Two different application protocols were used to study the effect of exposure time on glycine receptor pharmacology for cells expressing hGlyRα1. As shown from the raw data traces and corresponding Hill plots, the highest concentration, 3 mM glycine, did not elicit the maximum peak response during long exposures (22 s), in contrast to stacked applications (1 s). Cells were kindly provided by AstraZeneca.

In this example both Na⁺ and Ca²⁺ mediate the action potential. When nifedipine is applied in the current clamp mode, the action potential is shortened significantly due to block of the calcium channels.
The stem cell-derived cardiomyocytes (iCell) were kindly supplied by CDI.

With suction the GUVs are attracted to the aperture. As soon as one GUV hits the glass substrate, it bursts and forms a bilayer across the aperture. Shown are single channel recordings from gramicidin which was incorprated into the bilayer after its formation. Traces were recorded in 100 mM HCl at −100 mV.

The image shows current response of an individual cell in the presence of increasing cadmium concentrations. The IC₅₀ was calculated from the Hill fit to be 3.6 ± 0.4 μM (n = 5).

Cells were kindly provided by Millipore.

Representative current responses of an individual cell expressing Cav2.2 to a I/V voltage protocol. The average peak current at 30 mV of all recorded cells was -698 ± 115 pA (n = 6).

Cells were kindly provided by Millipore

Representative current responses of an individual cell expressing Ca 2.2 to a standard voltage protocol. The average mean current at -20 mV of all recorded cells was -785 ± 110 pA (n = 12).
Cells were kindly provided by Millipore

Current responses of a double pulse protocol with varying test potentials between the pulses (5 s) was used to determine the half inactivating potential. Peak current responses to the second pulse are expressed relative to the response to the first pulse. Both curves in Figure 5 were fitted to the Boltzmann equation and revealed a half-inactivating potential of -65 mV and a half-activating potential of -33 mV.

Dose dependent block by Mibefradil on current traces from an individual cell expressing Ca 3.2. Data was averaged and fitted to the Hill equation where the IC₅₀ value was determined as 1.7 µM. Error bars corresponds to S.E.M.
Cells were kindly provided by Millipore.

The left picture shows a typical action potential from Cor.At^® cardiomyocytes. Whole cell currents recorded in the voltage clamp mode reveal cardiomyocyte-typical ion channels (right). The traces represent mERG-, L-type Ca²⁺- (blue, block by 50 μM nifedipine), Na⁺- and K⁺-currents (from top left to bottom right).

Cells were kindly provided by Axiogenesis.

The membrane of erythrocytes contains different ion channels like Ca²⁺-activated K⁺ channels, or the volume-sensitive Na⁺/K⁺ pump. Studies also revealed the participation of a Ca²⁺-permeable non-selective cation channel in the regulation of erythrocyte 'apoptosis'. Shown are single channel fluctuations as recorded from an erythrocyte in the cell attached configuration on the Patchliner^®.

The figure shows current responses of a hGlyRα1 expressing L-tk cell to alternating exposures to 100 μM glycine and 100 μM glycine + 1 μM strychnine.

Cells were kindly provided by Astrazeneca, Södertälje, Sweden.

Original traces of one application of 20 μM glycine followed by 6 applications of 20 μM glycine in conjunction with increasing concentrations of a positive modulator. 1 mM glycine was used as a second positive control.

Cells and the positive modulator were kindly provided by Astrazeneca, Södertälje, Sweden.

Even sticky compounds pose no problem for the Patchliner^®. IC₅₀ measurements of well known sticky substances were determined on the Patchliner^®: Terfenadine IC₅₀ = 11.0 ± 3 nM, Flunarizine IC₅₀ 163.7 ± 19 nM and Cisapride IC₅₀ 8.9 ± 3 nM.

hERG expressing HEK293 cells were kindly provided by Cytomyx/Millipore, UK.

The effects of erythromycin on hERG currents were tested at different temperatures. Erythromycin has been shown to block hERG channels at physiological temperature with an IC₅₀ of approx. 40 µM. However, at RT erythromycin is much less potent. For more details on these experiments please refer to the Application Note. Cells were kindly provided by Cytomyx/Millipore, UK.

Shown is a screenshot of a pharmacology experiment performed with a Patchliner^® Octo. Eight full dose response curves from hERG expressing CHO cells were performed simultaneously. Original current traces are displayed as well as the the peak current plotted over time. Cells were kindly provided by Cytomyx/Millipore, UK.

We have great data analysis tools! - Especially programmed routines in Igor make dose response curves, raw data and current time courses easily accessible. Also, creating average dose response curves over multiple experiments - even conducted on different days - remains easy.

Full dose response curves at different holding potentials were recorded for each cell (hNav1.5 in HEK293). Currents were elicited by a 10 ms voltage step to 0 mV. Plotted are average peak currents as a function of holding potential and lidocaine concentration. Cells were kindly provided by Cytomyx/Millipore, UK.

Whole cell currents from rat cortical astrocytes (primary culture) were evoked by 500 ms long depolarizing voltage steps (-100 mV to +40 mV). Currents were blocked by administration of internal Cs⁺, and recovered when switching back to Cs⁺-free internal solution. Averaged data are presented as mean ± S.E.M. (n=35). For more information, see Nature 254, 4 (2), 2009. Data courtesy of C. Peers, University of Leeds, Leeds, UK.

A unique feature of the Patchliner^® is its ability to exchange the internal solution during the recording. The figure shows recordings of Kv1.3 from two Jurkat cells (simultaneoulsy recorded) in the presence of control internal solution, after the exchange of the internal solution with a Cs⁺ solution, and subsequent washout (left to right).

The top images show dose dependent block of GABA_A currents by bicuculline. The IC₅₀ was determined as 1.2 ± 0.2 μM (n=11). The lower graph shows the positive modulation of glycine activation of hGlyRα1. Here, six co-applications of 20 μM glycine and increasing concentrations of a positive modulator are shown. Cells were kindly provided by AstraZeneca.

The basophilic leucaemia cells (RBL) exhibit an inwardly rectifying potassium current, Kir. Changing the external K⁺ concentration (here between 4.5 mM and 143 mM) leads to a change of the current amplitude of the inward current (holding: -100 mV). This gives us a convenient tool to study the speed of the external solution exchange which was determined as ~50 ms.

An illustrative typical family of traces obtained during construction of a current voltage relationship from a human lymphpblast recording using amphotericin B to access th whole cell. Currents were evoked by voltage steps from holding (−80 mV) to potentials varying from −100 mV to +60 mV.
Data are taken from Milligan C.J. et al., Nature Protocols, 2009, 4(2), 244-255.

The pharmacology of dibucaine was investigated by the application of 0.3, 1, 3, 10 μM in the presence of 10 μM nifedipine (L-type Ca²⁺-current blocker). Two control additions of nifedipine (10 μM) were made before the addition of increasing concentrations of dibucaine. The IC₅₀ value was determined as 355 ± 40 nM (n=3), which is in accordance with the literature. Cells were kindly provided by Axiogenesis.

Complete nicotine dose response curves were obtained by applying increasing concentrations of nicotine to a HEK293 cell expressing human nicotinic α7 acetyl choline receptors. The stacked application protocol was used.

Cells were kindly supplied by Galantos Pharma GmbH.

Stable whole-cell current amplitudes were obtained by repeated nicotine stimulation of HEK293 cells expressing human nAChR a7 receptors. The stacked application protocol was used. Cells were kindly supplied by Galantos Pharma GmbH.

BzATP concentration response curve obtained from wild-type P2X₇ receptors. Data are fite to the Hill equation with an EC₅₀ of 69.7 ± 7.5 μM. The inset show representative currents evoked by BzATP (3 − 100 μM). Holding potential was −60 mV.

Data are taken from Milligan C.J. et al., Nature Protocols, 2009, 4(2), 244-255.

Application of 5 μM quinidine leads to about 50 % block of the Kv1.3 currents (blue). After washout, the current is fully recovered (grey). The lower graph shows corresponding Imax (+40 mV) in the absence and presence of 5 μM quinidine, for two different cells with eight consecutive application and washout steps. The recording lasted over 40 minutes!

The I/V-characteristics of Nav1.5 currents (HEK293) are shown together with the repeated dose dependent block by TTX (lower panel). Five concentrations of TTX
(0.3, 1, 3, 10, 30 μM) were applied, followed by washout with antagonist-free buffer and re-application of the same TTX concentrations. Cells were kindly provided by Millipore.

Ligand gated ion channels often display receptor desensitization. A method was developed to minimize ligand exposure times and intervals between ligand exposures. The pipette first aspirates buffer, then compound. When expelling this stack, the cell is first exposed to ligand and then buffer. Exposure times as low as 400 ms are possible with this method. A GABA dose response curve, aquired in this manner, is shown on the left.

The current responses of a CHO cell expressing TRPV1 (ramp -100 mV to +100 mV) at increasing temperatures is shown. The ET₅₀ value was determined as 64°C. Challenge of the same cell with capsaicin (1 μM) and temperature (70°C) allows comparison of the responses. IC₅₀s for ruthenium red block of capsaicin- and heat-responses were determined as 1.6 ± 0.2 μM (n = 3) and 7.4 ± 1.3 μM (n = 3), respectively.

Shown is the time course of the current measured at +95 mV from an individual cell (these are data from a similar experiment as shown in TRP A1). The legend indicates the time periods in which the activator AITC was applied. Here we demonstrate the reproducibility of the current responses for TRP A1.

Cells were kindly supplied by Millipore.

Current voltage relationships showing activaion of TRPC5 by extracellular application of Gd³⁺ (100 μM). Voltage ramps (−100 mV to 100 mV, holding potential 0 mV) were for 1s at 0.1 Hz.

Data are taken from Milligan C.J. et al., Nature Protocols, 2009, 4(2), 244-255.

Left: Illustrative time series showing currents at +80 mV and −80 mV in a smooth muscle cell exposed to extracellular Gd³⁺ (100 μM) and then 2-APB (75 μM). Right: Membrane resistance (R_m), series resistance (R_S) and membrane capacitacne (C_m) values for successful recordings from smooth muscle cells.
Data are taken from Milligan C.J. et al., Nature Protocols, 2009, 4(2), 244-255.

A Current responses to a voltage ramp protocol from -150 mV to 150 mV over 200 ms in response to 100 μM pregnenolone sulphate (PS) and in combination with increasing concentrations of compound C. B Current responses elicited by PS, enhancement by 30 μM compound C and block of the current by co-application of antagonist (10 μM). Cells were kindly provided by Prof. Thomas Voets, KU Leuven, Belgium.

Recordings from a HEK cell expressing TRPV3 were made with the Patchliner showing activation by heat. External solution was heated inside the Patchliner pipette to the temperature shown and applied to the cells. TRPV3 was activated at temperatures ≥ 38°C.

Cells were kindly supplied by Millipore.