Nanion Technologies
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Data and Applications


Accurate Voltage Control


p14_4_Nav

To perform recordings of fast events, such as the activation and inactivation of Na currents, it is essential to have accurate voltage control. The image shows currents and I/V characteristics of Nav1.5 expressed in HEK293 cells.

Cells were kindly provided by Millipore.

Action Potentials in Cor.At® Cells


p14_2_CorAT

Cor.At® cardiomyocytes are derived from mouse embryonic stem (ES) cells. Whole cell currents recorded in voltage clamp mode reveal cardiomyocyte-typical ion channels (K+, Ca2+ and Na+). Traces on the lower left show prolongation of the action potential upon application of Dofetilide (1 μM).

Cells were kindly provided by Axiogenesis. Download: Application Note

Action Potentials in hESC Cells


p14_3_hESC

The left picture shows a typical action potential in hESC-derived cardiomyocytes (human embryonic stem cell-derived cardiomyocytes). The traces represent K+- (top left), Ca2+- (bottom left) and Na+-currents (top right). The lower right panel shows the corresponding I/V-relationships.

Cells were kindly provided by Geron/GE.

Alamethicin


application_alameticin_2

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. Alamethicin single channel conductances. Recordings from a GUV prepared bilayer in 85 mM KCl at -140 mV. Data were kindly provided by M. Sondermann/Prof. Behrends, Univ. Freiburg.

Bacterial Cytolysin


application_bacterial_3

Traces were recorded in 100 mM KCl, 10 mM Hepes, 0.1 mM EDTA, pH 7 at -40 mV. Lipid: Diphtanoyl-PC.

Recordings were kindly supplied by ITC & CNR-Istituto di Biofisica, Trento, Italy.

Bilayer - Low Noise


application_Low-noise-holder

Typical recording of the Irms noise level of a bilayer (DPhPC) on the Low Capacitance Holder. Typical values are 220 fA

(10 kHz filter frequency, 50 kHz sampling rate. Cf. 2.3 pA RMS on the standard Port-a-Patch®).

BK (KCa1.1)


application_kca11_1

Single channel recordings of BK channels as recorded from CHO cells in the cell attached mode at +60 mV (top), +40 mV (middle) and 0 mV (lowest trace).

BY2 Protoplasts


by2cells_2

Tobacco (Nicotiana tabacum L. cv Bright Yellow 2 [BY2]) cells are the most widely used plant cell culture. Depolarisation-activated outward currents in BY2 cell protoplasts. Shown are whole cell current in response to voltage steps (left) and the corresponding IV curve (right). From a holding potential of −40 mV, 500 ms voltage steps were applied from −60 mV to 60 mV in 20 mV increments, with 3 s intervals.


cAMP-Regulation of a Plant Ion Channel


p15_4_cAMP

The image shows internal cAMP-activation of a transiently expressed plant CNG channel and subsequent external block by lanthanum. Activators and blockers were manually added to the intracellular and extracellular sides of the membrane.

Data were kindly provided by A. Kugler & P. Dietrich, Univ. Erlangen, Germany.

Cardiomyocytes


VentricularMyocyte1

Ca2+ current recorded from a ventricular myocyte. Raw current voltage relationship and peak current data are plotted (top). From the IV-plot the half-activating voltage was determined as -16 mV. The current was later blocked by the L-type Ca2+ channel blocker, nifedepine (10 µM). Currents were elicited by stepping to +10 mV for 200 ms from a holding potential of -40 mV every 5 seconds.

Thanks to S. Rakovic and D. Terrar from University of Oxford, for preparing the cardiomyocytes used in these experiments.

Cav2.2 Current-to-Voltage Relationship


Cav22IV_PaP

Representative current responses of an individual HEK-293 cell expressing Cav 2.2 to a standard voltage protocol. Average current-voltage relationship (n = 10). The error bars reflect the standard error of the mean (S.E.M.).
Cells were kindly provided by Millipore.

Cav3.2 - Channel Blockers


Cav32_Block

Shown are raw current traces (top) and average dose response curves (bottom) of CaV3.2 (T-type Ca2+- Channel) block by compounds as indicated. CaV3.2 is stably expressed in HEK293 cells. IC50s were 863 nM (Mibefradil), 27 μM (Nifedipine) and 52 μM Amiloride.

Cells were kindly provided by Cytomyx Millipore, UK.

Cav3.2 - T-Type Ca Channels


Cav32_IV

Shown are raw current traces (top left) and average peak current data (top right) of the current voltage relationship of CaV3.2 (T-type Ca2+- Channel) stably expressed in HEK293 cells. Activation and Inactivation plots were constructed (bottom). Half-activation and half-inactivating potentials were determined as -32 mV and -65 mV, respectively.

Cells were kindly provided by Cytomyx Millipore, UK.

Continuous Internal Perfusion


p24_1_internalPerf

KV1.3 currents (blue), endogenously expressed in Jurkat cells, were rapidly blocked by internal perfusion of Cs+ (light blue), and fully recovered after washout with K+ (grey). Internal solution replacement was repeated 19 times and the recording was stable for over 35 minutes, as shown in the lower graph.

Currents in Differentiated IMR32 Cells


Cori_IMR32_Seite_61_67

Whole cell current responses of undifferentiated (left) and for 12 days differentiated (right) IMR32 cells to a voltage step protocol (holding −90 mV, stepping for 20 ms from −60 mV to +60 mV in 20 mV increments) are shown. The change in current shape indicating increased expression of KV and NaV are apparent.

Erythrocytes - Port-a-Patch®


application_erythro_1

Erythrocytes lack mitochondria and nuclei and consist mainly of hemoglobin. The membrane contains different ion channels, for example, a Ca2+-activated K+ channel and a volume-sensitive Na+/K+ pump. Here, single channel activity recorded from an erythrocyte in the cell attached configuration is shown.

Gramicidin


application_gramicidin_1

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 two gramicidin derivates (94.88 pS and 28.28 pS) can be distinguished.

Recordings were kindly supplied by Tohoku University, Tohoku, Japan.

hEAG (hKv10.1)


application_heag_1

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 - Port-a-Patch


p14_1_hERG

Sticky compounds, such as some hERG blockers, exhibit expected IC50 values with the Port-a-Patch®. The concentrations of the half maximal block were: 1.27 nM (astemizole), 8.9 nM (cisapride), 163.7 nM (flunarizine) and 11.0 nM (terfenadine).

Cells were kindly provided by Cytomyx/Millipore, UK.

hERG - Temperature Control


DATA-Temp-Control-small

The image shows example traces for hERG mediated currents at 25 ± 2 °C (black) and 35 ± 2 °C (blue). The peak current amplitude was increased at 35 °C, and the rise time and decay time constants were faster at physiological temperature compared with those obtained at room temperature. Cells were kindly provided by Cytomyx/Millipore, UK.

hKv1.3 in Perforated Patch


altapplication_hkv13_2

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 (right).

hNav1.5


application_hnav15_1

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.


 

Internal Application of CaM to TRPV1


p24_3_TRPV1

TRPV1 currents were elicited by the external application of 20 μM capsaicin. After capsaicin activation, the currents were partially blocked by the internal application of Ca2+-calmodulin (50 μM Ca2+ / 500 nM CaM). TRPV1 channels were expressed in CHO cells.

Intracellular Second Messengers


p24_2_InternalApp

Currents mediated by BK channels expressed in CHO cells were studied using the Internal Perfusion System. Currents were elicited by a voltage step from -80 mV to +80 mV before and after adding an internal solution containing a higher concentration of free Ca2+.

Kir2


application_kir2_1neu

Current response in RBL cells to a voltage ramp from –150 mV to +80 mV in the presence of a low and a high external K+ concentration (left). Voltage dependent block of the inward K+ current after the external additon of 50 µM Ba2+ (middle). Same block in response to a continuous voltage step to –150 mV (right) from 0 mV.  Low K+ (4.5 mM K+), high K+ (143 mM K+).


kNBCs-1 (NBCe1-A)


application_kncb1_1

Recordings from HEK 293 cells expressing the electrogenic Na+/bicarbonate cotransporter (NBC1). Current responses to 200 ms voltage ramps in high external Na+ initially after establishment of the whole cell configuration (control), after application of HCO3- (bicarbonate), and after wash-out (wash-out). Data were kindly provided by Ira Kurtz, UCLA, USA.

Kv Currents in Differentiated IMR32 Cells


Cori_IMR32_Seite_64_70

Whole cell current responses of undifferentiated (a) and for 12 days differentiated (b) IMR32 cells to a voltage step protocol (holding −90 mV, stepping for 500 ms from −60 mV to +60 mV in 10 mV increments) are shown. In (c) and (d) the corresponding current voltag relationships are shown (closed circles). Open circles are the correspondig current voltage relationships after the cells have been exposed to internal Cs+.

Kv1.3 Pharmacological Experiments


p25_1_Kv1,3

KV1.3 current was blocked by the internal application of increasing concentrations of quinidine (left) or TEA+ (right). For quinidine the IC50 was determined as 15 μM (literature value external application 14 μM) and for TEA+ the IC50 was determined as 0.9 ± 0.3 mM (n = 3) (literature value internal application 0.6 mM).

Lysosomes Recordings


Lysosomes_both

Typical chloride currents in four isolated lysosomes.Chloride currents were determined as difference currents between IV curves recorded in the absence and presence of extralysosomal high (64 mM) Cl-. On the left a sketch is illustrating the methodology.

Data are taken from Schieder M. et al., The Journal of Biological Chemistry, 2010, 285(28), 21219-21222.


Membrane Movements and Kv1.3 Currents


Porti_AFM_JurkatWeb

Voltage induced membrane movements of a Jurkat cell. (a) Deflection signal of the cantilever resting on the cell membrane at an indenting force of 1.0 nN. (b) Corresponding whole cell current. Upon depolarization of the membrane, the characteristic response of the voltage geate potassium channel KV1.3 in Jurkat cells is observed. (c) Below the applied voltage protocol is displayed.

Date are taken from Pamir E., et al., Ultramicroscopy, 2008, 108, 552-557.

Mesophyll Protoplasts (Tabacco Leaves)


mesophyll-cells_2

Mesophyll protoplasts are enzymatically isolated from tobacco leaves. Shown are whole cell recordings which demonstrate the hyperpolarization-activated inward currents in the protoplasts. A voltage ramp from −40 mV to −120 mV was applied.

Data were kindly provided by Berghöfer; Eing; Flickinger; Frey (Forschungszentrum Karlsruhe, Germany).

Mitochondria Recordings


application_mitochondria_1

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.

P2X3


PortiP2X3

Shown are raw current traces from 1321 N1 cells expressing P2X3 receptors demonstrating the reproducability of the whole cell responses. Currents were activated by 100 ms application of 10 µM ATP. The waiting time between sweeps was 120 s (holding −60 mV).

Cells were kindly supplied by Evotec AG, Hamburg, Germany

Pharmacological Experiments at 35 °C


p28_4_fullDoseResp

A full dose response curve of quinidine acting on the hERG channel was obtained at physiological temperature (35 °C). The IC50 for quinidine at physiological temperature was 1.3 ± 0.2 μM (n = 5), similar to that obtained at room temperature (1.0 ± 0.03 μM, n = 3).

Cells were kindly provided by Cytomyx/Millipore, UK.

Pharmacology on hGlyRα1


p20_3_hGlyRalpha1

The Laminar Flow Chamber can also be used for studying glycine receptors. The pharmacology of the hGlyRα1, expressed in a mouse fibroblast cell line (L-tk), was investigated. The EC50 for glycine was determined as 89 ± 2.7 μM (n = 10) which is in accordance with the literature.
Cells were kindly provided by AstraZeneca, Sweden.

Primary Hippocampal Granule Cells


p15_2_Primary_Hippocampal_granules

Recordings from acutely isolated hippocampal granule cells show BK- and CaV currents. Whole cell currents were obtained by depolarizing voltage pulses from a holding potential of −80 mV.

Primary Neuronal Stem Cells


p15_2_Primary_Stem_2

Patch clamp recordings from cultured mouse primary neuronal stem cells were made in the whole cell configuration (holding potential −60 mV). Currents were evoked by 500 ms depolarizing voltage pulses, showing outwardly rectifying K+- currents.

Data courtesy of Dr. Gavin Dawe, National Uni­versity of Singapore.

Rapid Internal Exchange


p24_4_InternExch

Switching of internal solutions during gramicidin recordings from a lipid bilayer was obtained within seconds. A lipid bilayer was formed using giant unilamellar vesicles. Currents were recorded at a holding potential of +150 mV. The internal solution was switched from HCl to KCl, resulting in lower channel conductance.

Reliable Compound Application


p20_2_GABA

Ligand dependent activation of GABAA receptors can be recorded with the Laminar Flow Chamber with approx. 100 ms time constant for 10 μM GABA. Concentration dependent activation and desensitization of GABAA (α1β2γ2) receptors was obtained by applying 1, 3 and 10 μM GABA for 2.5 s at a time interval of 20 s.

Shaker I


application_shaker_1

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.

Data were kindly provided by Dr. Kenton Swartz, NIH, Bethesda, USA.


Shaker II


application_shaker_2

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.

Single Channel recordings of Sec 61


Sec61_Ramp

Microsomes were fused with GUVs within the VesiclePrepPro. The resulted Proteoliposomes formed a bilayer onto the NPC-1 Chip of the Port-a-Patch. Nice single channel openings could be recorded in symmetrical KCl solution applying a ramp from -110 mV to +100 mV for 400 ms. Data were kindly provided by A. Cavalié, M. Jung, R. Zimmermann, Universität des Saarlandes, Homburg, Germany.


Sonoporation


application_sonoporation_2

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 5 MHz. Pulses of 50 cycles were repeated for 2 s every 10 ms. Data were kindly provided by Dr. Cheri Deng, University of Michigan, USA.

TRPM7


mTRPPM7InternalPerfPorty_sml

Shown are mTRPM7 (in HEK293 cell) raw current responses to a voltage ramp voltage procotol  (−100 mV to 100 mV over 200 ms) recorded under control conditions and after subsequent internal perfusion with Mg2+ (left). Initally, currents showed some run-up before they became stable. The arrows in the timecourse on the right mark the timepoints for which the raw current traces are shown.

TRPM8 Activated by Menthol


AN_Port-a-Patch_TRPV1M8-2

Menthol activated the TRPM8 channels expressed in HEK293 cells.

TRPM8/TRPV1


p15_1_TRP

TRP channels recorded in HEK293 cells. Menthol (dark blue) activated the TRPM8 channels (shown on the left) and could be inhibited by genistein (blue). TRPV1 channels expressed in HEK293 cells (right) were stimulated by application of capsaicin.
TRPV1 data were kindly supplied by Dr. David Cohen, Oregon Health and Service University, Portland, OR, USA.

TRPV1


application_trpv1_1

Whole cell current responses from HEK 293 cells transiently expressing TRPV1 to a ramp voltage protocol (‑100 mV to +100 mV). Capsaicin at a concentration of 2 μM reversibly activated the channel.

Data were kindly provided by David Cohen, Oregon Health & Science University, Portland, USA.

TRPV1 - Heat Activated Channels


p28_2_heatActivChan

Raw traces of TRPV1 current responses to voltage ramps from –100 mV up to +100 mV. TRPV1 current at RT (28°C), during two stimulations by application of heated solution (34°C and 36°C), and after cooling down to RT (29°C). The picture on the right-hand side shows the current amplitudes as recorded at -100 mV and +100 mV plotted against time.

Bilayer Recordings of KcsA


KCSA-Vesicle-prep-pro

The image shows a representative current trace recorded from a planar lipid bilayer in which purified KcsA channels were reconstituted (100 mM KCl, asymmetric pH; pH 4/pH 7). A ramp from -100 mV to +100 mV was used to evoke the single channel events.

Download: Application Note

Bilayer Recordings of OmpF


OMPFPolyamine_bea

Channels were activated in the presence of spermine (0.5 mM). The downward spikes represent the closure events due to the effect of spermine.

Data were kindly supplied by Prof. Mathias Winterhalter, Jacobs University, Bremen, Germany.

DNA Translocation in Hemolysin


p45_4_AlphaHemolysinNeu

Reconstituted Alpha hemolysin channels are constantly open at positive and negative membrane potentials. Gating is observed as a result of the passage of a single stranded DNA molecule through the pore. Recordings were performed on the Port-a-Patch®.

Data were kindly supplied by Prof. Fritz Simmel, Technical University of Munich, Munich, Germany.

Reconstitution of IP3 Receptors


IP3_bea_vereinfacht

Representative traces of the IP3 (inositol-triphosphate) receptor were recorded at different voltages in the presence of IP3 (0.2 mM free Ca2+ and 1 mM Na2ATP, 140 mM KCl). Addition of 2 μM IP3 on the cytosolic side evoked openings of the IP3 receptor.

Data were kindly supplied by Prof. Colin Taylor, University of Cambridge, Cambridge, UK.

TRPM8


TrpM8_420x420

Shown are single channel events of TRPM8 reconstituted in a planar lipid bilayer. The recordings were made with the with the Port-a-Patch®. The channel was activated by PIP2 and methanol. Currents were recorded at 100 mV.

Data kindly provided by Dr. Zakharian and Dr. Rohac From UMDNJ New Jersey Medical School, Newark, NJ 07103, USA