Patch-clamp single-channel recording

Patch clamp single channel recording experiments with neuroblastoma (N1E-115) cells provided additional support to the above hypothesis (22.). The single sodium channel conductance of neuroblastoma cells was reduced by increasing the external calcium concentration from 0.18 to 9.0 mM. The dissociation constant for calcium block was estimated to be 32.4 1.05 mM. 

Sigworth, F. J. (1995). Electronic design of the patch clamp, Single-Channel Recording, 2nd ed. (Sackman, B., Neher, E., eds.). Plenum Press, New York. 

The flow of ions through a single membrane channel (channels are shown in red in the illustration at the left) can he detected hy the patch clamp technique, which records current changes as the channel transits between the open and closed states. [(Left) After E. Neher and B. Sakmann. The patch clamp technique. Copyright 1992 by Scientific American, Inc. All rights reserved. (Right) Courtesy of Dr. Mauricio Montal.]... 

Figure 16.14 Original Kca1-1 (BK) single channel recordings and the inhibitory effect of iberiotoxin. Currents were recorded from a guinea pig urinary bladder smooth muscle cell using the whole cell (cell-attached), perforated patch-clamp technique at 0 mV holding potential (Section...

Patch-clamp—Technique for recording tiny electrical currents flowing through a single or multiple ion channels. 

Penner R (1995) A practical guide to patch clamping. In Sakmann B, Neher E (eds) Single-channel recording. Plenum, New York, pp 3-30... 

Readers who want to have an extended theoretical background on the properties of ionic channels are referred to the textbook Ionic Channels of Excitable Membranes by Hille [5]. More details on the patch-clamp technique are described in the book Single Channel Recording by Sak-mann and Neher [4]. 

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... 

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.)...

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. 

Selected entries from Methods in Enzymology [vol, page(s)] Analysis, software for, 207, 717 barrier models, 207, 818 closed and open time estimation, 207, 755 data acquisition, 207, 747 modal behavior analysis, 207, 757 multiple channel problem, 207, 756 single-channel [extraction of kinetic information, 207, 765 measurement in tissue slices, 207, 220] synaptic, resolution improvement in patch clamp recording, 207, 216 whole-cell recording in calcium channel, 207, 181 fluctuation analysis, 207, 192. 

Figure 4. (a) Planar bilayer membrane system for single-channel currents measurement. Soybean lecithin in n-decane was applied to a hole separating two aqueous chambers. Chambers were filled with metal chloride salt at pH 7.2. The voltage was applied to the outer cell with respect to the inner. The currents across the bilayer were recorded on a PCM recorder through a patch-clamp amplifier and a lowpass filter, (b) Typical records of current observed at -t-50.0 mV (symmetrical 0.5 M solution). Currents increase upward from the zero level shown by the dotted line in each panel. 

In a record obtained by the patch clamp technique, the channel is closed for much of the time (i.e. no current flows across the patch of membrane that contains it), but at irregular intervals the channel opens for a short time, producing a pulse of current. Successive current pulses are always of much the same size in any one experiment, suggesting that the channel is either open or closed, and not half open (there are exceptions to this rule). The durations of the pulses, however, and the intervals between them, vary in an apparently random fashion from one pulse to the next. Hence the openings and closings of channels are stochastic events. This means that, as with many other molecular processes, we can predict when they will occur only in terms of statistical probabilities. But one of the most useful features of the patch clamp method is that it allows observation of these stochastic changes in single ion channels as they actually happen individual protein molecules can be observed in action. 

In the outside-out model, the pipette is attached to the entire cell as in the whole cell model, followed by a sharp pull that causes the cell membrane to break and reseal with the pipette tip (Fig. 3b). With the extracellular region exposed, channel activity as a response to different external stimuli can be probed. This configuration is less common than the inside-out method. Using an outside-out method, single-channel opening activity has been recorded while various neurotransmitters were released. For example, this patch clamp method was used as a detector for capillary electrophoresis separations of GABA, glutamate, and NMDA (7). 

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