[show abstract][hide abstract] ABSTRACT: Unlike the genomics revolution, which was largely enabled by a single technological advance (high throughput sequencing), rapid advancement in proteomics will require a broader effort to increase the throughput of a number of key tools for functional analysis of different types of proteins. In the case of ion channels -a class of (membrane) proteins of great physiological importance and potential as drug targets- the lack of adequate assay technologies is felt particularly strongly. The available, indirect, high throughput screening methods for ion channels clearly generate insufficient information. The best technology to study ion channel function and screen for compound interaction is the patch clamp technique, but patch clamping suffers from low throughput, which is not acceptable for drug screening. A first step towards a solution is presented here. The nano patch clamp technology, which is based on a planar, microstructured glass chip, enables automatic whole cell patch clamp measurements. The Port-a-Patch is an automated electrophysiology workstation, which uses planar patch clamp chips. This approach enables high quality and high content ion channel and compound evaluation on a one-cell-at-a-time basis. The presented automation of the patch process and its scalability to an array format are the prerequisites for any higher throughput electrophysiology instruments.
Current Drug Discovery Technologies 02/2004; 1(1):91-6.
[show abstract][hide abstract] ABSTRACT: In evaluating ion channel function, electrophysiology, e.g., patch clamping, provides the highest information content. For the analysis of ion channel-modulating compounds, one variant of the patch-clamp technique, the whole-cell configuration, is particularly useful. We present here patch-clamp recordings in the whole-cell configuration and single channel recordings performed with planar patch-clamp chips, which are microstructured from borosilicate glass substrate. The chips are used in the Port-a-Patch, an ion channel research/screening instrument that enables automated patch-clamp experiments on a single cell. A software runs the experiment by executing user-determined protocols for cell positioning, as well as for electrical stimulation and current readout. In various electrophysiological experiments, the high quality of recordings and the versatility of the perfusion of the recorded cells are demonstrated. Quantitative pharmacological experiments are performed on sodium channels expressed in HEK cells using solution volumes in the low microliter range. The exceptionally low volume consumption in the experiments make the system attractive for work on rare or expensive compounds. Due to the low volumes necessary, a rapid solution exchange is facilitated, which is shown on RBL cells. The patch-clamp chip enables a rapid and precise perfusion, allowing sophisticated investigations on ion channel function with the Port-a-Patch.
Assay and Drug Development Technologies 11/2003; 1(5):665-73. · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: Metabolic activity of cultured cells can be monitored by measuring changes in the pH of the surrounding medium caused by metabolic products such as protons, carbon dioxide or lactic acid. Although many systems designed for this purpose have been reported, almost all of them are based on bulk measurements, where the average metabolic activity of all cells in contact with the device is recorded. Here, we report on a novel biosensor, based on a modified light-addressable potentiometric sensor (LAPS) device, which enables the metabolic activity of cultured cells to be measured with spatial resolution. This is demonstrated here by detecting the differential sensitivity to a cholinergic receptor agonist of two different co-cultured cellular populations. By making simultaneous measurements of the metabolic activity of different cell types seeded on different segments of one sensor, this device not only provides a rapid means of assessing cellular specificity of pharmaceutical compounds but also has the potential of being used to non-invasively monitor humoral as well as synaptic communication between different cell populations in co-culture. The temporal and spatial resolution of the device were investigated and are discussed.
Biosensors and Bioelectronics 02/2003; 18(1):31-41. · 5.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: We report here an approach for simultaneous fluorescence imaging and electrical recording of single ion channels in planar bilayer membranes. As a test case, fluorescently labeled (Cy3 and Cy5) gramicidin derivatives were imaged at the single-molecule level using far-field illumination and cooled CCD camera detection. Gramicidin monomers were observed to diffuse in the plane of the membrane with a diffusion coefficient of 3.3 x 10(-8) cm(2)s(-1). Simultaneous electrical recording detected gramicidin homodimer (Cy3/Cy3, Cy5/Cy5) and heterodimer (Cy3/Cy5) channels. Heterodimer formation was observed optically by the appearance of a fluorescence resonance energy transfer (FRET) signal (irradiation of Cy3, detection of Cy5). The number of FRET signals was significantly smaller than the number of Cy3 signals (Cy3 monomers plus Cy3 homodimers) as expected. The number of FRET signals increased with increasing channel activity. In numerous cases the appearance of a FRET signal was observed to correlate with a channel opening event detected electrically. The heterodimers also diffused in the plane of the membrane with a diffusion coefficient of 3.0 x 10(-8) cm(2)s(-1). These experiments demonstrate the feasibility of simultaneous optical and electrical detection of structural changes in single ion channels as well as suggesting strategies for improving the reliability of such measurements.
[show abstract][hide abstract] ABSTRACT: We present a technique by which it is possible to produce a planar sensor for ion channel electrophysiology from glass substrates. Apertures with diameters in the low micrometer to submicrometer range are achieved by irradiation of a glass chip with a single heavy ion and subsequent wet track etching. The function of the device is demonstrated by recordings of single channel currents mediated by the model ion channel gramicidin A in lipid bilayers spanning the micromachined aperture.
[show abstract][hide abstract] ABSTRACT: An essential requirement for successful long-term coupling between neuronal assemblies and semiconductor devices is that the neurones must be able to fully develop their electrogenic repertoire when growing on semiconductor (silicon) substrates. While it has for some time been known that neurones may be cultured on silicon wafers insulated with SiO2 and Si3N4, an electrophysiological characterisation of their development under such conditions is lacking. The development of voltage-dependent membrane currents, especially of the rapid sodium inward current underlying the action potential, is of particular importance because the conductance change during the action potential determines the quality of cell-semiconductor coupling. We have cultured rat striatal neurones on either glass coverslips or silicon wafers insulated with SiO2 and Si3N4 using both serum-containing and serum-free media. We here report evidence that not only serum-free culture media but also growth on semiconductor surfaces may negatively affect the development of voltage-dependent currents in neurones. Furthermore, using surface-charge measurements with the atomic force microscope, we demonstrate a reduced negativity of the semiconductor surface compared to glass. The reduced surface charge may affect cellular development through an effect on the binding and/or orientation of extracellular matrix proteins, such as laminin. Our findings therefore suggest that semiconductor substrates are not entirely equivalent to glass in terms of their effects on neuronal cell growth and differentiation.
European Biophysics Journal 02/2001; 29(8):607-20. · 2.27 Impact Factor
[show abstract][hide abstract] ABSTRACT: The effect of benzodiazepines (BZs) on GABA(A)-ergic synaptic responses depends on the control receptor occupancy: the BZ-induced enhancement of receptor affinity can lead to greater peak amplitudes of quantal responses only when, under normal conditions, receptors are not fully saturated at peak. Based on this fact, receptor occupancy at the peak of spontaneous miniature inhibitory postsynaptic currents (mIPSCs) has been assessed in various mammalian neuronal preparations. To use the same principle with compound (or multiquantal), action potential-evoked IPSCs, complications introduced by quantal asynchrony in conjunction with the BZ-induced increase in the decay time of the quantal responses have to be overcome. We used a simple analytic convolution model to calculate expected changes in the rise time and amplitude of postsynaptic currents when the decay time constant, but not the peak amplitude, of the underlying quantal responses is increased, this being the expected BZ effect at saturated synapses. Predictions obtained were compared with the effect of the BZ flunitrazepam on IPSCs recorded in paired pre- and postsynaptic whole cell voltage-clamp experiments on striatal neurons in cell culture. In 22 pairs, flunitrazepam (500 nM) reliably prolonged the decay of IPSCs (49 +/- 19%, mean +/- SE) and in 18 of 22 cases produced an enhancement in their peak amplitude that varied markedly between 3 and 77% of control (26.0 +/- 5.3%). The corresponding change in rise time, however (+0.38 +/- 0.11 ms, range -0.8 to +1.3 ms) was far smaller than calculated for the observed changes in peak amplitude assuming fixed quantal size. Because therefore an increase in quantal size is required to explain our findings, postsynaptic GABA(A) receptors were most likely not saturated during impulse-evoked transmission at these unitary connections. The peak amplitudes of miniature IPSCs in these neurons were also increased by flunitrazepam (500 nM, +26.8 +/- 6.6%), and their decay time constant was increased by 26.3 +/- 7.3%. Using these values in our model led to a slight overestimate of the change in compound IPSC amplitude (+28 to +30%).
Journal of Neurophysiology 09/2000; 84(2):771-9. · 3.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: The light-addressable potentiometric sensor (LAPS) measures localized photo-induced currents from a silicon wafer, which are dependent on the local surface potential and on the intensity of the light pointer. In this study the ability of the LAPS to record extracellular potentials of adherent cells was investigated. Time dependent LAPS photocurrent signals that correlated in time with contractions were recorded from beating cardiac myocytes cultured on LAPS surfaces. Signals could be recorded both when the LAPS was biased to working points where the photocurrent was maximally sensitive to potential changes and when it was biased to working points where the photocurrent was insensitive to changes in surface potential. Therefore, signals could not be predominantly created by changes in extracellular potential and might be related to mechanical contractions. One possible explanation might be, that the cell-induced modulation of photocurrents arose as a result of cell shape changes. Such alterations in cell shape might have focused and defocused the light pointer and, thus, modulated its intensity. To further test this hypothesis, height changes of beating cardiac myocytes were measured with an atomic force microscope (AFM). They were found to match well with signals derived from LAPS measurements. Therefore, it can be concluded, that LAPS signals were mainly determined by the periodic changes in shape of beating heart cells, and this interference precludes the measurements of extracellular electrophysiological potentials from these cells.
[show abstract][hide abstract] ABSTRACT: We present a method for integrating an isolated cell membrane patch into a semiconductor device. The semiconductor is nanostructured for probing native cell membranes for scanning probe microscopy in situ. Apertures were etched into suspended silicon-nitride layers on a silicon substrate using standard optical lithography as well as electron-beam lithography in combination with reactive ion etching. Apertures of 1 μm diam were routinely fabricated and a reduction in size down to 50 nm was achieved. The stable integration of cell membranes was verified by confocal fluorescence microscopy in situ.
[show abstract][hide abstract] ABSTRACT: 1. T-butyl-bicyclo-phosphorothionate (TBPS) is a prototypical representative of the cage-convulsants which act through a use-dependent block of the GABA(A)-receptor-ionophore complex. Using current recordings from cultured neurones of rat striatum the manner was investigated in which two antagonists, bicuculline and penicillin, presumably acting at the agonist binding site and in the ionic channel, respectively, modify the rate of block by TBPS. 2. Penicillin (5 or 10 mM) did not slow the rate of block by TBPS, but produced a significant enhancement of block rate, which, however, was inversely related to the degree of antagonism by penicillin of the GABA-induced current. 3. Bicuculline (10 microM) reduced the rate of block by TBPS. However, this effect was 3 fold weaker than its GABA-antagonistic action. The slowing of block rate and the current antagonism exhibited a biphasic, positive-negative relationship. Co-application of bicuculline (100 microM) in a concentration that produced nearly complete antagonism and TBPS (10 microM) resulted in a marked ( approximately 40%) reduction of subsequent GABA response amplitudes compatible with a direct, bicuculline-induced conformational change in the receptor required for the binding of and block by TBPS. 4. The lack of protection afforded by the channel blocker penicillin as well as the lack of correlation between bicuculline antagonism of the Cl(-)-current and its efficiency in protecting against TBPS block is evidence against an open channel blocking mechanism for TBPS. TBPS does, therefore, not appear to gain access to its binding site via the open pore but through alternative routes regulated from the agonist binding site.
British Journal of Pharmacology 02/2000; 129(2):402-8. · 5.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: We investigate the microscopic contact of a cell/semiconductor hybrid. The semiconductor is nanostructured with the aim of single channel recording of ion channels in cell membranes. This approach will overcome many limitations of the classical patch-clamp technique. The integration of silicon-based devices 'on-chip' promises novel types of experiments on single ion channels.
[show abstract][hide abstract] ABSTRACT: In testing various designs of cell-semiconductor hybrids, the choice of a suitable type of electrically excitable cell is crucial. Here normal rat kidney (NRK) fibroblasts are presented as a cell line, easily maintained in culture, that may substitute for heart or nerve cells in many experiments. Like heart muscle cells, NRK fibroblasts form electrically coupled confluent cell layers, in which propagating action potentials are spontaneously generated. These, however, are not associated with mechanical disturbances. Here we compare heart muscle cells and NRK fibroblasts with respect to action potential waveform, morphology, and substrate adhesion profile, using the whole-cell variant of the patch-clamp technique, atomic force microscopy (AFM), and reflection interference contrast microscopy (RICM), respectively. Our results clearly demonstrate that NRK fibroblasts should provide a highly suitable test system for investigating the signal transfer between electrically excitable cells and extracellular detectors, available at a minimum cost and effort for the experimenters.
[show abstract][hide abstract] ABSTRACT: 1. At striatal inhibitory synapses in cell culture, replacement of extracellular Ca2+ with Sr2+ desynchronized inhibitory postynaptic currents (IPSCs), reducing their peak amplitude and producing a succession of late, asynchronous synaptic events (late release). In the averaged IPSC waveform this resulted in an increase in both the fast and the slow decay time constant as well as in the time to peak. 2. Rapid removal of extracellular Sr2+ during late release was without effect on the time course of the averaged IPSC. Thus, late release is not dependent on continuous Sr2+ influx, but must be related to the way in which Sr2+, as opposed to Ca2+, interacts with constituents of the intracellular space. 3. After application of the membrane-permeant acetoxymethyl ester (AM) form of the Ca2+-chelator BAPTA, Sr2+-induced late release was greatly reduced and the kinetics of the Sr2+-dependent IPSC approached those of the Ca2+-dependent response. EGTA AM had a similar but less pronounced effect. 4. Using rapid solution exchange, we stimulated synapses first in Sr2+- or Ca2+- and 100-300 ms afterwards in Ca2+-containing solution. Paired-pulse facilitation of late release was the same whether the conditioning pulse induced a presynaptic influx of Sr2+ or of Ca2+. 5. It is concluded that Sr2+-mediated asynchrony is probably due to a less efficient intraterminal buffering of Sr2+ as opposed to Ca2+, allowing for Sr2+ ions to activate release in an area less confined to the immediate vicinity of the presynaptic Ca2+ channel. This hypothesis explains both the action of endogenous buffers and the apparent lack of specific facilitatory interaction between Ca2+-mediated and Sr2+-induced late release.
The Journal of Physiology 02/1999; 514 ( Pt 2):447-58. · 4.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Postsynaptic inhibitory gamma-aminobutyric acid-A (GABAA)-receptor-mediated current responses were measured using simultaneous pre- and postsynaptic whole cell recordings in primary cell cultures of rat striatum. Substitution of Sr2+ for extracellular Ca2+ strongly desynchronized the inhibitory postsynaptic currents (IPSCs), resulting in a succession of asynchronous IPSCs (asIPSCs). The rise times and decay time constants of individual evoked asIPSCs were not significantly different from those of miniature IPSCs that are the result of spontaneous vesicular release of GABA. Thus asIPSCs reflect quantal transmission at the individual contacts made by one presynaptic neuron on the recorded postsynaptic cell. Increasing the concentration of Sr2+ from 2 to 10 mM and decreasing that of Mg2+ from 5 to 1 mM produced an increase in the frequency of asIPSCs consistent with an enhancement of the mean probability of release (Pr). At the same time the amplitude distribution of asIPSCs was shifted toward larger values, whereas responses to exogenously applied GABA on average were slightly decreased in amplitude. Application of the GABAB-receptor agonist baclofen (3-10 microM) strongly reduced the frequency of asIPSC, consistent with a decrease in Pr, and led to a shift of the amplitude distribution toward smaller values. Baclofen had no effect on responses to exogenously applied GABA. In summary, our data suggest that at striatal inhibitory connections the weight of single contacts may be controlled presynaptically by variation in the amount of transmitter released.
Journal of Neurophysiology 07/1998; 79(6):2999-3011. · 3.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: 1. The effect of cholinergic receptor activation on gamma-aminobutyric acid (GABA)-mediated inhibitory synaptic transmission was investigated in voltage-clamped CA1 pyramidal neurons (HPNs) in the guinea pig hippocampal slice preparation. 2. The cholinergic agonist carbachol (1-10 microM) induced a prominent and sustained increase in the frequency and amplitudes of spontaneous inhibitory postsynaptic currents (IPSCs) in Cl(-)-loaded HPNs. The potentiation of spontaneous IPSCs was not dependent on excitatory synaptic transmission but was blocked by atropine (1 microM). 3. Monosynaptically evoked IPSCs were reversibly depressed by carbachol (10 microM). 4. The frequency of miniature IPSCs recorded in the presence of tetrodotoxin (0.6 or 1.2 microM) was reduced by carbachol (10 or 20 microM) in an atropine-sensitive manner. 5. We conclude that, while cholinergic receptor activation directly excites hippocampal GABAergic interneurons, it has, in addition, a suppressant effect on the synaptic release mechanism at GABAergic terminals. This dual modulatory pattern could explain the suppression of evoked IPSCs despite enhanced spontaneous transmission.
Journal of Neurophysiology 03/1993; 69(2):626-9. · 3.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: Effects of GABAA-, barbiturate- and benzodiazepine receptor agonists and GABAB agonist, baclofen, on voltage-dependent Ca2+ current (ICa) were studied in isolated frog sensory neurons after suppression of Na+ and K+ currents using single-electrode voltage-clamp. GABA, muscimol, taurine and pentobarbital (PB) dose-dependently induced a transient Cl- current (ICl), while baclofen and diazepam (DZP) did not elicit any currents. With GABAA agonists such as GABA, muscimol and taurine, ICa was suppressed transiently, and the maximum inhibition of ICa occurred within 1 min. The suppression of ICa by all GABAA agonists was neither voltage dependent nor attenuated in the presence of either bicuculline or picrotoxin. In addition, there was no correlation between GABA- and baclofen-induced suppressions of ICa. The results suggest that the inhibition of ICa by GABAA receptor agonists is not due to either GABAA or GABAB receptor activation at least. The inhibition of ICa by baclofen, PB and DZP was persistent. PB suppressed the amplitude of ICa and also facilitated the inactivation process, suggesting that PB behaves as a Ca channel blocker. However, the mechanisms of ICa suppression by baclofen and DZP are the subject for a future study. The potency order of the drugs in reducing ICa was muscimol greater than GABA = DZP greater than baclofen greater than PB greater than taurine.
Neuroscience Research 01/1989; 6(2):97-105. · 2.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: A suppressant effect of intracellular free Ca2+ on the gamma-aminobutyric acid (GABA)-induced chloride inward current (ICI(GABA)) was studied in isolated frog sensory neurones under whole cell voltage clamp. Voltage-dependent Ca2+ influx elicited during the steady state of ICI(GABA)-induced a fast, slowly recovering current relaxation in the outward direction, the amplitude of which was dependent on total Ca2+ influx. This suppressant effect showed specificity for different divalent cations, suggesting action at a specific intracellular effector. Single channel recording revealed a Ca2+-dependent decrease in the duration of the open time of the GABA-gated Cl- channel without change in single channel conductance.
[show abstract][hide abstract] ABSTRACT: Apertures with diameters in the low micrometer to submicrometer range are achieved by irradiation of a glass chip with a single heavy ion and subsequent wet track etching. By this technique it is possible to produce a planar sensor for ion channel electrophysiology from a glass substrate. The function of the device is demonstrated by recordings of single-channel currents mediated by the model ion channel Gramicidin A in artificial lipid bilayers spanning the micromachined aperture. Ion channels play key roles in most functions and dysfunctions of all cells. The most direct and accurate methods for studying ion channel behaviour record the transmembrane current that results when the channels open to allow ions to flow, whilst keeping the transmembrane voltage constant. The most precise of these voltage-clamp techniques is patch clamping (1), where a tight, high resistance seal is formed between the tip of an electrolyte-filled glass pipette and the cell membrane. This high resistance increases the resolution of recording so that currents mediated by few or even single open channels can be directly observed. The minimal requirement for an electrophysiological ion channel sensor is a small (micron-sized) aperture in an insulating material that separates two electrolyte-containing compartments. Pulling out a glass pipette to provide a micro- sized orifice is an elegant way of producing an aperture of small dimensions at the tip of a device that can be handled. However, this also produces a relatively long pathway through which current must flow to the opening, leading to a considerable access resistance and capacitance of the device. The fabrication of submicron apertures using the ion-track etching technique allows on-chip single channel recording with low access resistance and low capacitance. Such an 'on-chip pore' in glass, fulfils in particular the desired low-noise requirement (2).