Patch-clamp technique

Researchers at the MoneU Center (Philadelphia, Pennsylvania) are using a variety of electrophysical and biochemical techniques to characterize the ionic currents produced in taste and olfactory receptor cells by chemical stimuli. These studies are concerned with the identification and pharmacology of the active ion channels and mode of production. One of the techniques employed by the MoneU researchers is that of "patch clamp." This method aUows for the study of the electrical properties of smaU patches of the ceU membrane. The program at MoneU has determined that odors stimulate intraceUular enzymes to produce cycUc adenosine 3, 5 -monophosphate (cAMP). This production of cAMP promotes opening of the ion channel, aUowing cations to enter and excite the ceU. MoneU s future studies wiU focus on the connection of cAMP, and the production of the electrical response to the brain. The patch clamp technique also may be a method to study the specificity of receptor ceUs to different odors, as weU as the adaptation to prolonged stimulation (3). 

Precise kinetic electroanalytical data permit to describe quantitatively the kinetics of the whole process with a precision that has never been achieved before by patch-clamp techniques or spectroscopic near-field methods. This enables to investigate finely these events and to identify the exact physicochemical nature of all the individual physicochemical and biological factors which concur to produce vesicular release. 

The patch-clamp technique is based on the formation of a high resistance seal (109-10lon) between the tip of a glass micropipette and the cell membrane it touches (gigaohm-seal). This technique allows recordings of ionic currents through single ion channels in the intact cell membrane and in isolated membrane patches at a... 

In a different context, a micropipette has been applied to monitor the current through a single-ion channel in a biological membrane. The patch-clamp technique invented by Sackmann and Neher [119] led to their Nobel Prize in medicine. The variations in channel current with voltage, concentration, type of ions, and type of channels have been explored. While the functions of specific channels, in particular their ionic selectivity, have been well known, only a handful of channels have the internal geometry and charge distribution determined. The development of a theory to interpret the mass of channel data and to predict channel action is still lacking. 

Using the cell-attached patch clamp technique on frog muscle fibers (79), one can observe only two conditions the open, conducting state of the receptor and a nonconducting state of unknown identity. The transitions behave according to stochastic principles the lifetimes of any particular condition are distributed exponentially. The open state has a mean duration that is the inverse of the rate of channel closing. Because channel open time depends only upon a conformational shift, agonist concentration does not influence the parameter. It is, however, influenced... 

The primary characteristic of a sequential blocker, as observed with the patch clamp technique, is that the reciprocal of the mean duration of the lifetime equals the normal channel closing rate plus the rate constant of channel blockade times the drug concentration. Therefore, increasing the drug concentration shortens the mean channel open time. 

The electrophysiological experiments reported here and done with patch-clamp techniques support this idea. The external application of MTX to isolated cardiac myocytes caused a sustained inward current which was carried by Ca . MTX did not increase the voltage-dependent Ca channel current, and both the time dependence and voltage dependence of the MTX-induced current were clearly different from those of the usual Ca channel current. These results suggest that the MTX-induced steady current is different from the usual voltage-dependent Ca channel current, and that this is possibly a current which flows through a new type of Ca -permeable channel. Tbe steady current described here may be responsible for the highly enhanced Ca influx induced by MTX and could account for the excitatory action of MTX on smooth and cardiac muscles. 

Techniques for Tentatively Identifying Mechanisms of Action. Once the mechanism by which a toxin kills has been assessed, and toxin reasonably purified, it becomes relevant to try and ascertain as efficiently as possible the cellular mechanisms "tar-getted" by the toxin. This is a necessary step before final analysis of action using pure toxin and site-specific procedures such as the patch-clamp technique. 

The field of ion-channel research has met with intense interest during the past ten years. One reason for this development has been the advent of new methods such as the patch clamp technique invented by Sakmann and Neher [1] and new approaches to the cloning of the complete amino acid sequencing of ion channels as it was introduced by Numa and collaborators [2]. 

Even beyond this, it should be clear that the large clamp voltage of say 100 mV may already lead to the inactivation of larger channels, and, still worse, the excision of the cell membrane itself may inactivate channels. It is not surprising then that the current literature gives a grossly distorted view of the world of Cl -channels. It is full of large and intermediate channels, but much less data are available on small channels. This analytical problem can be overcome by other patch clamp techniques. 

It is clearly impossible to give a comprehensive overview of this rapidly expanding field. I have chosen a few experts in their field to discuss one (class of) transport protein(s) in detail. In the first five chapters pumps involved in primary active transport are discussed. These proteins use direct chemical energy, mostly ATP, to drive transport. The next three chapters describe carriers which either transport metabolites passively or by secondary active transport. In the last three chapters channels are described which allow selective passive transport of particular ions. The progress in the latter field would be unthinkable without the development of the patch clamp technique. The combination of this technique with molecular biological approaches has yielded very detailed information of the structure-function relationship of these channels. 

Figure 1 Schematic diagrams illustrating the patch-clamp technique. (A) Overall setup for isolating single ionic channels in an intact patch of cell membrane. P = patch pipet R = reference microelectrode I = intracellular microelectrode Vp = applied patch potential Em = membrane potential Vm = Em — Vp = potential across the patch A = patch-clamp amplifier. (From Ref. 90.) (B) Five different recording configurations, and procedures used to establish them, (i) Cell attached or intact patch (ii) open cell attached patch (iii) whole cell recording (iv) excised outside-out patch (v) excised inside-out patch. Key i = inside of the cell o = outside of the cell. (Adapted from Ref. 283.)...

OP Hamill, A Marty, E Neher, B Sakman, FJ Sigworth. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluegers Arch 391 85-100. 

The opening of masses of ion channels in nematode muscle membranes may be detected using the two-microelectrode voltage-clamp technique. In contrast, the opening of single ion channels may be recorded using the vesicle preparation and patch-clamp technique. These techniques are both described below. 

Fig. 21.4. Vesicle formation and patch-clamp techniques used to record levamisole receptor channel currents from Ascaris muscle. (A) Muscle membrane vesicles bud-off from the bag membrane following a 10 min collagenase treatment and incubation for 1 h at 37°C in Ascaris saline. (B) Levamisole is applied to the outside surface of the membrane to activate receptor channels cell-attached patches are usually used but it is also possible to make inside-out and outside-out patch recordings.

The advantage of the patch-clamp technique for recording from single levamisole receptors is that biophysical properties of individual receptors... 

Cahalan, M. and Neher, E. Patch clamp techniques an overview. Methods Enzymol. 207 3-14,1992. 

Thus, a 10 1 transmembrane gradient of a single monovalent ion, say potassium, will generate a membrane potential of 58 mV. See Resting Potential Action Potential Depolarization Threshold Potential Nernst Equation Goldman Equation Patch-Clamp Technique... 

PERMEABILITY PERMEABILITY CONSTANT MEMBRANE POTENTIAL ACTION POTENTIAL DEPOLARIZATION GOLDMAN EQUATION NERNST EQUATION RESTING POTENTIAL THRESHOLD POTENTIAL PATCH-CLAMP TECHNIQUE Membrane protein dynamics,... 

The flow of ions across the plasma membrane is recorded as current. An extensive description of the patch-clamp technique is given in ref (30). 

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