what is whole cell voltage clamp recording

Whole-cell voltage clamp recording This unit describes the use of whole-cell voltage clamping to study voltage-gated channels. Stepwise changes in voltage produced by this technique cause channels to interconvert between different states, and these transitions are monitored as changes in membrane current.

Full Answer

What is whole cell clamping used for?

Whole-cell voltage clamp recording This unit describes the use of whole-cell voltage clamping to study voltage-gated channels. Stepwise changes in voltage produced by this technique cause channels to interconvert between different states, and these transitions are monitored as changes in membrane current.

What is a voltage clamp?

The voltage clamp is an experimental method used by electrophysiologists to measure the ion currents through the membranes of excitable cells, such as neurons, while holding the membrane voltage at a set level.

What is whole-cell recording?

Whole-cell recording allows the measurement of the overall electrical properties of a cell membrane and, specifically, either the total current through all the channels on the membrane or the membrane potential. This configuration is achieved from the cell-attached configuration by breaking the membrane patch within the pipette tip.

What current is needed to clamp the voltage in a cell?

The current that is needed to clamp the voltage is opposite in sign and equal in magnitude to the current through the membrane. Alternatively, the cell can be current clamped in whole-cell mode, keeping current constant while observing changes in membrane voltage.

What is whole-cell voltage clamp?

The voltage clamp is an experimental method used by electrophysiologists to measure the ion currents through the membranes of excitable cells, such as neurons, while holding the membrane voltage at a set level.

What does whole-cell patch clamp record?

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the electrical properties of a substantial part of the neuron.

What is whole-cell current clamp?

The current-clamp method detects transmembrane voltage change resulting from ion channel activity. This technique allows the investigator to control the amount of current injected into the cell, thereby controlling the transmembrane potential. Figure 1.

What is a voltage clamp used for?

The voltage clamp is a technique used to control the voltage across the membrane of a small or isopotential area of a nerve cell by an electronic feedback circuit.

What is the difference between patch clamp and voltage clamp?

In the voltage-clamp configuration, a current is injected into the cell via a negative feedback loop to compensate changes in membrane potential. Recording this current allows conclusions about the membrane conductance. The patch-clamp technique allows the investigation of a small set or even single ion channels.

How does patch clamp recording work?

0:444:30The Patch Clamp Method - YouTubeYouTubeStart of suggested clipEnd of suggested clipThis arrangement is the cell attached patch clamp recording method. A record of the current flowingMoreThis arrangement is the cell attached patch clamp recording method. A record of the current flowing through a single ion channel reveals when the channel is in an open or closed State.

What is cell attached recording?

Cell-attached recording, in which a patch electrode is attached to the cell but the membrane is not broken, has been widely used for recording single channel currents, for recording the summed current of many single channels in a patch of membrane, and for recording spontaneous cell firing activity.

What does the current clamp measure?

A current clamp measures electric current flowing through a wire, cable,busbar, or other conductor. The analog output of the clamp can be read by a voltmeter, oscilloscope, power analyzer or DAQ. It allows you to measure the current in a conductor without having to alter or disconnect the cable.

What is the advantage of current clamp?

The advantage of recording in current clamp is that the membrane potential remains unaffected by the recording technique and requires fewer adjustments during the experiment. In contrast, using voltage-clamp mode, the command potential would have to be adjusted frequently due to the changed parameters.

How does a whole cell voltage clamp work?

During whole-cell voltage clamp the interior of the cell is exchanged by diffusion with the solution contained within the recording electrode. If channels are regulated intracellularly their activity may decrease or „wash out” as second messengers (e.g., cAMP) or cofactors (GTP, ATP, etc.) are lost from the cell's interior. For example, intracellular regulation of calcium (Ca 2+) channels has been demonstrated using this technique. Ca 2+ currents wash out, but this can be reversed by including ATP and the catalytic subunit of cAMP-dependent protein kinase in the dialysis solution ( 10 ). Furthermore, nucleotides, activated kinases, and protease inhibitors (e.g., calpastatin) may be required to sustain channel activity in membrane patches ( 11 ). This suggests that some Ca 2+ channels might be directly phosphorylated before they can demonstrate full activity. However, the washout or rundown of channel activity is likely to be much more complex than the previous statements imply. In some cases, a transitory „runup” of channel activity has been observed ( 12 ). Furthermore, a number of processes which may be completely independent of dephosphorylation can contribute to channel rundown ( 13–15 ). In particular, whole-cell recordings may fail to maintain low and stable concentrations of intracellular calcium concentration ( [Ca 2+] i) even though high concentrations of buffers are employed. Elevated intracellular Ca 2+ may then contribute to Ca 2+ channel inactivation and/or trigger a number of calcium-dependent process.

What is whole cell recording?

Whole cell recording can be used to monitor the activity of macroscopic currents (or in current clamp, to monitor changes in voltage) and it is currently the most commonly used configuration of the patch-clamp recording method.

How does patch clamp recording improve temporal resolution?

They applied gentle suction inside the recording pipette when it was pressed against the cell membrane (see Fig. 13.12 ). This action increased the seal resistance between pipette and cell membrane substantially (from 10 8 Ω to 10 10 Ω). The higher seal resistance improved the signal-to-noise ratio and also made it possible to apply voltage steps to the inside of the patch electrode, without the need to voltage-clamp the entire cell with microelectrodes. In addition to achieving seal resistances of >1 GΩ, they also decreased stray capacitance from the electrode wall by coating the outside of the recording pipette with Sylgard® (silicon elastomer).

What is the purpose of the patch-clamp method?

Figure 13.12. Different recording conformations using the patch-clamp method. The patch-clamp recording method, originally used to monitor single channel currents in cell-attached patches, is highly versatile ( Hamill et al., 1981; Sigworth and Neher, 1980; Sigworth, 1986 ). As described in detail by Hamill et al. (1981), the first step in all recording configurations is the formation of a gigaohm seal. This high resistance seal greatly limits current flow between the inside of the patch pipette and the bath. In cell-attached patch recordings, currents through single ion channels can be resolved (see Figs. 13.14, 13.15) or, depending on the size of the patch electrode and the density of ion channels in the membrane, currents through a large number of ion channels (macropatch). The gigaohm seal between the patch pipette and the cell membrane is electrically as well as mechanically stable. The patch pipette can be withdrawn and, if the gigaohm seal remains intact, as it usually does, an inside-out patch is formed. In the inside-out patch configuration the inside of the patch of membrane is accessible and exposed to the bath (see Fig. 13.12 ). Alternatively, after forming a gigaohm seal, negative pressure (suction) can be applied to the inside of the patch electrode; the membrane will rupture to give electrical (and physical) access to the inside of the cell. Currents can be monitored at constant voltage from the whole cell (see Fig. 13.12 ). Whole cell recording can be used to monitor the activity of macroscopic currents (or in current clamp, to monitor changes in voltage) and it is currently the most commonly used configuration of the patch-clamp recording method. Finally, from the whole-cell configuration, withdrawing the patch electrode can often result in the formation of an isolated outside-out patch. In this configuration, the outside of the patch of membrane faces the bath and solutions such as agonists can be applied to activate and measure single ligand-gated ion channel currents ( Hamill et al., 1981 ).

What is the recording configuration of a whole cell?

13.12 ). In this configuration, the single patch electrode is used to control the membrane voltage (under ideal conditions) and to measure the aggregate membrane currents of the whole cell. After forming a gigaohm seal, the patch of membrane is ruptured by suction in the pipette giving physical as well as electrical access to the inside of the cell. As the membrane is ruptured, there is a large increase in the capacitance reflecting the addition of the whole cell membrane capacitance to the circuit. For cells of ~10–15 μm in diameter the membrane capacitance is ~5–10 pF ( Hamill et al., 1981; Fenwick et al., 1982a; Fenwick et al., 1982b ). A series of whole cell Ca V currents evoked by a series of step-depolarizations in 10 mV increments are shown in Fig. 13.8A. Ca V currents activate and inactivate with faster kinetics as the size of the voltage step increases.

What is the rundown of glutamate receptor channel activity?

The rundown of glutamate receptor-channel activity (and other channels for that matter) either in whole-cell recordings or in recordings from patches likely results from a wide variety of cellular- , membrane- , and receptor-dependent changes. Knowledge about these alterations can potentially provide information about pathological processes in the cells. However, it should be remembered that in most cases the objective of such experiments is not to fully characterize washout but rather to determine the mechanisms that are functionally important for regulating glutamate receptors.

What is a patch clamp recording?

Whole-cell patch clamp recording is a powerful technique for interrogating the cellular response to stimulation of inputs. This approach, combined with pharmacological manipulations, can be used to infer the relative contribution of inhibitory, excitatory, modulatory, and peptidergic inputs. However, individual neurons commonly receive inputs from a wide variety of brain regions. Stimulation of these inputs with metal or glass electrodes can resolve individual input pathways only under the exceptional circumstance in which these inputs are physically segregated (eg, Schafer collateral versus perforant path inputs to the CA1 region of the hippocampus). In the vast majority of brain regions, inputs are intermingled. Furthermore, recognition of transmitter co-release and novel, molecularly defined parallel pathways ( Graves et al., 2012; Tritsch, Ding, & Sabatini, 2012; Varga et al., 2009) highlight the importance of molecular isolation of inputs for stimulation. The combination of optogenetic manipulations with patch clamp recordings from brain slice preparations has greatly advanced the possibilities of analyzing pathway-specific synaptic properties and connectivity ( Hjelmstad, Xia, Margolis, & Fields, 2013; Lammel et al., 2012; Matsui, Jarvie, Robinson, Hentges, & Williams, 2014; Stuber et al., 2011 ). With further advances it should soon be possible to express opsins with sufficiently different activation parameters in different brain regions so that multiple different inputs onto an individual cell can be independently activated with great molecular specificity. This type of approach can also be used in vivo while making extracellular or even whole cell recordings from awake behaving animals so the detailed electrophysiological consequences of activating specific inputs in vivo can be elucidated.

What is the function of the voltage clamp in excitable cells?

The voltage clamp allows the membrane voltage to be manipulated independently of the ionic currents, allowing the current–voltage relationships of membrane channels to be studied.

What is a voltage clamp?

The voltage clamp is an experimental method used by electrophysiologists to measure the ion currents through the membranes of excitable cells, such as neurons, while holding the membrane voltage at a set level. A basic voltage clamp will iteratively measure the membrane potential, and then change the membrane potential (voltage) ...

What is a TEVC?

The two-electrode voltage clamp (TEVC) technique is used to study properties of membrane proteins, especially ion channels. Researchers use this method most commonly to investigate membrane structures expressed in Xenopus oocytes. The large size of these oocytes allows for easy handling and manipulability.

How did Cole develop the voltage clamp technique?

Cole developed the voltage clamp technique before the era of microelectrodes, so his two electrodes consisted of fine wires twisted around an insulating rod.

How does a voltage clamp work?

A basic voltage clamp will iteratively measure the membrane potential, and then change the membrane potential (voltage) to a desired value by adding the necessary current. This "clamps" the cell membrane at a desired constant voltage, allowing the voltage clamp to record what currents are delivered.

How is transmembrane voltage recorded?

Transmembrane voltage is recorded through a "voltage electrode", relative to ground, and a "current electrode" passes current into the cell. The experimenter sets a "holding voltage", or "command potential", and the voltage clamp uses negative feedback to maintain the cell at this voltage.

Why do we use current readings?

Current readings can be used to analyze the electrical response of the cell to different applications. This technique is favored over single-microelectrode clamp or other voltage clamp techniques when conditions call for resolving large currents.

Why is the voltage clamp method used?

The voltage clamp method allows researchers to study voltage-gated ion channels by controlling the membrane potential of a neuron.

What is the purpose of a voltage clamp experiment?

To conduct a voltage clamp experiment, a portion of the axon, which would include the cell membrane and all the voltage-gated ion channels located there, is removed from a neuron and placed into a solution that mimics that of physiological extracellular solution. The ion concentrations across the membrane, as well as the electrochemical gradients, would remain the same.

What does voltage clamping do to the membrane potential?

This influx of positive ions would normally cause change the membrane potential to depolarize, but the voltage clamp equipment will measure the ion flow and inject a current of equal strength and opposite charge into the axon to maintain the membrane potential at 0 mV.

Why do sodium channels open during a voltage clamp?

Since the ion channels function as expected during the voltage clamp experiment, the voltage-gated sodium channels will inactivate , and the delayed voltage-gated potassium channels will open because, like the sodium channels, they are also activated when the membrane potential reaches threshold. This causes the ion flow to change from inward to outward. Normally, potassium efllux would cause a repolarization of the membrane potential, but the voltage clamp equipment will again inject a current that is equal in strength and opposite in charge to the potassium flow to keep the membrane potential steady at 0 mV.

How to move an axon from resting membrane potential to 0 mV?

To make the axon move from its resting membrane potential to 0 mV, the current electrode will pass positive current into the cell, depolarizing the cell until the membrane potential reaches the set value.

What is the first step in the voltage clamp method?

The initial step in the voltage clamp method is to measure the membrane potential of the axon. A recording electrode is placed into the axon, and a reference electrode is placed into the extracellular solution. The voltage difference between these two electrodes is the membrane potential of the axon.

Why does the voltage clamp work?

The voltage clamp equipment will inject current equal in strength and opposite in charge to the sodium influx in order to keep the membrane potential of the axon at 0 mV. The membrane potential will remain at 0 mV because the injected current offsets any change that would normally occur due to ion flow.

What is the advantage of cell-attached current-clamp over whole-cell recording?

An advantage of cell-attached current-clamp over whole-cell recording is that it accurately depicts whether a synaptic potential is hyperpolarizing or depolarizing without the risk of changing its polarity.

What is cell attached recording?

Cell-attached recording provides a way to record the activity of - and to stimulate - neurons in brain slices without rupturing the cell membrane. This review uses theory and experimental data to address the proper application of this technique and the correct interpretation of the data. Voltage-clamp mode is best-suited for recording cell firing activity, and current-clamp mode is best-suited for recording resting membrane potential and synaptic potentials. The magnitude of the seal resistance determines what types of experiments can be accomplished with a cell-attached recording: a loose seal is adequate for recording action potential currents, and a tight seal is required for evoking action potentials in the attached cell and for recording resting and synaptic potentials. When recording action potential currents, if the researcher does not want to change the firing activity of the cell, then it is important that no current passes from the amplifier through the patch resistance. In order to accomplish this condition, the recording pipette should be held at the potential that gives a holding current of 0. An advantage of cell-attached current-clamp over whole-cell recording is that it accurately depicts whether a synaptic potential is hyperpolarizing or depolarizing without the risk of changing its polarity.

What is a whole cell voltage clamp?

Voltage-clamp protocols (see Box 1 for a glossary) used with recombinant sodium channels expressed in neurons are generally similar to those used when recording from channels expressed in heterologous cell lines, but because native neurons in culture tend to grow neurites, special attention must be paid to space clamp. Although the following sections will provide advice on aspects of patch-clamp studies, a more comprehensive discussion of this powerful technique can be found in other publications 11, 12, 13.

Why is it important to have a good current-clamp recording?

It is vital that high-quality cultures are maintained at all times, to allow sufficient healthy cells to survive the transfection process. It helps if the personnel involved routinely performs these tasks and with that, the processing time can be reduced to an absolute minimum. Using well-tested materials is also important, in particular the medium used for maintaining the cells in culture; media and sera can vary across batches; hence, it is important to validate and purchase sufficient quantities to complete a given study. Full testing of new medium stocks has also proved to be helpful, even to the extent of completing some electrophysiological recording to examine normal and expected expression of specific ion channels and also to show successful current-clamp recordings with the new reagents.

What are voltage-gated ion channels?

Voltage-gated ion channels underlie action potential initiation and propagation in eukaryotic excitable membranes. Thus, determination of biophysical properties of individual channel isoforms can lead to a better understanding of the contribution of these channels to electrogenesis in the cells that house these channels. However, published studies have provided compelling examples of cell-type-specific effects on voltage-gated ion channels such as sodium channels, e.g., the Na v 1.6 sodium channel has been shown to be the main source of a resurgent current 1 in response to repolarization after depolarization to positive potentials in cerebellar Purkinje cells, but this current is not produced by Na v 1.6 in hippocampal CA3 neurons 2. Similarly, resurgent sodium current has been recorded from large- but not small-diameter neurons of dorsal root ganglion (DRG), even though both groups of cells express Na v 1.6 (ref. 3 ). Na v 1.8 (refs. 4, 5, 6) exhibits different slow inactivation properties in two distinct groups (IB4 + and IB4 −) of DRG neurons, and these different properties endow these two groups of neurons with distinct firing properties 7. Importantly, sensory and autonomic symptoms of an autosomal-dominant painful sodium channelopathy, which is caused by mutations in Na v 1.7, inherited erythromelalgia 8, appear to be explained by the differential response of sensory (DRG) and sympathetic ganglion neurons, e.g., superior cervical ganglion (SCG), to the expression of the same mutant Na v 1.7 sodium channel 9. These examples underscore the need to study the functional properties of sodium channels within the cells in which they are normally expressed.

What are the pitfalls of recording ion channel activity?

Artifacts and pitfalls. A source of concern when recording any type of ion channel activity is that the gating properties can change during the recording period. Time-dependent changes in current properties can occur for a number of reasons, including dilution of specific modulators due to diffusion of the recording pipette or the activation of second messenger pathways by components of the pipette solution. Some of these changes can be minimized by careful design of pipette solutions (e.g., GTP-γS can help stabilize some ionic currents) or controlled for by running time-dependent controls and starting specific protocols at consistent times after establishing the whole-cell recording configuration.

What is Na v1.7?

Na v 1.7 is normally expressed in both sensory DRG neurons and sympathetic SCG neurons (Table 1s in Dib-Hajj et al. 10) and it is important to examine the effects of the mutated channel on firing behavior of both types of neuron . Voltage-clamp experiments indicate that the mutation shifts the voltage dependence of activation in a hyperpolarized direction and enhances the current in response to small, slow depolarization (ramp current) 35. Moreover, using current-clamp recordings it is possible to show that the mutant channel has opposing effects on excitability of the two types of neurons. These recordings show that the mutant channel produced a 5-mV depolarization in RMP in both cell types. However, the mutant channel produces hyperexcitability (decreased current threshold for single action potentials; increased firing frequency in response to suprathreshold stimulation) in DRG neurons ( Fig. 2a–c) and hypoexcitability ( Fig. 2d–f) (increased current threshold for single action potentials; decreased firing frequency in response to suprathreshold stimuli) in SCG neurons. This is due, at least in part, to the selective presence of the sensory neuron-specific Na v 1.8 sodium channel, which is relatively resistant to inactivation by depolarization 4, 6, 36, in DRG neurons, and its absence in SCG neurons 9.

How long does it take to record DRG neurons firing?

Current-clamp recording from DRG neurons has typically been carried out on the same day of isolation. Studying native DRG neuron firing by current clamp after 24 h in culture is more challenging than acutely isolated neurons, although both TTX-S and TTX-R currents can be studied by voltage-clamp methods in these cultures (see above). Studying action potential firing in transfected neurons can only be undertaken after >24 h in culture to allow adequate expression of recombinant channels. We found that supplementing the culture media with nerve growth factor (NGF) and glial cell-line-derived neurotrophic factor (GDNF) (see Experimental Design) was necessary to reproducibly record action potentials in transfected DRG neurons, which is consistent with the published data that NGF and GDNF are necessary to maintain the expression of Na v 1.8 and Na v 1.9 channels in cultured DRG neurons 16, 17, and NGF also regulates the expression of Na v 1.7 channel 32, 33.

How are passive currents subtracted from measured currents?

Passive current components (linear leak currents and capacitive currents) are subtracted from the measured currents by recording a series of scaled voltage pulses just before, or after, the voltage pulse used to elicit voltage-gated sodium currents. These scaled pulses are typically one-fourth or one-fifth the amplitudes of the experimental pulses, and the subtraction process is referred to as P/4 or P/5 leak subtraction. Care must be taken in the design of the leak subtraction protocol. Initiation of the scaled subtraction pulses from a relatively negative holding potential and the use of a negative P/4 or P/5 protocol can ensure that the leak subtraction pulses do not inadvertently elicit voltage-dependent currents. It is important to include sufficient time between the leak pulses and the experimental pulse to ensure that the leak subtraction protocol is not inadvertently altering the sodium channel properties (e.g., by changing the proportion of channels that are fast- or slow-inactivated).

What is patch clamp recording?

A patch clamp recording of current reveals transitions between two conductance states of a single ion channel: closed (at top) and open (at bottom).

What is a patch clamp?

During a patch clamp recording, a hollow glass tube known as a micropipette or patch pipette filled with an electrolyte solution and a recording electrode connected to an amplifier is brought into contact with the membrane of an isolated cell. Another electrode is placed in a bath surrounding the cell or tissue as a reference ground electrode. An electrical circuit can be formed between the recording and reference electrode with the cell of interest in between.

What is an electrical circuit?

An electrical circuit can be formed between the recording and reference electrode with the cell of interest in between. Schematic depiction of a pipette puller device used to prepare micropipettes for patch clamp and other recordings. Circuit formed during whole-cell or perforated patch clamp.

How is patch clamping performed?

Patch clamping can be performed using the voltage clamp technique. In this case, the voltage across the cell membrane is controlled by the experimenter and the resulting currents are recorded. Alternatively, the current clamp technique can be used. In this case, the current passing across the membrane is controlled by the experimenter and the resulting changes in voltage are recorded, generally in the form of action potentials .

How are ligand-gated ion channels modulated?

For ligand-gated ion channels or channels that are modulated by metabotropic receptors, the neurotransmitter or drug being studied is usually included in the pipette solution, where it can interact with what used to be the external surface of the membrane. The resulting channel activity can be attributed to the drug being used, although it is usually not possible to then change the drug concentration inside the pipette. The technique is thus limited to one point in a dose response curve per patch. Therefore, the dose response is accomplished using several cells and patches. However, voltage-gated ion channels can be clamped successively at different membrane potentials in a single patch. This results in channel activation as a function of voltage, and a complete I-V (current-voltage) curve can be established in only one patch. Another potential drawback of this technique is that, just as the intracellular pathways of the cell are not disturbed, they cannot be directly modified either.

Why are patch clamps used?

Automated patch clamp systems have been developed in order to collect large amounts of data inexpensively in a shorter period of time. Such systems typically include a single-use microfluidic device, either an injection molded or a polydimethylsiloxane (PDMS) cast chip, to capture a cell or cells, and an integrated electrode.

Who invented the patch clamp?

Erwin Neher and Bert Sakmann developed the patch clamp in the late 1970s and early 1980s. This discovery made it possible to record the currents of single ion channel molecules for the first time, which improved understanding of the involvement of channels in fundamental cell processes such as action potentials and nerve activity. Neher and Sakmann received the Nobel Prize in Physiology or Medicine in 1991 for this work.

Overview

Variations of the voltage clamp technique

History

Technique

Mathematical modeling

Further reading