Ligand Gated
CNG
The model plant cell Arabidopsis thaliana has been shown to express putative cyclic nucleotide gated channels with cytoplasmic N- and C-termini. In contrast to some animal CNG channels they lack the typical pore motif, GYGD, which is responsible for the K+-selectivity. Thus, they are believed to be non-selective for monovalent cations and Ca2+. Plant CNG channels, therefore, may play a role in pathogen defense and other Ca2+-dependent signalling cascades.
cAMP-regulation of a plant ion channel. (left) Whole cell currents in the absence and presence of 200 µM internal cAMP in response to voltage steps from +60 mV to -140 mV in 20 mV decrements. Vhold was -40 mV. (right) Averaged I-V curves of 4 cells as shown left. 100 µM external LaCl3 inhibits the current (n=2). Internal K+ concentration was 150 mM, external K+ concentration was 30 mM.
Data were kindly provided by A. Kugler & P. Dietrich, Univ. Erlangen, Germany.
GABAA Receptor
GABAA (α1, β2, γ2) receptors belong to the family of fast ligand gated receptors. They are anion permeable and the ligand-induced currents show multiple sublevels. However, the pharmacology of the receptors within this family varies greatly. GABA is the primary inhibitory neurotransmitter in the mammalian CNS. Activation of GABAA (α1, β2, γ2) receptors by GABA tends to decrease neuronal excitability.
Ligand-dependent activation of GABAA (α1, β2, γ2) receptors. 10 µM GABA was applied for 1.2 s and, in the consecutive sweep (interval 30 s), for 0.5 s. Traces were recorded at -60 mV.
Concentration dependent activation and desensitization. 1, 3 and 10 µM GABA was perfused for 2.5 s (time interval 20 s).
Reproducibility of activation. Overlay of traces of 10 consecutively performed activations of the current by 10 µM GABA (time interval 30 s).
Glycine Receptor
Glycine receptors belong to the family of fast ligand gated receptors. Glycine is a major inhibitory neurotransmitter that acts on postsynaptic glycine receptors located mainly in the spinal cord and brainstem.
Ligand-dependent activation of hGlyRα. (left) Overlay of raw data from one hGlyRα1-expressingL(tk)-cell (holding potential -80 mV). The cell was exposed to increasing concentrations of glycine for 800 ms, where each agonist exposure was followed by a 10 s wash step. The bar in the graph indicates drug application. (right) EC50 of Glycine. Hill plot of normalized and averaged peak currents (n=7) for the determination of EC50-value for glycine activation of hGlyRα1-channels expressed in L(tk)-cells. Error bars are the standard error around the mean for each concentration. Cells were kindly provided by Astrazeneca, Söldertälje.
HCN2
The hyperpolarization-activated, cyclic nucleotide gated (HCN) cation channels are members of the superfamily of voltage gated channels. HCN channels open upon hyperpolarisation and close at positive potential. The cyclic nucleotides, cAMP and cGMP enhance HCN channel activity by shifting the activation curve of the channels to more positive voltages. The current produced by HCN channels is found in a variety of excitable cells including neurones, cardiac pacemaker cells and photoreceptors.
HCN2 whole cell current of stably transfected CHO cell before and after internal cAMP application. Subsequent block by external Cs+ was also performed on the same cell. Holding potential was –40 mV. HCN2 clone was kindly provided by M. Biel, LMU, Germany.
Whole cell currents of a HCN2-transfected cell in the presence of internal 10 nM cAMP (control). Block of one component of the current by 2 mM external CsCl. The instantaneous component is not blocked. Voltage steps from -160 mV to 0 mV in 20 mV increments. Vhold was -40 mV.
Voltage Gated
hKv1.3
Kv1.3 is a voltage-gated potassium channel with roles in human T cell activation/proliferation, cell-mediated cytotoxicity, and volume regulation and is thus a target for therapeutic control of T cell responses. The channel activity is induced upon membrane depolarization to voltages more positive than –40 mV. Activation kinetics are rapid and strongly voltage-dependent, whereas inactivation is much slower and shows no significant voltage dependence.
Kv1.3 has been described as showing a shift in its voltage dependence to more positive potentials when recorded in the perforated whole cell configuration. In our recordings performed with the Port-a-Patch we were able to confirm this observation.
Since the internal solution is so easily exchanged using the Port-a-Patch and Patchliner, both platforms are ideally suited for experiments using the perforated patch technique. Here, pore forming molecules are used to perforate the membrane patch in order to obtain electrical control over the membrane. Thus, the cell´s interior remains intact, and the cytosolic constituents that can be important for ion channel function are preserved. The cell can be sealed without the interference of the pore formers, which then are added automatically to the internal side of the chip. Electrical access is obtained quite rapidly since the pore forming agent has very short distances to diffuse compared to a conventional patch pipette.
Perforated and conventional whole cell configuration and derived Kv1.3-currents. Voltage-dependence was shifted significantly to more negative potentials in the whole cell configuration compared to the perforated whole cell configuration. Whole cell currents after a conventional membrane breakthrough (left) and perforated whole cell currents (middle). Nystatin was added to the internal solution after the cell attached configuration was reached. (right) I-V curves of peak current of 3 (conventional) and 5 (perforated) cells.
heag (hKv10.1)
Ether a go-go (eag) channels obtain a permeability to potassium and calcium which is dependent on voltage and cyclic AMP. They are primarily expressed in neuronal tissue but their appearance in various tumour entities is also indicative of an oncogenic role. Block of hEAG whole cell currents after the internal application of 200 µM TEA. Data were kindly provided by Walter Stühmer, MPI for exp. Med., Göttingen.
hERG (hKv11.1)
The hERG gene (KCNH2) encodes a potassium ion channel responsible for the repolarizing IKr current in the cardiac action potential. Abnormalities in this channel may lead to either Long QT syndrome (LQT2) (with loss-of-function mutations) or Short QT syndrome (with gain-of-function mutations), both potentially fatal cardiac arrhythmia, due to repolarization disturbances of the cardiac action potential.
hERG whole cell currents as recorded in CHO cells. Cells were kindly provided by Cytomyx/Millipore, UK.
hERG pharmacology. The figure displays each Hill fit of the accumulated data. Error bars reflect the standard error of the mean. The concentration of the half maximal block is IC50 = 11.0 ± 3 nM (Terfenadine), IC50 = 8.9 nM (Cisapride) and IC50 = 163.7 nM (Flunarizine).
hNav1.5
Voltage gated sodium channels (Nav are important elements of action potential initiation and propagation in excitable cells because they are responsible for the initial depolarization of the membrane.
Whole cell recordings performed with the Port-a-Patch. Whole cell currents of HEK293 cells stably expressing hNav1.5 (left). Current-voltage relation of peak currents (right). Cells were kindly provided by Cytomyx Millipore, UK.
Raw currents with increasing TTX concentrations, which were activated by a voltage pulse from –100 mV to –20 mV (left). Corresponding dose-response curve with a calculated IC50 of 1.8 ± 0.9 µM, n=6, (Lit. value: 1-2 µM) (right).
Shaker
The Shaker K channel is the A-type potassium channel in Drosophila Melanogaster. It got its name from its phenotype (shaking legs under ether anaesthesia). Here we show recordings from HEK293 cells transiently expressing a Shaker K mutant that has its inactivation removed (Shaker-IR). The holding potential was -80 mV.
The current-voltage relationship of the tail currents reveals a half-activating potential of -27 mV.
Block of Shaker-IR by Agitoxin2. Shown are current recordings from HEK293 transiently expressing Shaker-IR. Currents were elicited by a 500 ms step to +50 mV from a holding potential of -80 mV. K+ gradients are the same as for the IV curve experiment shown above. 200 nM Agitoxin2 almost fully blocks the Shaker K+ current.
Data were kindly provided by Dr. Kenton Swartz, NIH, Bethesda, USA
Others
Kir2
The basophilic leucaemia cells (RBL) exhibit an inwardly rectifying potassium current, IKIR. Besides this, there is also a small outward current component flowing through these channels. The macroscopic conductance reveals characteristics typical of classical K+ inward rectifiers of the IRK type. The channel gating is steeply voltage dependent. The current is susceptible to a concentration- and voltage-dependent block by extracellular Ba2+.
Current response in RBL cells to a voltage ramp from –150 to +80 mV in the presence of a low and a high external K+ concentration (left, above). Voltage dependent block of the inward K+ current after the external additon of 50 µM Ba2+ (middle, above). Same block in response to a continuous voltage step to –150 mV (right, above) from a holding potential of 0 mV. The solutions were changed using a Gilson pipettor. Low K+ (4.5 mM K+), high K+ (143 mM K+).
Currents of RBL cells endogeneously expressing K+ permeable channels (holding potential -100 mV). The external K+ concentration was changed using the Perfusion System. Exchange times for all solution exchanges were approx. 150 ms. Low K+ (4.5 mM K+), high K+ (143 mM K+).
kNBCs-1 (NBCe1-A)
Sodium-bicarbonate cotransporter kNBC-1 (NBCe1-A) Sodium-bicarbonate co-transporters are homologous membrane proteins that play an important role in the regulation of the intracellular pH as well as transepithelial HCO3-transport in various tissues. NBC1 and NBC4 are the members of the characterized HCO3- transporter superfamily that are known to be electrogenic.
Current responses to 200 ms voltage ramps (-100 mV to + 100 mV) were recorded in high external Na+ initially after establishment of the whole cell configuration (control), after application of HCO3- (bicarbonate), and after wash-out (wash-out).
Currents were recorded from HEK 293 cells transiently expressing the electrogenic Na+/bicarbonate cotransporter (NBC1). Data were kindly provided by Ira Kurtz, UCLA, USA.
TRP V1
TRP V1 is a member of the transient receptor potential channel family. It is mainly expressed in sensory nerves. Its presence is essential for transduction of nociception as well as inflammatory and hypothermic effects of vanilloid compounds. It also contributes to acute thermal nociception and thermal hyperalgesia following tissue injury. TRP V1 forms a relatively Ca2+ selective ion channel with outwardly rectifying properties. It is activated by the vanilloids such as capsaicin, by protons, increased temperatures, lipoxygenase products, as well as anandamide.
Whole cell current responses from HEK 293 cells transiently expressing TRP V1 to a ramp voltage protocol (-100 mV to + 100 mV). Two μM capsaicin reversibly activated the channel. Data were kindly provided by David Cohen, Oregon Health & Science University, Portland, USA.
Cardiomyocyte
Ventricular and atrial myocytes acutely isolated from adult animals are notoriously difficult to patch using conventional patch clamp, and have thus far proved almost impossible to patch using planar patch clamp. During a recent demonstration of the port-a-patch in the Department of Pharmacology at the University of Oxford, UK, we successfully demonstrated the possibility of using the port-a-patch for this cell type.
Ventricular and atrial myocytes were acutely isolated from adult guinea pig heart at the Department of Pharmacology on the day of recording. Despite a low success rate for giga-seal formation and whole cell recordings, the traces below show for the first time the potential for the use of the port-a-patch for cardiomyocyte electrophysiology. The voltage-activated Ca2+ or K+ currents shown below were recorded using an Axon Axopatch 200B amplifier and were elicited using a voltage protocol stepping from -40mV to +30mV, from a holding potential of -40mV, for 200ms in 10mV increments. Nifedepine, an L-type Ca2+ channel blocker, was also used to successfully block the current in one cell.
Voltage-activated Ca2+ currents recorded from a ventricular myocyte are shown on the left (above). Currents were elicited by stepping from -40mV to +30mV, from a holding potential of -40mV, for 200ms in 10mV increments. Experiments were performed at room temperature. On the right (above), the corresponding current-voltage relationship is shown. In this cell, the maximum current was elicited at approximately -10mV.
Voltage-activated Ca2+ currents recorded from another ventricular myocyte are shown here on the left. Currents were elicited using the same step protocol described above. The corresponding current-voltage relationship is shown on the right. In this cell, the maximum current was elicited at approximately +10mV.
The Ca2+ current recorded from a ventricular myocyte was blocked by the L-type Ca2+ channel blocker, nifedepine. In this recording (from the same cell shown in figure 2), currents were elicited by stepping to +10mV for 200ms from a holding potential of -40mV every 5 seconds. Once a stable recording was achieved, 10µM nifedepine was applied which blocked the current. For this figure, 3-5 traces from control and 3-5 traces in the presence of nifedepine were averaged and the resultant traces are shown.
An outward K+ current recorded from an atrial myocyte. Currents were elicited using the same voltage protocol described above, stepping from -40mV to +30mV, from a holding potential of -40mV, for 200ms in 10mV increments. No voltage-activated Ca2+ currents were observed in this cell, only an outward K+ current.
We are very grateful to the laboratories of Stevan Rakovic and Derek Terrar at the Department of Pharmacology, University of Oxford, for providing the equipment needed for the demonstration, and for preparing the cardiomyocytes used in these experiments.
Plant Cells – BY2 Cells
Cell lines such as HEK293 cells play an important role in the basic understanding of the molecular and cellular biology of mammalian cells. Also, cell lines from plants have been obtained from various tissues and species of higher plants. Among these, the tobacco (Nicotiana tabacum L. cv Bright Yellow 2 [BY2]) cell line, isolated by Kato and coworkers (1972), is rather unique and is well characterized. This cell line is highly homogeneous and shows an exceptionally high growth rate, multiplying 80- to 100-fold in 1 week. Furthermore, a high cell cycle synchrony can be obtained and enzymatic isolation of BY2 cell protoplast make them applicable to patch clamp experiments. These features make this cell line a powerful tool for exploring the molecular and cellular biology of plant cells.
Tobacco (Nicotiana tabacum L. cv Bright Yellow 2 [BY2] (above) cells are the most widely used plant cell culture cells. Depolarisation-activated outward currents in BY2 cell protoplasts. Whole-cell current in response to voltage steps (below, left) and the corresponding IV-curve (below, right). From a holding potential of – 40 mV, 500-ms voltage steps were applied from -60 mV to 60 mV in 20mV increments, with a 3-s interval.
Plant Cells – Tobacco mesophyll Cells
Mesophyll protoplasts are enzymatically isolated from the mesophyll tissue of tobacco leaves. During incubation of mesophyll tissue in the enzyme solution, the cell wall is digested. Subsequent filtering and washing steps by centrifugation in iso-osmotic solutions enables the accumulation of vital and cell wall-free protoplasts.
Hyperpolarization-activated inward currents in tobacco mesophyll cell protoplasts. Whole-cell current in response to a voltage ramp. From a holding potential of – 40 mV, a 800-ms voltage ramp was applied from + 40 mV to -120 mV. Internal solution contained 10 mM Ba2+, external solution contained 40 mM Ca2+.
Data were kindly provided by Berghöfer, Thomas; Eing, Christian; Flickinger, Bianca; Frey, Wolfgang. Institut für Hochleistungsimpuls- und Mikrowellentechnik, Forschungszentrum Karlsruhe, Karlsruhe, Germany
Sonoporation
Ultrasound-induced membrane permeability (sonoporation) is being studied as a possible methodology for intracellular drug delivery and non-viral gene transfection. Despite recent progress in the field the mechanism of sonoporation on a cellular level has not been studied extensively due to a lack of methods allowing observation of sonoporation in real-time. The combination of a patch clamp set-up with an ultrasonic system overcomes this obstacle and allows the study of sonoporation as well as the resealing of the pores in the cell membrane in real-time. Both processes need to be understood to achieve consistent delivery of the desired compound into the cell. To apply the ultrasound to the cell an ultrasound probe was immersed in the extracellular solution. Sonoporation only occurred when the contrast agent (lipid encapsulated microbubbles of gas) was present.
The picture (above) shows the simple set-up of the Port-a-Patch with the Ultrasound probe immersed in the external solution.
Current response of a non-transfected Chinese hamster ovary (CHO) cell to a train of ultrasound pulses (US) in the absence (A) and presence (B) of contrast agent. The holding potential was -80 mV. Ultrasound signals had a centre frequency of 5MHz. Pulses of 50 cycles were repeated for 2 s every 10 ms.
Shown is another current response of the same cell as above on a longer time scale demonstrating the re-sealing process of the membrane.
Data were kindly provided by Dr Cheri Deng, University of Michigan, USA.
Mouse Neural Stem Cell
Stem cells are gaining more interest for drug discovery purposes because of their authentic cellular environment compared to using immortalized cell lines as carrier of over-expressed ion channels. The Port-a-Patch and Patchliner have been used for voltage- and current-clamp recordings from mouse embryonic stem cell derived cardiomyocytes. In these studies I/V-characteristics were evaluated, as well as recordings of action potentials. Read More >>
Mouse primary neural stem cells have also been investigated using the Port-a-Patch. These cells were extracted from E14 fetuses and then cultured. Before the experiments, cells were detached from the surface, and cells in suspension were used for recordings on the Port-a-Patch. The cells easily formed giga-Ohm seals, and patch clamp recordings were obtained from the whole-cell configuration (membrane holding potential -60 mV), applying 500 ms depolarizing voltage pulses. K-currents were identified in the neural mouse stem cells as shown in the raw data traces below.
Data provided by Dr. Gavin Dawe, National University of Singapore, Singapore.
Single Channel Recordings
BK (KCa1.1)
BK channels are large conductance Ca2+ and voltage-activated K+ channels, which allow K+ to leave the cytoplasm under physiological conditions when activated by membrane voltage and/or intracellular Ca2+. This results in hyperpolarization or a decrease in cell excitability. BK channels are essential for key physiological processes, for example, they are important for controlling the contraction of smooth muscle cells. Single channel recordings performed with the Port-a-Patch. Single channel recordings of BK channels as recorded from CHO cells in the cell attached mode at +60 (top), +40 (middle) and 0 mV (lowest trace).
Erythrocytes
Erythrocytes lack mitochondria and nuclei and consist mainly of hemoglobin, a complex molecule containing heme groups whose iron atoms temporarily link to oxygen molecules in the lungs or gills and release them throughout the body. The membrane contains different ion channels, for example, a Ca2+-activated K+ channel and a volume-sensitive Na+/K+ pump. Studies have also revealed the participation of ion channels in the regulation of erythrocyte ‘apoptosis’. Osmotic shock, oxidative stress and energy depletion all activate a Ca2+-permeable non-selective cation channel in the erythrocyte cell membrane. Single channel fluctuations as recorded in an erythrocyte in the cell attached configuration.
Mitochondria
Mitochondria were isolated from rat cardiac tissue. The patch clamp measurements were performed in the cell attached configuration with an Axopatch 200B. Single channel currents were elicited by a voltage ramp from 0 mV to – 80 mV. Due to the cell attached configuration inward currents are displayed as a positive current.
Bilayer Recordings
Gramicidin
Gramicidin is a barrel shaped molecule, which forms a conducting channel in the membrane by dimerization. Plotting the current amplitude vs. the voltage reveals conductances of 94.88 pS and 28.28 pS, which correspond to two different Gramicidin derivates present in the bilayer. Traces were recorded in 100 mM HCl at the indicated potentials. Clearly 2 gramicidin derivates (94.88 pS and 28.28 pS) can be distinguished. Lipid: 2 μM diameter Diphtanoyl-PC bilayer prepared from a GUV. Recordings: Tohoku University, Tohoku, Japan.
Alamethicin
Alamethicin is a channel forming peptide and, when patch clamped, reveals multiple non-equidistant conductance levels due to formation of Alamethicin oligomers in the bilayer. After pipetting a stock solution onto the chip, the molecules insert in the lipid bilayer and channel formation can be investigated. Alamethicin single channel conductances. Recordings from a GUV prepared bilayer in 85 mM KCl at -140 mV. Histogram of ~4 x 106 points revealing the diferent conductance levels of Alamethicin. Lipid: Diphtanoyl-PC. Data were kindly provided by M. Sondermann/Prof. Behrends, Univ. Freiburg.
Bacterial Cytolysin
Another example shows Cytolysin from a bacterial strain as recorded in a painted bilayer. In contrast to the well known gating behavior of Gramicidin and Alamethicin, the Cytolysin shows stepwise conductance increases without any gating. Bacterial Cytolysin. Traces were recorded in 100 mM KCl, 10 mM Hepes, 0.1 mM EDTA, pH 7 at -40 mV. Lipid: Diphtanoyl-PC. Recordings: ITC & CNR-Istituto di Biofisica, Trento, Italy.
MscL
MscL gating recorded at 30mV and the indicated negative hydrostatic pressure, in symmetrical recording solution (200mM KCl, 40mM MgCl2, 5mM Hepes, pH 7.2/KOH)
LEFT: MscL reconstituted in azolectin (PC), added during GUV formation (1:200) (w/w) in Sorbitol.
RIGHT: MscL reconstituted in azolectin (PC), added during GUV formation (1:200) (w/w) in Sucrose.
Data were kindly provided by Courtesy of Andrew Battle and Boris Martinac, University of Queensland, Brisbane, Australia.