Patch clamp

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... 

Electrophysiological studies (mainly using voltage-clamp and patch clamp) revealed the essential properties of the sodium channels kinetics of channel gating and selective ion permeation. Sodium channels are... 

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. 

Electrophysiological Experiments. Guinea pig myocardial cells prepared as described previously 24) were superfused at 37 C with a Tyrode solution. Electrical properties of the myocytes were examined by the patch-clamp methods (25) using fire-polished pipettes. The current was measured by means of a patch-clamp amplifier, stored on the tape through a digital PCM data recording system, and analyzed with a computer. 

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. 

These approaches to receptor identification and classification were, of course, pioneered by studies with peripheral systems and isolated tissues. They are more difficult to apply to the CNS, especially in in vivo experiments, where responses depend on a complex set of interacting systems and the actual drug concentration at the receptors of interest is rarely known. However, the development of in vitro preparations (acute brain slices, organotypic brain slice cultures, tissue-cultured neurons and acutely dissociated neuronal and glial cell preparations) has allowed more quantitative pharmacological techniques to be applied to the action of drugs at neurotransmitter receptors while the development of new recording methods such as patch-clamp... 

Figure 13.3 Whole-cell patch-clamp recordings of excitatory postsynaptic currents (EPSCs) from dorsal horn neurons of rat (prenatal P2-13) spinal cord slices. The normal evoked EPSC of about 160pA obtained by focal stimulation of nearby tissue was dramatically reduced by addition of a cocktail (CABS) of CNQX 10 pM, D-APV 50 pM, bicuculline 10 pM and strychnine 5 pM to block glutamate, GABAa and glycine receptors. The small residual EPSC shown was blocked by the ATP P2 receptor antagonist suramin and is therefore probably mediated by released ATP. (Prom Bardoni et al. 1997 and reproduced by permission of the Journal of Neuroscience)...

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]. 

The main problem has been a methodological one. The patch clamp analysis of single channels views the world of channels through a very small analytical window [10]. A single channel event (opening) needs to be sufficiently long-lived and sufficiently large to be picked up within the current noise band under optimized conditions, and with the low-pass filter set to say 2 kHz. The open time needs to be close to a millisecond and the current amplitude close to 0.5 pA to permit detection. 

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. 

In our patch clamp studies in excised membrane patches in which we attempted to characterize the Cl we have noted that Cl did not only inhibit the probability of the ICOR channel being open but we also found that the input conductance of the patch was reduced at the same time and with the same time course [72]. We have followed up on this observation and we were able to show that this reduction in input conductance is caused by an inhibition of small ( lOpS) Cl -channels. Hence, we postulate that the same patches containing ICOR channels also contain small (unresolved, cf. section 2.4) Cl -channels which are inhibited reversibly by CL It cannot be excluded at this stage that these small Cl -channels are responsible for the defect in CF. 

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. 

Goldhaber, J.I. and Weiss, J.N. (1993). Hydrogen peroxide increases sodium-calcium exchange in patch-clamped guinea pig ventricular myocytes loaded with Fura-2. Circulation, 88(4), 724, abstract. 

Trotier D., Dpving K., Ore K. and Shalchian-Tabrizi C. (1998). Scanning electron microscopy and gramicidin patch clamp recordings of microvillous receptor neurons dissociated from the rat vomeronasal organ. Chem Senses 23, 49-57. 

A BLM can even be prepared from phospholipid monolayers at the water-air interface (Fig. 6.10B) and often does not then contain unfavourable organic solvent impurities. An asymmetric BLM can even be prepared containing different phospholipids on the two sides of the membrane. A method used for preparation of tiny segments of biological membranes (patch-clamp) is also applied to BLM preparation (Fig. 6.10C). 

Fig. 6.21 Joint application of patch-clamp and voltage-clamp methods to the study of a single potassium channel present in the membrane of a spinal-cord neuron cultivated in the tissue culture. The values indicated before each curve are potential differences imposed on the membrane. The ion channel is either closed (C) or open (O). (A simplified drawing according to B. Hille)...

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