, Hill coefficient n=12), but not by the gap junction channel blocker 18alpha-glycyrrhetinic acid. On the other hand, minimizing the osmotic imbalance while [Ca(2+)](o) was reduced led to a current typical for Cx45 hemichannels; I (hc) activated/deactivated at positive/negative voltage. Furthermore, it was reversibly inhibited by 18alpha-glycyrrhetinic acid or palmitoleic acid, but not by mibefradil. Computations based on g (VRAC,ss)=f(V (m)) and g (hc,ss)=f(V (m)) indicated that the concomitant operation of both currents results in a bell-shaped conductance-voltage relationship. The functional implications of the data presented are discussed. Conceivably, VRAC and hemichannels are involved in a common signaling pathway.

Whole Proteome Analysis of the Protozoan Parasite Trypanosoma brucei using Stable Isotope Labeling by Amino Acids in Cell Culture and Mass Spectrometry

The single-celled protozoan Trypanosoma brucei spp. is the causative agent of human African trypanosomiasis and nagana in cattle. Quantitative proteomics for the first time allowed for the characterization of the proteome from several different life stages of the parasite (1-3). To achieve this, stable isotope labeling by amino acids in cell culture (SILAC; (4)) was adapted to T. brucei spp. cultures. T. brucei cells grown in standard media with dialyzed fetal calf serum containing heavy isotope-labeled amino acids (arginine and lysine) show efficient incorporation of the labeled amino acids into the whole cell proteome (8-12 divisions) and no detectable amino acid conversions. The method can be applied to both of the major life stages of the parasite and in combination with RNAi or gene knock-out approaches.

Neuronal precursor cell-expressed developmentally down-regulated 4-1 (NEDD4-1) controls the sorting of newly synthesized Ca(V)1.2 calcium channels

Neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) proteins are ubiquitin ligases, which attach ubiquitin moieties to their target proteins, a post-translational modification that is most commonly associated with protein degradation. Nedd4 ubiquitin ligases have been shown to down-regulate both potassium and sodium channels. In this study, we investigated whether Nedd4 ubiquitin ligases also regulate Ca(v) calcium channels. We expressed three Nedd4 family members, Nedd4-1, Nedd4-2, and WWP2, together with Ca(v)1.2 channels in tsA-201 cells. We found that Nedd4-1 dramatically decreased Ca(v) whole-cell currents, whereas Nedd4-2 and WWP2 failed to regulate the current. Surface biotinylation assays revealed that Nedd4-1 decreased the number of channels inserted at the plasma membrane. Western blots also showed a concomitant decrease in the total expression of the channels. Surprisingly, however, neither the Ca(v) pore-forming α1 subunit nor the associated Ca(v)β and Ca(v)α(2)δ subunits were ubiquitylated by Nedd4-1. The proteasome inhibitor MG132 prevented the degradation of Ca(v) channels, whereas monodansylcadaverine and chloroquine partially antagonized the Nedd4-1-induced regulation of Ca(v) currents. Remarkably, the effect of Nedd4-1 was fully prevented by brefeldin A. These data suggest that Nedd4-1 promotes the sorting of newly synthesized Ca(v) channels for degradation by both the proteasome and the lysosome. Most importantly, Nedd4-1-induced regulation required the co-expression of Ca(v)β subunits, known to antagonize the retention of the channels in the endoplasmic reticulum. Altogether, our results suggest that Nedd4-1 interferes with the chaperon role of Ca(v)β at the endoplasmic reticulum/Golgi level to prevent the delivery of Ca(v) channels at the plasma membrane.

Biologicals and fetal cell therapy for wound and scar management

Few biopharmaceutical preparations developed from biologicals are available for tissue regeneration and scar management. When developing biological treatments with cellular therapy, selection of cell types and establishment of consistent cell banks are crucial steps in whole-cell bioprocessing. Various cell types have been used in treatment of wounds to reduce scar to date including autolog and allogenic skin cells, platelets, placenta, and amniotic extracts. Experience with fetal cells show that they may provide an interesting cell choice due to facility of outscaling and known properties for wound healing without scar. Differential gene profiling has helped to point to potential indicators of repair which include cell adhesion, extracellular matrix, cytokines, growth factors, and development. Safety has been evidenced in Phase I and II clinical fetal cell use for burn and wound treatments with different cell delivery systems. We present herein that fetal cells present technical and therapeutic advantages compared to other cell types for effective cell-based therapy for wound and scar management.

Ca(2+) release from the sarcoplasmic reticulum activated by the low affinity Ca(2+) chelator TPEN in ventricular myocytes

The Ca(2+) content of the sarcoplasmic reticulum (SR) of cardiac myocytes is thought to play a role in the regulation and termination of SR Ca(2+) release through the ryanodine receptors (RyRs). Experimentally altering the amount of Ca(2+) within the SR with the membrane-permeant low affinity Ca(2+) chelator TPEN could improve our understanding of the mechanism(s) by which SR Ca(2+) content and SR Ca(2+) depletion can influence Ca(2+) release sensitivity and termination. We applied laser-scanning confocal microscopy to examine SR Ca(2+) release in freshly isolated ventricular myocytes loaded with fluo-3, while simultaneously recording membrane currents using the whole-cell patch-clamp technique. Following application of TPEN, local spontaneous Ca(2+) releases increased in frequency and developed into cell-wide Ca(2+) waves. SR Ca(2+) load after TPEN application was found to be reduced to about 60% of control. Isolated cardiac RyRs reconstituted into lipid bilayers exhibited a two-fold increase of their open probability. At the low concentration used (20-40muM), TPEN did not significantly inhibit the SR-Ca(2+)-ATPase in SR vesicles. These results indicate that TPEN, traditionally used as a low affinity Ca(2+) chelator in intracellular Ca(2+) stores, may also act directly on the RyRs inducing an increase in their open probability. This in turn results in an increased Ca(2+) leak from the SR leading to its Ca(2+) depletion. Lowering of SR Ca(2+) content may be a mechanism underlying the recently reported cardioprotective and antiarrhythmic features of TPEN.

Calcium indicator loading of neurons using single-cell electroporation

Studies of subcellular Ca(2+) signaling rely on methods for labeling cells with fluorescent Ca(2+) indicator dyes. In this study, we demonstrate the use of single-cell electroporation for Ca(2+) indicator loading of individual neurons and small neuronal networks in rat neocortex in vitro and in vivo. Brief voltage pulses were delivered through glass pipettes positioned close to target cells. This approach resulted in reliable and rapid (within seconds) loading of somata and subsequent complete labeling of dendritic and axonal arborizations. By using simultaneous whole-cell recordings in brain slices, we directly addressed the effect of electroporation on neurons. Cell viability was high (about 85%) with recovery from the membrane permeabilization occurring within a minute. Electrical properties of recovered cells were indistinguishable before and after electroporation. In addition, Ca(2+) transients with normal appearance could be evoked in dendrites, spines, and axonal boutons of electroporated cells. Using negative-stains of somata, targeted single-cell electroporation was equally applicable in vivo. We conclude that electroporation is a simple approach that permits Ca(2+) indicator loading of multiple cells with low background staining within a short amount of time, which makes it especially well suited for functional imaging of subcellular Ca(2+) dynamics in small neuronal networks.

β1- and β3- voltage-gated sodium channel subunits modulate cell surface expression and glycosylation of Nav1.7 in HEK293 cells

Voltage-gated sodium channels (Navs) are glycoproteins composed of a pore-forming α-subunit and associated β-subunits that regulate Nav α-subunit plasma membrane density and biophysical properties. Glycosylation of the Nav α-subunit also directly affects Navs gating. β-subunits and glycosylation thus comodulate Nav α-subunit gating. We hypothesized that β-subunits could directly influence α-subunit glycosylation. Whole-cell patch clamp of HEK293 cells revealed that both β1- and β3-subunits coexpression shifted V ½ of steady-state activation and inactivation and increased Nav1.7-mediated I Na density. Biotinylation of cell surface proteins, combined with the use of deglycosydases, confirmed that Nav1.7 α-subunits exist in multiple glycosylated states. The α-subunit intracellular fraction was found in a core-glycosylated state, migrating at ~250 kDa. At the plasma membrane, in addition to the core-glycosylated form, a fully glycosylated form of Nav1.7 (~280 kDa) was observed. This higher band shifted to an intermediate band (~260 kDa) when β1-subunits were coexpressed, suggesting that the β1-subunit promotes an alternative glycosylated form of Nav1.7. Furthermore, the β1-subunit increased the expression of this alternative glycosylated form and the β3-subunit increased the expression of the core-glycosylated form of Nav1.7. This study describes a novel role for β1- and β3-subunits in the modulation of Nav1.7 α-subunit glycosylation and cell surface expression.

Force-controlled patch clamp of beating cardiac cells.

From its invention in the 1970s, the patch clamp technique is the gold standard in electrophysiology research and drug screening because it is the only tool enabling accurate investigation of voltage-gated ion channels, which are responsible for action potentials. Because of its key role in drug screening, innovation efforts are being made to reduce its complexity toward more automated systems. While some of these new approaches are being adopted in pharmaceutical companies, conventional patch-clamp remains unmatched in fundamental research due to its versatility. Here, we merged the patch clamp and atomic force microscope (AFM) techniques, thus equipping the patch-clamp with the sensitive AFM force control. This was possible using the FluidFM, a force-controlled nanopipette based on microchanneled AFM cantilevers. First, the compatibility of the system with patch-clamp electronics and its ability to record the activity of voltage-gated ion channels in whole-cell configuration was demonstrated with sodium (NaV1.5) channels. Second, we showed the feasibility of simultaneous recording of membrane current and force development during contraction of isolated cardiomyocytes. Force feedback allowed for a gentle and stable contact between AFM tip and cell membrane enabling serial patch clamping and injection without apparent cell damage.

Nedd4-2-dependent ubiquitylation and regulation of the cardiac potassium channel hERG1

The voltage-gated cardiac potassium channel hERG1 (human ether-à-gogo-related gene 1) plays a key role in the repolarization phase of the cardiac action potential (AP). Mutations in its gene, KCNH2, can lead to defects in the biosynthesis and maturation of the channel, resulting in congenital long QT syndrome (LQTS). To identify the molecular mechanisms regulating the density of hERG1 channels at the plasma membrane, we investigated channel ubiquitylation by ubiquitin ligase Nedd4-2, a post-translational regulatory mechanism previously linked to other ion channels. We found that whole-cell hERG1 currents recorded in HEK293 cells were decreased upon neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2) co-expression. The amount of hERG1 channels in total HEK293 lysates and at the cell surface, as assessed by Western blot and biotinylation assays, respectively, were concomitantly decreased. Nedd4-2 and hERG1 interact via a PY motif located in the C-terminus of hERG1. Finally, we determined that Nedd4-2 mediates ubiquitylation of hERG1 and that deletion of this motif affects Nedd4-2-dependent regulation. These results suggest that ubiquitylation of the hERG1 protein by Nedd4-2, and its subsequent down-regulation, could represent an important mechanism for modulation of the duration of the human cardiac action potential.

Role of KCNMA1 in breast cancer

KCNMA1 encodes the α-subunit of the large conductance, voltage and Ca(2+)-activated (BK) potassium channel and has been reported as a target gene of genomic amplification at 10q22 in prostate cancer. To investigate the prevalence of the amplification in other human cancers, the copy number of KCNMA1 was analyzed by fluorescence-in-situ-hybridization (FISH) in 2,445 tumors across 118 different tumor types. Amplification of KCNMA1 was restricted to a small but distinct fraction of breast, ovarian and endometrial cancer with the highest prevalence in invasive ductal breast cancers and serous carcinoma of ovary and endometrium (3-7%). We performed an extensive analysis on breast cancer tissue microarrays (TMA) of 1,200 tumors linked to prognosis. KCNMA1 amplification was significantly associated with high tumor stage, high grade, high tumor cell proliferation, and poor prognosis. Immunofluorescence revealed moderate or strong KCNMA1 protein expression in 8 out of 9 human breast cancers and in the breast cancer cell line MFM223. KCNMA1-function in breast cancer cell lines was confirmed by whole-cell patch clamp recordings and proliferation assays, using siRNA-knockdown, BK channel activators such as 17ß-estradiol and the BK-channel blocker paxilline. Our findings revealed that enhanced expression of KCNMA1 correlates with and contributes to high proliferation rate and malignancy of breast cancer.

Structural plasticity of spines at giant mossy fiber synapses

The granule cells of the dentate gyrus give rise to thin unmyelinated axons, the mossy fibers. They form giant presynaptic boutons impinging on large complex spines on the proximal dendritic portions of hilar mossy cells and CA3 pyramidal neurons. While these anatomical characteristics have been known for some time, it remained unclear whether functional changes at mossy fiber synapses such as long-term potentiation (LTP) are associated with structural changes. Since subtle structural changes may escape a fine-structural analysis when the tissue is fixed by using aldehydes and is dehydrated in ethanol, rapid high-pressure freezing (HPF) of the tissue was applied. Slice cultures of hippocampus were prepared and incubated in vitro for 2 weeks. Then, chemical LTP (cLTP) was induced by the application of 25 mM tetraethylammonium (TEA) for 10 min. Whole-cell patch-clamp recordings from CA3 pyramidal neurons revealed a highly significant potentiation of mossy fiber synapses when compared to control conditions before the application of TEA. Next, the slice cultures were subjected to HPF, cryosubstitution, and embedding in Epon for a fine-structural analysis. When compared to control tissue, we noticed a significant decrease of synaptic vesicles in mossy fiber boutons and a concomitant increase in the length of the presynaptic membrane. On the postsynaptic side, we observed the formation of small, finger-like protrusions, emanating from the large complex spines. These short protrusions gave rise to active zones that were shorter than those normally found on the thorny excrescences. However, the total number of active zones was significantly increased. Of note, none of these cLTP-induced structural changes was observed in slice cultures from Munc13-1 deficient mouse mutants showing severely impaired vesicle priming and docking. In conclusion, application of HPF allowed us to monitor cLTP-induced structural reorganization of mossy fiber synapses.

Cellular mechanisms of burst firing-mediated long-term depression in rat neocortical pyramidal cells

During wakefulness and sleep, neurons in the neocortex emit action potentials tonically or in rhythmic bursts, respectively. However, the role of synchronized discharge patterns is largely unknown. We have recently shown that pairings of excitatory postsynaptic potentials (EPSPs) and action potential bursts or single spikes lead to long-term depression (burst-LTD) or long-term potentiation, respectively. In this study, we elucidate the cellular mechanisms of burst-LTD and characterize its functional properties. Whole-cell patch-clamp recordings were obtained from layer V pyramidal cells in somatosensory cortex of juvenile rats in vitro and composite EPSPs and EPSCs were evoked extracellularly in layers II/III. Repetitive burst-pairings led to a long-lasting depression of EPSPs and EPSCs that was blocked by inhibitors of metabotropic glutamate group 1 receptors, phospholipase C, protein kinase C (PKC) and calcium release from the endoplasmic reticulum, and that required an intact machinery for endocytosis. Thus, burst-LTD is induced via a Ca2+- and phosphatidylinositol-dependent activation of PKC and expressed through phosphorylation-triggered endocytosis of AMPA receptors. Functionally, burst-LTD is inversely related to EPSP size and bursts dominate single spikes in determining the sign of synaptic plasticity. Thus burst-firing constitutes a signal by which coincident synaptic inputs are proportionally downsized. Overall, our data thus suggest a mechanism by which synaptic weights can be reconfigured during non-rapid eye movement sleep.