Search

The state of the art technology for the study of ion channels is the patch clamp technique. Ion channels mediate electrical current flow, have crucial roles in cellular physiology, and are important drug targets. The most popular (whole cell) variant of the technique detects the ensemble current over the entire cell membrane. Patch clamping is still a laborious process, requiring a skilled experimenter to micromanipulate a glass pipette under a microscope to record from one cell at a time. Here we report on a planar, microstructured quartz chip for whole cell patch clamp measurements without micromanipulation or visual control. A quartz substrate of 200μm thickness is perforated by wet etching techniques resulting in apertures with diameters of ∼1μm. The apertures replace the tip of glass pipettes commonly used for patch clamp recording. Cells are positioned onto the apertures from suspension by application of suction. Whole cell recordings from different cell types (CHO, N1E-115 neuroblastoma) are performed with microstructured chips studying K+ channels and voltage gated Ca2+ channels.

Beside the ongoing efforts to determine structural information, detailed functional studies on transporters are essential to entirely understand the underlying transport mechanisms. We recently found that solid supported membrane-based electrophysiology (SSME) enables the measurement of both sugar binding and transport in the Na+/sugar cotransporter SGLT1 (Bazzone et al, 2022a). Here, we continued with a detailed kinetic characterization of SGLT1 using SSME, determining KM and KDapp for different sugars, kobs values for sugar-induced conformational transitions and the effects of Na+, Li+, H+ and Cl− on sugar binding and transport. We found that the sugar-induced pre-steady-state (PSS) charge translocation varies with the bound ion (Na+, Li+, H+ or Cl−), but not with the sugar species, indicating that the conformational state upon sugar binding depends on the ion. Rate constants for the sugar-induced conformational transitions upon binding to the Na+-bound carrier range from 208 s−1 for D-glucose to 95 s−1 for 3-OMG. In the absence of Na+, rate constants are decreased, but all sugars bind to the empty carrier. From the steady-state transport current, we found a sequence for sugar specificity (Vmax/KM): D-glucose > MDG > D-galactose > 3-OMG > D-xylose. While KM differs 160-fold across tested substrates and plays a major role in substrate specificity, Vmax only varies by a factor of 1.9. Interestingly, D-glucose has the lowest Vmax across all tested substrates, indicating a rate limiting step in the sugar translocation pathway following the fast sugar-induced electrogenic conformational transition. SGLT1 specificity for D-glucose is achieved by optimizing two ratios: the sugar affinity of the empty carrier for D-glucose is similarly low as for all tested sugars (KD,Kapp = 210 mM). Affinity for D-glucose increases 14-fold (KD,Naapp = 15 mM) in the presence of sodium as a result of cooperativity. Apparent affinity for D-glucose during transport increases 8-fold (KM = 1.9 mM) compared to KD,Naapp due to optimized kinetics. In contrast, KM and KDapp values for 3-OMG and D-xylose are of similar magnitude. Based on our findings we propose an 11-state kinetic model, introducing a random binding order and intermediate states corresponding to the electrogenic transitions detected via SSME upon substrate binding.

We present a new minaturized all-in-one Amplifier suitable to perform high-quality electrophysiology measurements. The instrument has been evaluated on a platform where ion channel proteins are reconstituted in solvent-free bilayers and multiple corresponding experimental data sets are shown.Characterization of reconstituted ion channels is still challenging as low noise current measurements in the pA range have to be combined with a large bandwidth of tens of kilohertz. Traditional transimpedance amplifiers use resistive or capacitive feedback to generate an analog output which needs to be digitized for further processing and display, resulting in complex, bulky and expensive systems. The amplifier shown here uses a switched capacitor gain stage combined with a sigma-delta digitizer which directly produces a digital signal. The actual layout is completely done in one silicon die (ASIC). This novel approach allows the integration of a multiple gain stage, filters, signal conditioning circuitry, digitizer and USB interface into the size of a pack of cigarettes while noise performance (≈100fA rms @ 1KHz) and bandwidth (up to 100kHz) are comparable to state-of-the-art patch clamp amplifiers.Combined with an already established platform using planar glass chips for semi-automatic formation of solvent-free BLMs an integrated and easy-to-use system has been set up offering students and researches an easy entry to high level electrophysiology research.

Chip based automated patch clamp technique provides an attractive biophysical tool to quantify solute permeation through membrane channels. Proteo-Giant Unilamellar Vesicles (GUVs) were used to form stable lipid bilayer across the micrometer sized hole and single channel recordings were achieved with very low background noise. Influx of antibiotics into the periplasm of gram-negative bacteria is facilitated by porins that form channel in the outer membrane. Influx of two major class of antibiotics- cephalosporin and fluroquinolones through major E.coli porins OmpF and OmpC was investigated. Ion current fluctuations through porins in the presence of penetrating antibiotics revealed thermodynamic and kinetic parameters of substrate binding from which we calculated flux. We have been able to show rapid and efficient screening of antibiotics through bacterial porins at a single-molecule scale. In vitro activity of antibiotics was determined by microbiological assays which correlates with the results obtained from lipid bilayer measurements. In addition, molecular modelling provided details on the interaction of the molecules with the channel surface, revealed the preferred orientation of the antibiotic along its pathway and the position of affinity sites. Our approach may contribute to the rational design of new antibiotics against clinical bacterial strains for the most efficient delivery to target sites.

Mechanosensitive (MS) ion channels are membrane proteins that detect and respond to membrane tension in all branches of life. In bacteria, MS channels prevent cells from lysing upon sudden hypoosmotic shock by opening and releasing solutes and water.Specially prepared giant E. coli spheroplasts of bacteria can be used to study the function of bacterial ion channels in patch clamp experiments. Several native mechanosensitive channels can be measured in E. coli. Although this technique has been used since 1987 it still needs a lot of practice to measure native E.coli membranes.View Large Image | View Hi-Res Image | Download PowerPoint SlideThe spheroplasts don't have a Cytoskeleton and are therefore very fragile. using a planar borosilicate substrate to patch these membranes stabilizes these membrane patches.The currents shown above are elicited by increasing pressure steps ranging from 0 to - 120 mbar.A characterization of endogenous mechanosensitive ion channels and overexpressed ones will be shown on the poster.

TRPA1 is an ion channel expressed on peripheral and spinal sensory neurons and it mediates pain signal transmission. It functions as a cellular sensor for detecting painful mechanical, biochemical and thermal stimuli that cause sensory nerve hyperactivity during chronic pathologies including chronic pain, inflammation, itch and cough. TRPA1 receptor is shown to induce pain hypersensitivity in animal models of diabetic neuropathic pain and its blockade attenuates pain hypersensitivity as well as later loss of the nerve fibers and their function.The Results show different methods of stimulating TRPA1 in high throughput patch clamp experiments with the SyncroPatch 384PE.

To meet the ever increasing demand for higher throughput in ion channel screening and safety testing, new platforms are being introduced allowing for yet more throughput whilst retaining data quality. A new platform recording from 96 cells at a time with giga-seals, with continuous recording during compound application and addition of multiple compounds to each of the 96 cells being recorded will be described. This new platform vastly increases throughput, while reducing the cost per data point. Borosilicate glass substrates containing microstructured apertures are used for parallel patch clamp experiments. In the figure below, a screenshot of an experiment on 96 cells in a parallel is displayed. Shown are raw current responses of CaV3.2 channels expressed in HEK293 cells to a current voltage relationship step protocol.Recordings from various ligand- and voltage-gated ion channels on this platform will be shown.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Bilayer recording is a well-established technique for in-depth studies of biophysical properties of ion channels and is particularly suited for functional studies on proteins residing in intracellular membranes. Moreover, this technique supports a host of powerful emerging analytical techniques using biological nanopores as molecular sensors. Despite its proven value, bilayer recording can be frustrating due to the capricious nature of lipid bilayers, which have to be formed manually one by one and which often lack stability. We here show a new approach and device, which speeds up the entire process by the rapid and simultaneous formation of 16, highly stable micrometer-sized bilayers using Micro-Electrode-Cavity-Array (MECA)-Chips. A study will be presented showing that the MECA supports high-resolution polymer sizing with a single biological nanopore in a parallel format (Fig.1). Additionally, data on a variety of channel proteins recorded from proteoliposomes will be shown. using a surface containing micron-sized apertures in glass substrates, the fusion of vesicles on the surface becomes an attractive method for electrophysiology and then to reconstitute membrane proteins into the lipid bilayer.Fig. 1MonoPEG-28-mediated blockages of HL-nanopore(s) on a MECA chip.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The use of cardiac myocytes is becoming increasingly important for drug safety testing. Unique features of certain planar patch clamp workstations, coupled with ease-of-use and higher data throughput, make these devices ideal tools for ion channel screening and safety testing. Using stem cell derived cardiac myocytes, recordings could be made not only in the voltage-clamp mode but also in the current-clamp mode on a planar patch clamp workstation. This demonstrates for the first time parallel current-clamp recordings on a planar patch clamp workstation. Ion channels important in drug discovery, such as hERG and voltage-gated Na+, Ca2+ and K+ channels in the voltage-clamp mode from stem cell derived cardiac myocytes will be shown. In addition, action potential recordings in the current-clamp mode at 35°C, and modulation of the action potentials by hERG active compounds, will also be shown.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Since the discovery of the possible solubilization of membrane proteins and their isolation from other membrane constituents (purification), different methods were developed to reconstitute ion channel proteins into artificial lipid bilayers. These membrane proteins were then fully functional when correctly oriented and inserted in a lipid bilayer. The reconstitution plays a central role in identifying and characterizing the mechanisms of action of membrane proteins. The activity of the membrane proteins is studied using electrophysiology using different methods e.g., the black lipid membrane. Therefore, the structure-function relationship can be investigated to better understand the biophysical properties of membrane proteins in vivo.Using a glass surface containing a micrometer hole, the fusion of vesicles on the surface becomes an attractive method for electrophysiology and then to reconstitute membrane proteins into the lipid bilayer without denaturation. Then stable lipid bilayers are formed by bursting a GUV on the glass surface, forming a free-standing portion above the hole.With this technique, we studied the biophysical and pharmacological properties of different ion channels, for example potassium channels (KcsA, Kv1.2), sodium channels (NachBac, NaVsp1) as well as other ligand-dependent (IP3 receptor, NMDA receptor), mechanosensitive channels (MscL, TRP channels) and non-specific channels (Cx43, VDAC). I will describe here our methods for incorporation of proteins into the bilayer and the recording of single ion channel current measurements by using a planar patch clamp platform.

Nicotinic acetylcholine receptors (nAChRs) belong to the superfamily of ligand-gated ion channels. The most abundant homomeric nAChR in the mammalian brain are the pentameric α7 nAChRs, which consist of five α7 subunits. Positive allosteric modulators (PAMs) of nAChRs have been implicated to be putative therapeutics in the pharmacological treatment of Alzheimer's disease, schizophrenia and Parkinson's disease.Recently, Sitzia et al. (2011) showed a striking temperature dependence for positive allosteric modulation of PNU-120596 on nAChRs. The aim of our study was to investigate the temperature dependence of other PAMs on nACHRs with automated patch clamp techniques. For this study, we have used GH4C1cells expressing human nAChR α7 subunits. Electrophysiological recordings were performed on the Patchliner, a robotic patch clamp device allowing temperature controlled recordings from up to 8 cells simultaneously.View Large Image | View Hi-Res Image | Download PowerPoint SlideFirst experiments on GABA A receptors allude to a similar but weaker effect on the temperature dependence of PAMs.These data suggest that experiments on the efficacy of PAMs conducted at room temperature have to be interpreted with caution.Sitzia et al (2011) FrontPharmacol;2:81.

Over the last several years ion channels have received more and more interest as potential drug targets, because of their known involvement in chronic and acute disease. Patch clamp is the gold standard for obtaining highly relevant information about ion channels and their effectors but it is a notoriously laborious technique. To meet the ever increasing demand for higher throughput in ion channel screening and safety testing we have developed a highly parallel patch clamp platform, the SyncroPatch 96. The platform supports giga-seal recordings, continuous recording during compound application and addition of multiple compounds at each of the 96 cells recorded from at a time.View Large Image | View Hi-Res Image | Download PowerPoint SlideThe figure shows 96 individually recorded IV's of the sodium channel hNav1.5 expressed in HEK 293 cells.Here we present high quality data of voltage gated and ligand gated channels expressed in various cell lines. Whole cell access was achieved using either pore forming agents such as Nystatin, or by short applications of suction pulses.The SyncroPatch 96 offers high throughput without compromising data quality.

Introduction Chip-based automated patch clamp systems are widely used in drug development and safety pharmacology, allowing for high quality, high throughput screening at standardized experimental conditions. The merits of automation generally come at the cost of large amounts of cells needed, since cells are not targeted individually, but randomly positioned onto the chip aperture from cells in suspension. While cell usage is of little concern when using standard cell lines such as CHO or HEK cells, it becomes a crucial constraint with cells of limited availability, such as primary or otherwise rare and expensive cells, like induced pluripotent stem (IPS) cell-derived cardiomyocytes or neurons. Methods We established application protocols for CHO cells, IPS cell-derived neurons (iCell® Neurons, Cellular Dynamics International), cardiomyocytes (Cor.4U®, Axiogenesis) and pancreatic islet cells, minimizing cell usage for automated patch clamp recordings on Nanion's Patchliner. Use of 5 μl cell suspension per well for densities between 55,000 cells/ml and 400,000 cells/ml depending on cell type resulted in good cell capture. Results We present a new cell application procedure optimized for the Patchliner achieving > 80% success rates for using as little as 300 to 2000 cells per well depending on cell type. We demonstrate that this protocol works for standard cell lines, as well as for stem cell-derived neurons and cardiomyocytes, and for primary pancreatic islet cells. We present recordings for these cell types, demonstrating that high data quality is not compromised by altered cell application. Discussion Our new cell application procedure achieves high success rates with unprecedentedly low cell numbers. Compared to other standard automated patch clamp systems we reduced the average amount of cells needed by more than 150 times. Reduced cell usage crucially improves cost efficiency for expensive cells and opens up automated patch clamp for primary cells of limited availability.