What became immediately apparent was that in order to record the currents through individual channels, it was necessary to delimit a small area of the membrane in which there would only be one or a limited number of active channels whose individual currents could be identified. Recordings of discrete, step-like ‘single-channel currents’ of a few picoamps, supposedly passing through the postulated membrane channels, were not possible at the time in living cells because of the excessively high background noise (estimated to be at least one order of magnitude higher than the single-channel currents measured in membrane bilayers or estimated through noise analyses).

Meanwhile, experiments were conducted to record elementary currents through single channels using purified proteins in artificial lipid membranes. These experiments focused on ACh receptors and Na⁺ channels. Unfortunately, these efforts also failed because the proteins used were not fully functional. However, they provided valuable insights when studying gramicidin A. This small peptide is a perfect model for these early studies because it spontaneously forms a transmembrane channel by dimerizing in the bilayer. Experiments with gramicidin A revealed discrete, random current steps, proving that ions pass through gated pores rather than flowing continuously through the membrane matrix (Bamberg & Läuger 1973; Hladky & Haydon 1973).

This crucial scientific objective of recording single-channel currents apparently fraught with insurmountable problems found a solution when it crossed the paths of two young German scientists, Erwin Neher and Bert Sakmann. Erwin Neher had returned to Germany in 1967 after a year on a Fulbright studentship at the University of Wisconsin, where he studied spectroscopy of macromolecules, and was now looking around for some PhD project in biophysics, possibly related to nerve excitation. This search brought him to Prof Dieter Lux’s laboratory at the Max-Planck-Institut für Psychiatrie in Munich to study synaptic mechanisms in snail motor neurons. To avoid space clamp problems, Lux recommended that his young postdoc use suction pipettes to record currents. This suggestion would be recalled a few years later when trying to record elementary currents through single-channel.

While at Prof Lux’s laboratory, Neher met Bert Sakmann who had come to learn the basics of voltage clamping synaptic currents in snail neurons before moving to University College London to work in Bernard Katz’s biophysics laboratory on that topic. Sakmann was eager to understand the fundamental mechanisms of the synapse that Neher was studying because he believed they were essential to comprehending the central nervous system’s excitability. For this reason, they had many lively discussions. Following his interests, Sakmann spent three years (1971–1973) at University College in London. There, he began to think that single-channel currents could be recorded directly from cell membranes while working in Bernard Katz’s department. It was in that period that Katz, in collaboration with Ricardo Miledi, suggested that elementary current events through individual ACh receptor channels caused the noise observed in the membrane potential recordings when ACh was applied to the end-plates of frog skeletal muscle (Katz & Miledi 1972).

Neher and Sakmann met again at the Max-Planck-Institut für Biophysikalische Chemie in Göttingen in 1973. Neher had joined the Institute to gain expertise in single-channel recording in artificial lipid bilayers, and Sakmann had returned from London and was offered by Prof Otto Creutzfeldt to run his independent laboratory. Given their clear communion of interests, they soon agreed on collaborating to measure single-channel currents.

Prologue

Neher and Sakmann knew that to record currents through individual channels, they had to electrically isolate a small area of the membrane containing a limited number of active channels. They tried to demarcate these areas by pressing fire-polished glass pipettes with an aperture size of 1–2 μm onto the membrane surface. Denervated muscle fibers were used for the experiments, which Sakmann was very familiar with from his work in Katz’s laboratory in London, particularly the enzymatic procedure for cleaning the cell surface. Despite repeated trials under different conditions, they could only achieve seal resistances of 50–100 MΩ. Although these seal resistances were modest, they were sufficient to convey a significant fraction of the current coming through the isolated patch into the recording pipette and to lower the background noise generated by ions passing through the seal resistance to an acceptable level.

These aspects can be appreciated by looking at the resistances involved in the establishment of the mechano-electric seal between the membrane and the recording pipette (Fig. 1A). Of the various sources of electrical noise in the patch clamp recordings, the one resulting from the seal resistance is the most important. This noise, known as thermal noise, results from random motion of thermally excited charges through a generic resistance. It is quantified by the following general relation,

where σn, the root-mean-square deviation of the current (the background noise) through a resistor, which depends inversely on its resistance R (and directly on the bandwidth, Δf, at which the measurement is done). Here, k and T are the Boltzmann constant and the absolute temperature. This relationship has general validity and works for any type of charge moving through a resistive pathway, including the seal resistance (leakage path) between the glass pipette and the membrane.

Fig. 1

Arrangement of patch pipette and cell membrane and the equivalent electrical circuit. A The cartoon illustrates the relationship between the patch pipette and the cell membrane, focusing on the critical role of seal resistance in determining the fraction of current that passes through the patch pipette to the current–voltage amplifier (Ipip). This current, which represents the signal of the recording, is what remains of the current passing through the active channel(s) in the membrane patch (Ipatch), after leakage through the seal resistance (Iseal). Icell is the current through the cell membrane other than the patch. This current is generally negligible because the voltage drop commanded experimentally falls virtually entirely on the patch membrane due to its much higher resistance compared to the rest of the cell membrane. B Equivalent electrical circuit of the arrangement between the patch pipette and the cell membrane shown in A). Note the crucial node where the current through the patch is divided between Rpip and Rseal in reverse proportion to their sizes. In other words, the bigger the seal resistance, Rseal, compared to the pipette resistance, Rpip, the bigger the current that will flow through the patch pipette and the stronger the signal it will bring to the current–voltage amplifier. [Modified from (Ogden & Stanfield 1994)]

In this regard, it is important to recall that current noise is generated by all the resistances encountered by the current from the ground to the amplifier input. These resistances include the cell membrane resistance, the patch resistance, the seal resistance, and the electrode access resistance (Rcell, Rpatch, Rseal and Rpip in Fig. 1B). However, since the seal noise appears to be, with regard to this discussion, the most important component of the overall noise of the recording system, we will briefly continue to dwell on it with a quantitative example.

To estimate the current noise that would originate from the seal resistance and determine how this would impact on single-channel current recording, it is convenient to use Eq. 1 above. Given that 4kT equals 1.6 × 10−20 Joules and that typical recordings are done at a filter setting of 1 kHz, a seal resistance of 100 MΩ – which was common during the initial attempts to record single-channel currents – will generate background noise of 0.40 pA (rms). However, some additional noise arising from the seal should also be considered, due to the frequency-dependent noise introduced by the capacitance of the glass pipette walls. This combined level of noise appeared rather high to record clear current events in the order of a few picoamps, as estimated by noise analyses (Almers & Levinson 1975; Levinson & Meves 1975) or by doping artificial bilayer membranes with antibiotics or proteins (Bean et al. 1969; Hladky & Haydon 1970).

First Single-channel Recording

In 1976 Neher and Sakmann reported the first recordings of discrete, step-like currents from a membrane patch of a denervated muscle fiber perfused with ACh (Fig. 2A) (Neher & Sakmann 1976). These were immediately interpreted as single-channel currents through the ACh receptor. Thus, they were considered conclusive evidence of the presence of ion channels in native membranes. In addition to the many lines of indirect evidence accumulated over the past decade and the recently proposed membrane model of (Singer & Nicolson 1972), which included integral proteins spanning the full membrane, these step-like currents occurring randomly with random current event lengths were perfectly congruent with the notion of ion channels that open and close in an all-or-none, stochastic manner.

Fig. 2

First single-channel recording from frog muscle. A Oscilloscope recording of single-channel current through ACh receptors present in the membrane patch from a denervated frog muscle fiber. The receptors were activated by 200 nM suberyldicholine, a more effective ACh analogue, which was placed in the patch pipette. Membrane potential was set at − 120 mV. C and O denote the closed and open levels, respectively. Note few brief double openings (not indicated). B Recording setup: It consisted of the denervated muscle fiber and the patch pipette (P), as well as the circuit diagrams for amplifying and recording the single-channel currents (VG) and the classic two-microelectrode voltage clamp system (VC), which controls the membrane patch potential. The patch pipette had an opening of 3–5 µm and a resistance of 2–5 MΩ. [Reproduced from (Neher & Sakmann 1976)]

These recordings, including those much better resolved, obtained with the gigaseal improvement (see below) “conclusively proved the existence and function of ion channels”, as stated by the Stockholm Committee when awarding the 1991 Nobel Prize in Physiology or Medicine to Neher and Sakmann for their studies. After recording single-channel currents, the general sentiment was that now channels could be seen to switch back and forth, from closed to open, instead of being assumed, as before the single-channel recordings.

However, these recordings showed substantial noise, primarily due to the poor seal between the glass pipette and the muscle membrane, which resulted in current dispersion through it. This situation, which continued as such until the end of the 1970s, did not allow for rigorous characterization of the channels’ biophysical properties. However, it immediately demonstrated the technique’s extraordinary potential in enabling real-time observation of conformational transitions in single channel in their native environments when they open or close in response to specific stimuli, such as changes in membrane potential, specific agonists, or mechanical stimuli.

It should be added that in those years that Neher and Sakmann were refining the technique to record elementary currents from muscle fibers, Neher spent good parts of 1975 and 1976 working as a postdoc in the laboratory of Charles (Chuck) Stevens at Yale. There, under his watch, Neher conducted experiments similar to those done in Göttingen, obtaining similar results. In fact, the first single-channel recording may even have been made at Yale. Stevens was certainly closely involved with this new technique if he was asked to coauthor the 1976 Nature paper (Zador et al. 2023). In any case, Neher did acknowledge the work done at Yale, particularly the single-channel recordings conducted there, which he regarded as a crucial reassuring proof, having been made in a different laboratory with a completely different set-up.

Gigaseal Formation: A Breakthrough in Patch Clamping

The excessive noise of the first single-channel recordings had to be reduced drastically if meaningful studies on ion channels were to be carried out. Neher and Sakmann knowingly focused on the seal resistance. They tried to improve cell surface cleaning to achieve a better seal and to coat the pipette tip to reduce capacitance, which is another source of noise. They also tried different types of pipette glass with different charges, but none of these changes resulted in major improvement. In the meantime, they had assembled an excellent team of scientists in Göttingen, including Owen Hamill, Alan Marty, and Fred Sigworth. This team turned out to be extremely creative, providing innovative ideas for technical improvements to the method, offering theoretical insights into how channels work, and developing sophisticated analyses of single-channel recordings.

Disappointed by continued failures, Neher, Sakmann, and the others were on the verge of abandoning any further attempts to reduce seal resistance when they noticed that high seal resistances in the range of 10 to 100 gigaohms (GΩ) would easily form by applying mild suction with a pipette (Baker 1981). These seals, in the gigaohm range, later called gigaseals to distinguish them from the earlier megaohm seals, had the crucial advantage of reducing the background noise of the recording by more than an order of magnitude, enabling single-channel currents in the picoamp range to be resolved and the membrane patch to be voltage clamped without the use of additional microelectrodes (Sigworth & Neher 1980).

The marked decrease in background noise made the single-channel current recordings much better resolved, as shown in Fig. 3A from the comparison before and after the formation of the gigaseal. The upper trace shows the time course of the development of a gigaseal in an enzymatically treated frog muscle fiber, that brings the seal resistance of about 150 MΩ when the tip is only pressed on the muscle surface to about 60 GΩ attained upon applying the negative pressure (between arrows). The reduction of background noise can be clearly seen by comparing the baseline thickness (when no channel is active). The lower recordings show single-channel currents on an expanded timescale and at higher resolution before (left) and after (right) formation of the gigaseal, as well as the marked reduction of background noise.

Fig. 3

Gigaseal formation between the pipette tip and the cell membrane. A Upper trace: Continuous single-channel recording before and after application of mild suction through the patch pipette. The elementary current events are arguably from ACh receptors’ activation (note double openings at the beginning of the recording) since the patch pipette contained suberyldicholine (100 nM). At the beginning (before the first arrow), the seal resistance between the patch pipette and the membrane, attained by only pressing the pipette against the membrane, was about 150 MΩ. Following slight suction through the patch pipette (applied between the two arrows), a gigaseal of about 60 MΩ resistance formed, and background noise decreased markedly in parallel. The clear decrease in channel activity following the gigaseal formation was ascribed to transient depletion of the agonist at the pipette tip as a result of suction. Lower traces: Single-channel currents at higher magnification before (left) and after (right) the formation of the gigaseal. All recordings were carried out at the resting potential of the cell. Filter setting was 1 kHz for the upper trace and 3 kHz for the lower traces. [From (Hamill et al. 1981)]. B) Schematic electrical circuit of the patch clamp and the headstage current/voltage amplifier showing the major resistances involved. Since the pipette current, which originates from the patched membrane and consists of what remains after leakage through the seal resistance, passes through the feedback resistance of the inverting amplifier, Rf, using large values of Rf minimizes the thermal current noise. This is important for resolving minute currents during single-channel recordings. [Modified from (Sigworth & Neher 1980)]

A clear benefit of high seal resistance is that more of the membrane current flows into the pipette instead of passing through the glass/membrane seal. As a result, the signal-to-noise ratio increases (cf Fig. 3B). However, a more important aspect of high seal resistance is the parallel reduction of the background noise originating from the thermal motion of ions through the seal resistance (cf. Equation 1). For example, with gigaseals of 10 GΩ, which are routinely attained today, we can estimate a background noise of σn = 0.04 pA (rms) from Eq. 1. Compared to the initial experiments, when the seal resistance achieved was only about 100 MΩ and σn = 0.4 pA (rms; still estimated from Eq. 1), there is a tenfold reduction of background noise.

The Physico-chemical Basis of Gigaseal Formation

The physical and chemical aspects of how gigaseals form remain unclear, despite the extensive research conducted in this regard. However, these studies have identified important factors and conditions that favor successful gigaseal formation. Van der Waals forces are thought to be the main forces involved in sealing the interior patch pipette and the membrane, consistent with their chemically unspecific nature and the observation that gigaseals form with different cell types as well as with pure lipid membranes (Parsegian 2006). The formation of the gigaseal has been suggested to result from van der Waals forces squeezing out the interposed water layer between the membrane and pipette glass. This occurs when the seals are in the hundreds of megaohm range and the membrane and glass are 20–50 Å apart (Nir & Bentz 1978; Parsegian et al. 1979). When the seal resistance approaches values in the order of 10 MΩ, the estimated distance between the patch membrane and the pipette glass is only a few angstroms. Evidence supporting the close contact between the membrane and the glass without interposed water includes the observation that small molecules cannot cross the gigaseal. For example, ACh fails to activate ACh receptors in the patch when placed in the bathing solution of a gigasealed patch membrane.

The Classic Configurations of the Patch Clamp and Their Use

The formation of the gigaseal not only lowers greatly the noise on the recordings, it also renders the pipette/patch interaction mechanically very strong and amenable to various sorts of manipulations that allow one to attain different patch configurations, each very versatile and useful for addressing specific scientific questions.

The ‘Cell-attached’ Configuration

The formation of the gigaseal obtained by applying a moderate negative pressure inside the patch pipette once it has been lowered to touch the cell membrane establishes the initial configuration referred to as ‘cell-attached’ for obvious reasons (Fig. 4, indicated). It is also the precursor configuration to all other variants of the patch clamp technique. This configuration is very stable and allows measurement of the unitary currents through the channel(s) located on the dome of the membrane patch sucked inside the pipette. It is possible to record only the unitary current through the channel(s) in the patch when there is a second membrane, the remaining cell membrane, between the two electrodes because this second membrane’s resistance is extremely low compared to the patch resistance and can be safely ignored. However, this configuration is greatly limited because it is not possible to change the solutions (i.e., the type as well as the concentrations of ions or other compounds) on either side of the patch membrane. Moreover, given the much higher resistance of the patch membrane compared to the rest of the cell membrane, with this configuration it is not possible to control the membrane potential of the cell (but only that of the membrane patch).

Fig. 4

Schematic illustration of the four different configurations of patch clamp (indicated) and how they are obtained. See text for description. [From (Hamill et al. 1981)]

The ‘Inside-out’ Configuration

Both these limitations are bypassed by quickly withdrawing the patch pipette from the cell after obtaining a cell-attached configuration. This maneuvre causes the membrane patch inside the tip of the patch pipette to be torn from the cell while maintaining a gigaohm seal with the pipette and mechanical stability. This configuration is referred to as ‘inside-out’ because the inside of the patch is now exposed to the bath solution. Noteworthy, this configuration allows the solution perfusing the cytoplasmic (inside) face of the patch membrane to be changed according to experimentation needs (Fig. 4, indicated). The inside-out configuration is appropriate for studying modulation of ion channels by agents or compounds that interact with the cytoplasmic side of the channel, such as modulation of Ca2+-activated K+ channels by Ca2+ ions. Furthermore, this configuration, in which there is only the patch membrane between the two electrodes, allows the control of the membrane potential to be accurate. Note that this configuration, on the other hand, is of no help in studying channel activation by neurotransmitters or, in general, any kind of modulation that occurs through binding sites located on the outer face of the membrane, for the reason that it is not possible to change the solution inside the patch pipette. Note that a vesicle sometimes forms at the end of the tip to attain this configuration. This vesicle can be broken by briefly exposing it to air. Using low Ca2⁺ solutions can be an alternative, as they have been suggested to reduce vesicle formation.

The ‘Whole-cell’ Configuration

If, instead of withdrawing the patch pipette from the cell after obtaining the cell attached configuration, a stronger negative pressure is applied to the patch pipette, the patch inside is ruptured thereby bringing the intracellular milieu in contact with the solution inside the pipette. This configuration, called ‘whole cell’ configuration, unlike all the others presented above, allows one to study macroscopic currents originating from all the channels present on the entire membrane of the cell (Fig. 4, indicated). An additional feature of whole cell configuration, which can be regarded either as an advantage or disadvantage, depending on the aim of the experiment, is that compounds easily diffuse from the patch pipette into the cell and their effects on ion channel function can be studied. However, at the same time, the pipette solution eventually replaces the cytoplasm of the cell and all its important factors, which are washed out through the patch pipette.

The ‘Outside-out’ Configuration

The whole cell configuration is not only an interesting way to study macroscopic currents with low-resistance electrodes. It also represents the intermediate step to the last classical patch clamp configuration, the ‘outside-out’ configuration. By slowly withdrawing the patch pipette from the cell, in the whole cell configuration, an elongated neck is initially formed, which rapidly collapses and then separates from the cell (Fig. 4, indicated). This configuration, like the inside-out configuration, has the patch exposed to the bath solution, which can now be changed as needed. Yet, it differs from it because the side of the patch now exposed to the bath solution is the extracellular portion of the membrane. This has interesting experimental properties because it allows one to study the effects of extracellular modulators of ion channels. The mind immediately goes to the myriad of neurotransmitters and associated ionotropic receptors that this configuration allows one to study. The drawback of this configuration is the much more laborious process to obtain it and its moderate stability.

Other Configurations of the Patch Clamp

Over the years, other patch clamp configurations have been developed, including the “perforated patch”, “loose patch” and “giant patch”. However, since they are all meant to record macroscopic currents, we will not describe them further. Readers interested in learning more can find descriptions of these configurations in the following papers (Pusch & Neher 1988; Rae et al. 1991; Roberts & Almers 1992; Stühmer 1992; Collins et al. 1992).