Conventional Patch-clamp Methods Research Articles

Identification of a helix-turn-helix motif for high temperature dependence of vanilloid receptor TRPV2.

Pain and thermosensation rely on temperature-sensitive ion channels at peripheral nerve endings for transducing thermal cues into electrical signals. Members of the transient receptor potential (TRP) family are prominent candidates for temperature transducers in mammals. These thermal TRP channels possess an unprecedentedly steep temperature dependence, allowing them to discriminate small temperature variations. Thermodynamically, it is understood that the strong temperature sensitivity of the channel arises because opening of the channel undergoes reactions involving large enthalpy and entropy changes. However, the underlying molecular mechanisms have remained elusive. Here we investigated the molecular basis for heat activation of TRPV2, a thermal TRP channel in the vanilloid subfamily with the strongest temperature dependence among TRP channels. We unravel a minimum molecular region in the proximal N-terminus which dictates the slope temperature sensitivity of the channel. Structurally, the region comprises a helix-turn-helix motif and is positioned among the TRP helix from the C-terminus, the S2-S3 linker from the transmembrane domain and the ankyrin repeats from the distal N-terminus. Chimeric exchanges of the subregion alone sufficed to diminish the high temperature dependence in the wild-type TRPV2. Our results support a pivotal role for the structural assembly around the TRP domain in the gating of thermal TRP channels by temperature. The findings also shed insight into how the proximal N-terminal domain plays its role in the heat activation of vanilloid receptors. KEY POINTS: The vanilloid receptor subtype 2 (TRPV2) is a heat-sensitive transient receptor potential (TRP) channel with the strongest temperature dependence among thermal TRP channels. The channel also has a high temperature activation threshold above 50°C which has rendered it difficult to study by conventional patch-clamp methods. Here we utilize fast laser temperature jumps to address the challenges of technical accessibility and explore the molecular basis underlying the high temperature dependence of the channel. We unravel a short helix-turn-helix motif in the proximal N-terminus, which controls the heat activation profile of the channel. Chimeric exchanges of the subregion alone sufficed to diminish the high temperature dependence in the wild-type TRPV2. Our results provide insights on how the proximal N-terminal domain plays its role in the heat activation of vanilloid receptors.

Electrophysiological measurement of ion channels on plasma/organelle membranes using an on-chip lipid bilayer system

Ion channels are located in plasma membranes as well as on mitochondrial, lysosomal, and endoplasmic reticulum membranes. They play a critical role in physiology and drug targeting. It is particularly challenging to measure the current mediated by ion channels in the lysosomal and the endoplasmic reticulum membranes using the conventional patch clamp method. In this study, we show that our proposed device is applicable for an electrophysiological measurement of various types of ion channel in plasma and organelle membranes. We designed an on-chip device that can form multiple electrical contacts with a measurement system when placed on a mount system. Using crude cell membranes containing ion channels extracted from cultured cells without detergents, we detected open/close signals of the hERG, TRPV1, and NMDA channels on plasma membranes, those of the TRPML1 channels on lysosomal membranes, and open/close signals of the RyR channels on SR membranes. This method will provide a highly versatile drug screening system for ion channels expressed by various cell membranes, including plasma, SR, mitochondrial, Golgi, and lysosomal membranes.

Integration of autopatching with automated pipette and cell detection in vitro.

Patch clamp is the main technique for measuring electrical properties of individual cells. Since its discovery in 1976 by Neher and Sakmann, patch clamp has been instrumental in broadening our understanding of the fundamental properties of ion channels and synapses in neurons. The conventional patch-clamp method requires manual, precise positioning of a glass micropipette against the cell membrane of a visually identified target neuron. Subsequently, a tight "gigaseal" connection between the pipette and the cell membrane is established, and suction is applied to establish the whole cell patch configuration to perform electrophysiological recordings. This procedure is repeated manually for each individual cell, making it labor intensive and time consuming. In this article we describe the development of a new automatic patch-clamp system for brain slices, which integrates all steps of the patch-clamp process: image acquisition through a microscope, computer vision-based identification of a patch pipette and fluorescently labeled neurons, micromanipulator control, and automated patching. We validated our system in brain slices from wild-type and transgenic mice expressing channelrhodopsin 2 under the Thy1 promoter (line 18) or injected with a herpes simplex virus-expressing archaerhodopsin, ArchT. Our computer vision-based algorithm makes the fluorescent cell detection and targeting user independent. Compared with manual patching, our system is superior in both success rate and average trial duration. It provides more reliable trial-to-trial control of the patching process and improves reproducibility of experiments.

Dynamic Action Potential Clamp Investigation of Pro-Arrhythmic Risk of Drugs Binding to hERG Potassium Channels

Many commonly used drugs can bind to and block the hERG channel and cause the potentially life threatening acquired long-QT syndrome.Whilst obtaining an IC50 for drug block of hERG is relatively straight forward, this is a poor surrogate for risk of pro-arrhythmia. Predicting the overall consequences of hERG drug block on cardiac electrical activity is complicated by the fact that the effect of hERG channel block varies in different cells (e.g. epicardial, mid-myocardial, endocardial, Purkinje) of the heart. Furthermore it is significantly altered by the electrical remodeling that occurs in many chronic heart conditions. With the aging of our population and an increasing proportion of people with chronic heart conditions it is especially important to understand how disease states affect the consequences of hERG drug block and risk of pro-arrhythmia.Here we describe a recently developed dynamic action potential clamp system (dAPC) to investigate the effect of hERG block on cardiomyocytes. The system consists of conventional whole cell voltage clamp study of ion channels in mammalian expression systems, coupled to a real time computer model of human cardiomyocyte action. The dAPC system integrates the current recordings from a patch clamped cell into an in-silico cell model, the output of which is then used to determine the voltage of the patch clamped cell. When used in combination with a state of the art drug perfusion system this integrative approach will permit testing of drugs on specified ion channels in a physiologically relevant environment, something that is not possible with conventional patch clamp methods.

Assay Development and Validation for the Muscle Nicotinic Receptor Using the Qpatch HTX™ Automated Patch Clamp System

Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research Inc., 250 Massachusetts Avenue Cambridge, MA 02139, USA.QPatch HTX is an automated patch clamp system with greatly increased throughput over conventional patch clamp methods. In this study, we evaluated the suitability of this system for the characterization of the fast desensitizing ligand-gated ion channel coupled with alpha1 nicotinic acetylcholine receptors (α1nAChR). HEK293 cells stably expressing human α1nAChR were obtained from Millipore. All experiments were carried out with the QPatch HTX, which performs 48 parallel and independent patch-clamp recordings on a disposable QPlate X with 10 holes per measurement site. Brief applications of acetylcholine (ACh) produced fast inward currents characteristic of the α1nAChR recorded by conventional patch clamp in heterologous expression systems and the native channel. The EC50 values for ACh ranged from 3 to 10 µM (n=10). Nicotine and epibatidine also elicited inward currents with EC50 values of 110 µM and 4 µM, respectively (n=6 & 12). The ACh-induced current was resistant to bath applications of atropine up to 1 µM and muscarine did not elicit a response, showing that the ACh-induced inward current is mediated by nAChR. Non expressing HEK293 cells did not respond to ACh. The ACh-mediated currents were blocked by α-bungarotoxin and pancuronium in a dose dependent manner (IC50= 80 and 0.8 nM, respectively).In conclusion, this study demonstrated that fast desensitizing ligand-gated ion channel can be efficiently targeted with QPatch HTX system for characterization of physiological and pharmacological properties.

Characterization of Two-pore Channel 2 (TPCN2)-mediated Ca2+ Currents in Isolated Lysosomes

Two-pore channels (TPCNs) have been proposed to form lysosomal Ca(2+) release channels that are activated by nicotinic acid adenine dinucleotide phosphate. Here, we employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes. We show that TPCN2 is a highly selective Ca(2+) channel that is regulated by intralysosomal pH. Using site-directed mutagenesis, we identify an amino acid residue in the putative pore region that is crucial for conferring high Ca(2+) selectivity. Our glass chip-based method will provide electrophysiological access not only to lysosomal TPCN channels but also to a broad range of other intracellular ion channels.

Development and Validation of a Secondary Screening Assay for TRPM8 Antagonists Using QPatch HT

QPatch HT is an automated patch clamp system with high data quality/content and greatly increased throughput over conventional patch clamp methods. To determine whether this platform is suitable for secondary screening of antagonists of TRPM8, a cold- and menthol-activated ion channel that belongs to the transient receptor potential channel family, we used QPatch HT to test a set of chemically diverse compounds identified as TRPM8 antagonists by FLIPR and conventional patch clamp. We found that most compounds exhibited slower inhibition kinetics compared with conventional patch clamp, requiring multiple applications to reach steady-state inhibition. For most compounds, there was a relatively small (< or =4-fold) right shift in potency compared with conventional patch clamp. Nonetheless, the compound potencies obtained from QPatch HT exhibited a highly significant correlation with those from either conventional patch clamp (r(2) = 0.98) or FLIPR (r(2) = 0.97), over a wide range of concentrations and cLogP values (approximately 4 orders of magnitude) and with virtually identical rank-order potency. The throughput by QPatch HT was at least 10-fold higher than that obtained by conventional patch clamp. Our results validate the use of QPatch HT for secondary screening of TRPM8 antagonists and, along with other recent studies, illustrate its utility as an important tool for ion channel drug discovery.

Population Patch-Clamp Electrophysiology Analysis of Recombinant GABAA α1β3γ2 Channels Expressed in HEK-293 Cells

Gamma-amino butyric acid (GABA)-activated Cl- channels are critical mediators of inhibitory postsynaptic potentials in the CNS. To date, rational design efforts to identify potent and selective GABA(A) subtype ligands have been hampered by the absence of suitable high-throughput screening approaches. The authors describe 384-well population patch-clamp (PPC) planar array electrophysiology methods for the study of GABA(A) receptor pharmacology. In HEK293 cells stably expressing human alpha1beta3gamma2 GABA(A) channels, GABA evoked outward currents at 0 mV of 1.05 +/- 0.08 nA, measured 8 s post GABA addition. The I(GABA) was linear and reversed close to the theoretical E(Cl) (-56 mV). Concentration-response curve analysis yielded a mean pEC(50) value of 5.4 and Hill slope of 1.5, and for a series of agonists, the rank order of potency was muscimol > GABA > isoguvacine. A range of known positive modulators, including diazepam and pentobarbital, produced concentration-dependent augmentation of the GABA EC( 20) response (1 microM). The competitive antagonists bicuculline and gabazine produced concentration-dependent, parallel, rightward displacement of GABA curves with pA(2) and slope values of 5.7 and 1.0 and 6.7 and 1.0, respectively. In contrast, picrotoxin (0.2-150 microM) depressed the maximal GABA response, implying a non-competitive antagonism. Overall, the pharmacology of human alpha1beta3gamma2 GABA(A) determined by PPC was highly similar to that obtained by conventional patch-clamp methods. In small-scale single-shot screens, Z' values of >0.5 were obtained in agonist, modulator, and antagonist formats with hit rates of 0% to 3%. The authors conclude that despite the inability of the method to resolve the peak agonist responses, PPC can rapidly and usefully quantify pharmacology for the alpha1beta3gamma2 GABA(A) isoform. These data suggest that PPC may be a valuable approach for a focused set and secondary screening of GABA(A) receptors and other slow ligand-gated ion channels.

Patch clamp technique: Review of the current state of the art and potential contributions from nanoengineering

The patch clamp technique permits high-resolution recording of the ionic currents flowing through a cell's plasma membrane. In different configurations, this technique has allowed experimenters to record and manipulate the currents that flow either through single ion channels or those that flow across the whole plasma membrane. Unfortunately, the conventional patch clamp method is laborious, requiring the careful fabrication of electrodes, skillful manipulation of the patch pipette towards a cell, and the clever design of electronics and apparatus to allow low-noise recordings. Advances in microfabrication offer promising technologies for high-throughput patch clamp recordings, particularly suitable for drug screening. This paper provides a review of the advances that have been made in the patch clamp technique over the years and considers where application of nanotechnology might provide significant contributions in the future.

Inhibitory interaction of cannabinoid CB1 receptor and dopamine D2 receptor agonists on voltage-gated currents of goldfish cones.

Dopamine is a light-adaptive signal that desensitizes the retina, while cannabinoids reportedly increase photosensitivity. The presynaptic membrane of goldfish retinal cones has dopamine D2 receptors and cannabinoid CB1 receptors. This work focused on whether dopamine D2 receptor agonist quinpirole and cannabinoid CB1 receptor agonist WIN 55212-2 (WIN) interacted to modulate voltage-dependent membrane currents of cones. A conventional patch-clamp method was used to record depolarization evoked whole-cell outward currents (Iout) and an inward calcium current (ICa) from the inner segment of cones in goldfish retinal slices. WIN had biphasic actions: low concentrations (<1 microM) increased the currents via Gs, while higher concentrations (>1 microM) decreased the currents via Gi/Go. Neither dopamine nor the D2 agonist quinpirole (1-20 microM) had a significant effect on either Iout or ICa. Quinpirole at 50 microM had a mild suppressive (approximately 20%) effect on Iout. However, quinpirole (<10 microM) completely blocked the enhancement of both currents seen with 0.7 microM WIN. The effect of quinpirole was blocked by sulpiride and by pertussis toxin, indicating that quinpirole was acting via a D2 receptor-Gi/o coupled mechanism. The suppressive action of 50 microM quinpirole (approximately 20%) was not additive with the suppressive effect of 3 microM WIN (approximately 40%). D2 agonists via Gi/o oppose the action of low concentrations of CB1 agonists acting via Gs to modulate cone membrane currents, suggesting a role in shaping the cone light response and/or sensitivity to changes in ambient light conditions. The nonadditive effect of high concentrations of WIN and quinpirole suggests that both decrease membrane currents via the same transduction pathway, Gi/Go protein kinase A (PKA).

Angiotensin II increases L-type Ca2+ current in gramicidin D-perforated adult rabbit ventricular myocytes: comparison with conventional patch-clamp method.

The effects of angiotensin II (Ang II) on L-type Ca2+ current (I(Ca,L)) remains controversial. We studied the effects of Ang II on I(Ca,L) in single adult rabbit ventricular myocytes using a perforated patch-clamp technique with gramicidin D. Ang II increased I(Ca,L) in a concentration-dependent manner (EC(50)=0.75 nM). In contrast, in conventional whole-cell patch-calmp, I(Ca,L)ran down gradually and the I(Ca,L) response to Ang II was variable, suggesting the potential loss of diffusible components crucial for the Ang II-induced signaling process. An AT(1) antagonist, CV11974 (0.1 microM), completely inhibited the increase in I(Ca,L) induced by Ang II (0.1 microM), whereas an AT(2) antagonist, PD123319 (10 microM), did not influence the I(Ca,L) increase. Neither pre- nor after-treatment with a Na+/H+ exchange (NHE) inhibitor HOE642 (1 microM) affected the Ang II-induced increase in I(Ca,L). The protein kinase C (PKC) inhibitor chelerythrine (1 microM) did not affect the Ang II-induced I(Ca,L) increase. The present findings indicate that Ang II increases I(Ca,L) via AT(1) receptors in adult rabbit ventricular myocytes. Neither the activation of NHE nor PKC may contribute to the Ang II-induced activation of I(Ca,L).

Cholinergic control of membrane conductance and intracellular free Ca2+in outer hair cells of the guinea pig cochlea

We have studied the action of cholinergic agonists on outer hair cells, both in situ and isolated from the cochlea of the guinea pig, combining new fast CCD technology for Ca2+imaging and conventional patch-clamp methods. Carbachol (1mM) activated a current with a reversal potential near –70mV and a bell-shaped I-V curve, suggesting that it was a Ca2+-activated K+current. In a few cells, this current was preceded by a transient inward current, probably owing to an influx of Ca2+and other cations through the acetylcholine (ACh) receptors. The amplitude of the Ca2+signal was maximal in a circumscribed region at the basal pole of the cell and decreased steeply towards the apical pole, compatible with Ca2+-influx and/or Ca2+-induced Ca2+-release at the cells base. The time course of the Ca2+-rise was fastest at the base, but it was still slightly slower, and more rounded, than that of the K+current. In some recordings the K+current was observed without any measurable change of intracellular Ca2+. The K+current was potentiated (18%) by caffeine (5mM), and decreased (19%) by ryanodine (0.1mM) in the majority of cells tested. The results are discussed in terms of a labile intracellular Ca2+store located at the base of the cell, close to the Ca2+-permeable ACh receptor channels and Ca2+-activated K+channels, whose contribution to the Ca2+rise occurring in the region of the channels is variable, and probably dependent on its ability to refill with Ca2+.

Reactive oxygen species and calcium homeostasis in cultured human intestinal smooth muscle cells.

Reactive oxygen species (ROS) significantly alter cell function. We examined the effects of hydrogen peroxide (H2O2) and xanthine/xanthine oxidase (X/XO) on isolated intestinal muscle cells. We assessed cell viability with the exclusion dye trypan blue and assayed the effects of H2O2 and X/XO on the intracellular redox state with the fluorescent probe 2',7'-dichlorofluorescein. Intracellular calcium concentration was measured in cells loaded with fura 2-acetoxymethyl ester, and we recorded whole membrane currents with conventional patch-clamp methods. Cells remained viable after a 5-min exposure to H2O2 and X/XO. H2O2 and X/XO led to a significant rise of the intracellular concentration of ROS. H2O2 (270 microM to 2.7 mM) as well as X/XO (0.25-16 mU; 0.5 mM xanthine) significantly increased intracellular calcium concentrations. Depletion of intracellular calcium with ryanodine or thapsigargin did not abolish the effect of ROS on the intracellular calcium concentration. In the absence of external calcium or in the presence of the calcium channel blocker nifedipine, H2O2 and X/XO still increased the intracellular calcium level. Thus calcium influx and calcium release from internal stores contributed to this rise in cytosolic calcium. Catalase and superoxide dismutase blunted or completely abolished the changes in calcium concentration elicited by H2O2 and X/XO. Exposure to ROS resulted in a rapid decline of the membrane resistance without significant changes in voltage-sensitive ion currents. We conclude that ROS disrupt the calcium homeostasis of cells at concentrations that do not lead to immediate cell death. The resulting elevation in cytosolic free calcium will activate a variety of biochemical reactions and may thus contribute to the cytotoxicity of reactive oxygen molecules.