Single-channel Current Recordings Research Articles

9] Single-channel electrophysiology: Use of the patch clamp

The patch clamp technique affords unparalleled resolution of the detailed properties of ion channel currents. Patch clamping is not difficult and has been used to record single-channel currents from many cell types. The number of artifacts associated with the method appears to be rather small. The main experimental difficulties arise from the need to process large amounts of data. With the development of inexpensive computers and mass storage devices, these problems should be alleviated. The study of membrane excitability is expanding rapidly owing to the introduction of high-resolution patch clamp techniques. The conceptual revolution that will inevitably follow is just beginning.

Cholecystokinin activation of single-channel currents is mediated by internal messenger in pancreatic acinar cells.

Cholecystokinin (CCK) is one of several active peptides that have been isolated from both the gut1 and the brain2. CCK evokes membrane depolarization and conductance increase in both pancreatic acinar cells3,4 and pyramidal neurones of hippocampus5. In the acinar cells, activation of cholinergic muscarinic and two different peptidergic receptor sites (for CCK and bombesin) result in the same type of conductance increase6. The application of intracellular Ca mimics the action of the agonists7 and Ca has been proposed as an intracellular mediator for the membrane conductance increase8. A cation channel activated by internal Ca has been directly demonstrated in single-channel current recording experiments on isolated patches of plasma membrane from the basolateral surface of pancreatic acini9. We now demonstrate that CCK and acetylcholine (ACh) can activate these inward current channels indirectly. Single-channel currents were observed in electrically isolated membrane patches in situ when micropipette application of CCK or ACh was made outside the patch area. Single-channel currents from the same membrane patches were subsequently recorded after they had been excised (inside-out) and identical values for the single-channel conductance were obtained (∼35 pS). No effect of CCK was obtained in the presence of the specific receptor antagonist dibutyryl cyclic GMP10–12 although in all such cases single-channel currents were subsequently recorded from the excised patch (Ca-activated). These results demonstrate directly that CCK and ACh control ion channel opening via an intracellular messenger (Ca).

Single channel potassium currents of the anomalous rectifier

The recording of single channel currents has revealed the discrete events underlying the conductance changes for the Na and delayed K currents of the action potential, as well as those underlying acetylcholine-evoked currents1–5. The existence of similar discrete events has been suggested by noise analysis of the current termed anomalous K rectifier6,7, in which a K conductance is increased by hyperpolarization or by an increased extracellular concentration of K ions8. I have now used the patch current recording technique to investigate the anomalous rectifier of the tunicate egg cell. The statistics of pulse-like events in the patch current were found to be consistent with the inactivation kinetics of the anomalous rectifier. The extrapolated zero-current potential of the pulse-like events was approximately equal to that of the total anomalous K current. The single channel conductance was 5.2 pS in 50 mM K solution.

Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches

1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.

Single postsynaptic channel currents in tissue cultured muscle.

Some of the most compelling evidence for the existence of ionic channels in cell membranes comes from direct recording of quantised current jumps generated by the opening and closing of individual channels. Single-channel jumps have been extensively studied for lipid bilayer membranes doped with various channel-forming additives. Recently agonist-induced single-channel currents were detected in denervated frog muscle by use of extracellular electrodes, which can isolate the current from a small area of membrane. The current jumps provide a means for the direct test of many of the inferences about ionic channels which have come from electrical noise analysis. In this report we present measurements of single-channel currents induced by the agonist carbamylcholine in tissue-cultured mammalian muscle. These measurements confirm the earlier noise studies on tissue culture preparations. Recordings of single-channel currents induced by the agonist, suberyldicholine, in avian muscle are presented by Nelson and Sachs.