Population patch clamp improves data consistency and success rates in the measurement of ionic currents

Present whole-cell patch-clamp methodology has only moderate consistency and throughput, rendering impractical functional measurements on large numbers of ion channel ligands or on large numbers of unknown or mutant channel genes. In the population patch clamp (PPC) described herein, a single voltage-clamp amplifier sums the whole-cell currents from multiple cells at once, each sealed to a separate aperture in a planar substrate well. The resulting ensemble currents are more consistent from well to well, and the success rate for each recording attempt is >95%. The PPC was implemented by modifying the PatchPlate substrate and amplifiers in the IonWorks patch-clamp instrument. The increased data consistency and likelihood of a successful recording in each well, combined with 384-well measurements in parallel, allow the direct electrophysiological recording of thousands of ensemble ionic currents per day. Therapeutic groups in drug discovery programs require this order of throughput to screen directed compound libraries against ion channel targets. The potential for studying the function of large numbers of ion channel mutants may be realized with the technique. The procedure incorporates subtraction methods that correct for expected distortions and also reliably produces data that agree with previous patch-clamp studies.     

Effects of perfluorooctane sulfonate on action potentials and currents in cultured rat cerebellar Purkinje cells

Recently, PFOS was reported to be ubiquitously detected in the environment, as well as in human serum, raising concerns regarding its health risks. We investigated the effects of PFOS on action potentials and currents in cultured rat cerebellar Purkinje cells using whole-cell patch-clamp recording. In current-clamp experiments, PFOS significantly decreased the action potential frequency during current injection, the maximum rate of fall and the threshold of action potential, and negatively shifted the resting membrane potential at doses over 30microM. In voltage-clamp experiments, PFOS shifted the half-activation and inactivation voltages of I(Ca), I(Na), and I(K) toward hyperpolarization at 30microM. I(HCN1) expressed in Xenopus oocytes was similarly affected. Incorporation of PFOS into the cell membrane probably increased the surface negative charge density, thereby reducing the transmembrane potential gradient and resulting in hyperpolarizing shifts of both the activation and inactivation of ionic channels. These findings indicate that PFOS may exhibit neurotoxicity.     

The interpretation of current-clamp recordings in the cell-attached patch-clamp configuration

In these experiments we have investigated the feasibility and accuracy of recording steady-state and dynamic changes in transmembrane potential noninvasively across an intact cell-attached patch using the current-clamp mode of a conventional patch-clamp amplifier. Using an equivalent circuit mimicking simultaneous whole-cell voltage-clamp and cell-attached current-clamp recordings we have defined both mathematically and experimentally the relationship between the membrane patch resistance, the seal resistance, and the fraction of the whole-cell potential recorded across an intact membrane patch. This analysis revealed a steep increase in the accuracy of recording of steady-state membrane potential as the seal/membrane ratio increases from 0. The recording accuracy approaches 100% as the seal/membrane ratio approaches infinity. Membrane potential measurements across intact cell-attached patches in rat basophilic leukemia cells and rat megakaryocytes revealed a surprisingly high degree of accuracy and demonstrated the ability of this noninvasive technique to follow dynamic changes in potential in nonexcitable cells.     

A novel voltage clamp technique for mapping ionic currents from cultured skeletal myotubes.

The biophysical properties and cellular distribution of ion channels largely determine the input/output relationships of electrically excitable cells. A variety of patch pipette voltage clamp techniques are available to characterize ionic currents. However, when used by themselves, such techniques are not well suited to the task of mapping low-density channel distributions. We describe here a new voltage clamp method (the whole cell loose patch (WCLP) method) that combines whole-cell recording through a tight-seal pipette with focal extracellular stimulation through a loose-seal pipette. By moving the stimulation pipette across the cell surface and using a stationary whole-cell pipette to record the evoked patch currents, this method should be suitable for mapping channel distributions, even on large cells possessing low channel densities. When we applied this method to the study of currents in cultured chick myotubes, we found that the cell cable properties and the series resistance of the recording pipette caused significant filtering of the membrane currents, and that the filter characteristics depended in part upon the distance between the stimulating and recording pipettes. We describe here how we determined the filter impulse response for each loose-seal pipette placement and subsequently recovered accurate estimates of patch membrane current through deconvolution.     

Voltage-dependent membrane capacitance in rat pituitary nerve terminals due to gating currents

We investigated the voltage dependence of membrane capacitance of pituitary nerve terminals in the whole-terminal patch-clamp configuration using a lock-in amplifier. Under conditions where secretion was abolished and voltage-gated channels were blocked or completely inactivated, changes in membrane potential still produced capacitance changes. In terminals with significant sodium currents, the membrane capacitance showed a bell-shaped dependence on membrane potential with a peak at approximately -40 mV as expected for sodium channel gating currents. The voltage-dependent part of the capacitance showed a strong correlation with the amplitude of voltage-gated Na+ currents and was markedly reduced by dibucaine, which blocks sodium channel current and gating charge movement. The frequency dependence of the voltage-dependent capacitance was consistent with sodium channel kinetics. This is the first demonstration of sodium channel gating currents in single pituitary nerve terminals. The gating currents lead to a voltage- and frequency-dependent capacitance, which can be well resolved by measurements with a lock-in amplifier. The properties of the gating currents are in excellent agreement with the properties of ionic Na+ currents of pituitary nerve terminals.     

氟哌啶醇和（R—）—NPA抑制C6神经胶质瘤细胞的钾电流

本工作用全细胞膜片箝方法,观察了氟啶醇（ｈａｌｏｐｅｒｉｄｏｌ以下简称ＨＡＬＯ）和Ｒ（－）－Ｐｒｏｐｙｌｎｏｒａｐｏｍｏｒｐｈｉｎｅ（以下简称ＮＰＡ）对Ｃ６神经胶质瘤细胞（Ｃ６ｇｌｉｏｍａｃｅｌｌｓ）的电压依赖性钾电流的作用,并初步分析了它们的作用机制。结果表明,ＨＡＬＯ和ＮＰＡ都能抑制Ｃ６细胞的Ｋ＾＋Ｊ电流中的慢成分,而对快成分的作用有所不同。它们的抑制作用不是由多巴胺Ｄ２受体介导的,也不是通过     

Cell permeabilization and inhibition of voltage-gated Ca(2+) and Na(+) channel currents by nanosecond pulsed electric field

Previous studies have found that nanosecond pulsed electric field (nsPEF) exposure causes long-term permeabilization of the cell plasma membrane. In this study, we utilized the whole-cell patch-clamp method to study the nsPEF effect on currents of voltage-gated (VG) Ca(2+) and Na(+) channels (I(Ca) and I(Na)) in cultured GH3 and NG108 cells. We found that a single 300 or 600 ns pulse at or above 1.5-2 kV/cm caused prolonged inhibition of I(Ca) and I(Na). Concurrently, nsPEF increased a non-inactivating "leak" current (I(leak)), presumably due to the formation of nanoelectropores or larger pores in the plasma membrane. The nsPEF effects were similar in cells that were exposed intact and subsequently brought into the whole-cell recording configuration, and in cells that were first brought into the whole-cell configuration and then exposed. Although both I(leak) and the inhibition of VG currents were enhanced at higher E-field levels, these two nsPEF effects showed relatively weak correlation with each other. In some cells, I(leak) increased 10-fold or more while VG currents remained unchanged. At longer time intervals after exposure (5-15 min), I(Ca) and I(Na) could remain inhibited although I(leak) had largely recovered. The causal relation of nsPEF inhibitory effects on VG currents and permeabilization of the plasma membrane is discussed.     

Action of tetraethylammonium on calcium-activated potassium channels in pig pancreatic acinar cells studied by patch-clamp single-channel and whole-cell current recording

Summary The effects of tetraethylammonium ions on currents through high-conductance voltage- and Ca²⁺-activated K⁺ channels have been studied with the help of patch-clamp single-channel and whole-cell current recording on pig pancreatic acinar cells. In excised outside-out membrane patches TEA (1 to 2 mM) added to the bath solution virtually abolishes unitary current activity except at very positive membrane potentials when unitary currents corresponding to a markedly reduced conductance are observed. TEA in a lower concentration (0.2 mM) markedly reduces the open-state probability and causes some reduction of the single-channel conductance. In inside-out membrane patches bath application of TEA in concentrations up to 2 mM has no effect on single-channel currents. At a higher concentration (10 mM) slight reductions in single-channel conductance occur. In whole-cell current recording experiments TEA (1 to 2 mM) added to the bath solution completely suppresses the outward currents associated with depolarizing voltage jumps to membrane potentials of 0 mV and blocks the major part (70 to 90%) of the outward currents even at very positive membrane potentials (30 to 40 mV). In contrast TEA (2 mM) added to the cell interior (pipette solution) has no effect on the outward K⁺ current. Our results demonstrate that TEA in low concentrations (1 to 2 mM) acts specifically on the outside of the plasma membrane to block current through the high-conductance Ca²⁺- and voltage-activated K⁺ channels     

Aftercurrent in submucous neurons of guinea pig

Membrane currents elicited by iontophoretic applications of acetylcholine (ACh currents) were recorded from neurons of the guinea pig submucous plexus using a whole-cell patch-clamp recording technique. The ACh currents declined to 5–20% of their peak amplitude during about 2 sec-long application of ACh. After the end of ACh application, a transient increase of the ACh current (the aftercurrent, AC) was observed. The most probable mechanisms responsible for the ACh current decline and for the appearance of the AC are transitions of nicotinic ACh receptors first to a desensitized state, and then to the state with an open ionic channel, respectively.Neirofiziologiya/Neurophysiology, Vol. 25, No. 4, pp. 291–296, July–August, 1993     

Voltage clamp limitations of dual whole-cell gap junction current and voltage recordings. I. Conductance measurements

Previous correction methods for series access resistance errors in the dual whole-cell configuration did not take into account the effect of nonzero resting potentials (E(rest)) and junctional reversal potentials (E(rev)). Dual whole-cell currents were modeled according to resistor-circuit analysis and two correction formulas for the measurement of junctional currents (I(j)) were assessed. The equations for I(j), derived from Kirchoff's law before and after baseline subtraction of the nonjunctional current, were assessed for accuracy under a variety of whole-cell patch-clamp recording conditions. Both equations accurately correct for dual whole-cell voltage-clamp errors provided that the cellular parameters are included in the nonbaseline subtracted I(j) derivations. Junctional conductance (g(j)) estimates are most reliable at high junctional resistance (R(j)) values and minimize the need for corrective methods based on electrode series and cellular input resistances (R(el) and R(in)). In the "open-cell" configuration, low R(j) values relative to R(in) are required for accurate g(j) estimates. These methods provide the basis for accurate quantitative measurements of junctional resistance (or conductance) of gap junction channels or connexin hemichannels in the dual whole-cell or open-cell configurations. Revaluation of V(j)-dependent gating of rat connexin40 g(j) produced nearly identical Boltzmann fits to previously published data. Continuous g(j)-V(j) curves generated by variable slope V(j) ramps provide for more accurate fits and assessment of the time-dependence of the half-inactivation voltage and net gating charge movement.     

Effects of cytosolic Ca2+ on membrane voltage and conductance of cultured mesangial cells from stroke-prone spontaneously hypertensive rats and WKY rats

Mesangial cells (MC) are considered to play an important role in the development of hypertension. The purpose of this study was to characterize the effects of cytosolic Ca2+ on membrane voltage and conductance of MC using stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar Kyoto rats (WKY). We applied the patch-clamp technique in the whole-cell configuration to measure membrane potential (Vm) and ion currents. There was no significant difference in resting Vm values between MC from WKY and SHRSP. The cytosolic Ca2+ increase induced membrane depolarization and the increase of Cl- currents in MC from WKY but not in MC from SHRSP. On the other hand, the Ca2+ increase induced membrane hyperpolarization and the increase of K+ currents in MC from SHRSP but not in MC from WKY. Such differences between MC from two rat strains may play an important role in the alterations in renal hemodynamics observed in hypertension.     

Supercharging: a method for improving patch-clamp performance.

Patch-clamp performance can be improved without altering the normal headstage configuration described by (Hamill, O. P., A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth, 1981, Pfluegers Arch. Eur. J. Physiol., 391:85-100). The "supercharging" method permits resolution of such fast events as calcium and sodium tail currents. Digital computer modeling and analog electronic simulation were used to identify appropriate shapes for the command voltage and the voltage applied to a capacitor tied to the input of the headstage. The voltage command pulse consists of a step with a brief (5-15 microseconds) rectangular spike on its leading edge. Spike amplitude is a function of the membrane capacitance and the access resistance. The spike drives current through the access resistance and speeds charging of the membrane capacitance, making it possible to complete a voltage step within 5-15 microseconds. Clamping speed is independent of the electrode and feedback resistance over a wide range. The second function of the patch clamp amplifier is current measurement, and good time resolution requires suppression of the capacity transient. This can be accomplished by applying an appropriately shaped voltage to the small capacitor tied to the input of the headstage. Series resistance compensation for ionic current transients does not interfere with supercharging. Although the focus of this paper is on whole cell recording, the supercharging concept may prove useful for single channel and bilayer recording techniques.     

不同牵张刺激对豚鼠胃窦环行肌细胞电压依赖性钙电流的影响

利用全细胞膜片钳技术,在胃窦环行肌细胞上观察了不同方式的牵张刺激对电压依赖性钙电流的影响,探讨牵张刺激对胃窦平滑肌细胞电压依赖性钙电流的作用。用低渗性溶液灌流细胞引起的牵张刺激首先增加电压依赖性钙电流,接着激活一种内向性钳制电流。钙电流的增加发生在灌流后１ｍｉｎ内,而内向性钳制电流在细胞明显膨胀之后缓慢激活。低渗和正压引起的细胞膨胀明显增加电压依赖性钙离子电流,而利用两个电极直接牵细胞则不出现钙电     

Membrane chloride conductance and capacitance in Jurkat T lymphocytes during osmotic swelling.

Video microscopy and whole-cell patch-clamp recording were used to monitor changes in relative cell volume (V/Vo), chloride conductance (gCl), and membrane capacitance (Cm) during osmotically induced swelling in Jurkat T lymphocytes. Cellular swelling was initiated with hyperosmotic pipette solutions. Simultaneous evaluation of V/Vo and gCl revealed a 59-s delay between the inception of swelling and the activation of outwardly rectifying, ATP-dependent Cl- channels. Following the delay, increases in V/Vo and gCl progressed in parallel. In contrast, Cm, a measure of cell surface area, fell gradually at a rate of approximately 150 fF/min after whole-cell access was achieved. The decline in Cm lasted 200 s and was followed by a rapid rise (approximately 750 fF/min). The rise in Cm coincided with a variable increase in "leak" current, gCl increased at a slower rate and reached lower peak values in experiments performed without ATP; ATP had no effect on the biphasic Cm time course. The temporal separation of conductance and capacitance during swelling suggests that gCl and Cm vary independently, supporting the hypothesis that a large portion, if not all, of the whole-cell Cl- conductance activated during swelling is provided by volume-sensitive Cl- channels preexisting in the plasma membrane.     

The store-operated calcium entry pathways in human carcinoma A431 cells: functional properties and activation mechanisms

Activation of phospholipase C (PLC)-mediated signaling pathways in nonexcitable cells causes the release of Ca2+ from intracellular Ca2+ stores and activation of Ca2+ influx across the plasma membrane. Two types of Ca2+ channels, highly Ca2+-selective ICRAC and moderately Ca2+-selective ISOC, support store-operated Ca2+ entry process. In previous patch-clamp experiments with a human carcinoma A431 cell line we described store-operated Imin/ICRACL plasma membrane Ca2+ influx channels. In the present paper we use whole-cell and single-channel recordings to further characterize store-operated Ca2+ influx pathways in A431 cells. We discovered that (a) ICRAC and ISOC are present in A431 cells; (b) ICRAC currents are highly selective for divalent cations and fully activate within 150 s after initiation of Ca2+ store depletion; (c) ISOC currents are moderately selective for divalent cations (PBa/PCs = 14.5) and require at least 300 s for full activation; (d) ICRAC and ISOC currents are activated by PLC-coupled receptor agonists; (e) ISOC currents are supported by Imin/ICRACL channels that display 8.5-10 pS conductance for sodium; (f) ICRAC single channel conductance for sodium is estimated at 0.9 pS by the noise analysis; (g) Imin/ICRACL channels are activated in excised patches by an amino-terminal fragment of InsP3R1 (InsP3R1N); and (h) InsP3 binding to InsP3R1N is necessary for activation of Imin/ICRACL channels. Our findings provide novel information about store-operated Ca2+ influx pathways in A431 cells.     

Comparison of potassium currents in human decidua before and after the onset of labor

The human decidua plays a prominent role in the signaling between maternal and fetal tissues. It also secretes a plethora of molecules that modulate uterine function. Ion-channel activity underpins many cellular functions; however, the channels in human decidua have not been characterized in any detail. We have used the whole-cell recording mode of the patch-clamp technique to carry out current-clamp and voltage-clamp recordings of membrane properties and whole-cell potassium (K+) currents of freshly isolated decidual stromal cells. Decidual tissue was obtained from women after spontaneous vaginal delivery (SVD) or elective cesarean section (CS). Cells from both groups generated action potentials, the overshoots and durations of which were dependent on extracellular calcium levels, inhibited by cobalt and enhanced by barium. Potassium current (IK) density was higher in the CS than in the SVD group. Outwardly directed currents were heterogeneous with respect to their activation/inactivation profiles and exhibited outward rectification. The main difference between the SVD and CS group was the presence of a sustained current component in CS cells that is tetraethylammonium chloride-resistant and appears to be unaffected by E-4031. No evidence for the activation of any calcium-activated K+ currents was obtained. We propose that human parturition is associated with subtle changes in K+ channel remodeling, reflecting the transition from uterine quiescence to activation and stimulation. An understanding of the signal transduction events underlying these process may eventually lead to novel approaches to prevent preterm labor via decidual rather than myometrial intervention.     

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets¹. The dopamine signal is terminated by diffusion^2-3, extracellular enzymes⁴, and membrane transporters⁵. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine⁵. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)⁵. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis⁶. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume⁷. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.     

Voltage-dependent effect of tetraethylammonium on the nicotinic acetylcholine receptors of rat superior cervical ganglion neurons

The effect of tetraethylammonium (TEA) on the currents evoked in neurons of the rat superior cervical ganglion by iontophoretic application of acetylcholine (ACh) was studied using a whole-cell patch-clamp recording technique. Tetraethylammonium was used at a concentration of about 20 µM, providing no blocking effect on the ACh-induced membrane currents at a range of positive membrane potentials and reducing these currents recorded at a range of negative membrane potentials by about half. The blocking effect of TEA increased with hyperpolarization within the –50 to –90 mV membrane potential range, and did not depend on the membrane potential level within a range of 0 to –50 mV. The analysis of dose dependence showed that both the voltage-dependent and the voltage-independent blocking effects are due to TEA competitive action on the ganglionic nicotinic acetylcholine receptors (nAChR). The results suggest that the TEA-induced competitive blockade is voltage-dependent.Neirofiziologiya/Neurophysiology, Vol. 27, No. 1, pp. 63–66, January–February, 1995.