A Practical Guide to Patch Clamping R. Penner. Tightseal Wholecell Recording A. Marty, E. Neher. Guide to Data Acquisition and Analysis S.H. Heinemann. Electronic Design of the Patch Clamp F.J. Sigworth. Low Noise Recording K. Benndorf. Voltage Offsets in Patch Clamp Experiments E. Neher. Techniques for Membrane Capacitance Measurements K.D. Gillis. Patch Pipette Recordings from the Soma, Dendrites and Axon of Neurons in Brain Slices B. Sakmann, G. Stuart. Patchclamp and Calcium Imaging in Brain Slices J. Eilers, et al. Fast Application of Agonists to Isolated Membrane Patches P. Jonas. Electrochemical Detection of Secretion from Single Cells R.H. Chow, L. von Ruden. Technical Approaches to Studying Specific Properties of Ion Channels in Plants R. Hedrich. The Giant Membrane Patch D.W. Hilgemann. A Fast Pressureclamp Technique for Studying Mechanogated Channels D.W. McBride, O.P. Hamill. Electrophysiological Recordings from Xenopus oocytes W. Stuhmer, A.B. Parekh. PCR Analysis of Ion Channel Expression in Single Neurons of Brain Slices H. Monyer, P. Jonas. 3 additional articles. Index.

Single-channel recording

Two metabolites of the steroid hormones progesterone and deoxycorticosterone, 3 alpha-hydroxy-5 alpha-dihydroprogesterone and 3 alpha, 5 alpha-tetrahydrodeoxycorticosterone, are potent barbiturate-like ligands of the gamma-aminobutyric acid (GABA) receptor-chloride ion channel complex. At concentrations between 10(-7) and 10(-5)M both steroids inhibited binding of the convulsant t-butylbicyclophosphorothionate to the GABA-receptor complex and increased the binding of the benzodiazepine flunitrazepam; they also stimulated chloride uptake (as measured by uptake of 36Cl-) into isolated brain vesicles, and potentiated the inhibitory actions of GABA in cultured rat hippocampal and spinal cord neurons. These data may explain the ability of certain steroid hormones to rapidly alter neuronal excitability and may provide a mechanism for the anesthetic and hypnotic actions of naturally occurring and synthetic anesthetic steroids.

https://science.sciencemag.org/content/sci/232/4753/1004.full.pdf

Steroid Hormone Metabolites are Barbiturate-Like Modulators of the GABA Receptor

A new method is described as an alternative to whole-cell recording in order to prevent "wash-out" of the muscarinic response to acetylcholine (ACh) in rat lacrimal gland cells. The membrane of a cell-attached patch is permeabilized by nystatin in the patch pipette, thus providing electrical continuity between the pipette and the cytoplasm of the cell without the loss or alteration of cytoplasmic compounds necessary for the maintenance of the response to ACh. With normal whole-cell recording in these cells, the response to ACh, seen as the activation of Ca-activated K and Cl currents, lasts for approximately 5 min. With the nystatin method, the response is not diminished after 1 h. Nystatin, applied extracellularly, is shown to cause a rapid and reversible increase of membrane conductance to cations. In the absence of wash-out, we were able to obtain dose-response curves for the effect of ACh on Ca-activated K currents. An increase of [ACh] caused an increase in the K current, with apparent saturation at concentrations above approximately 1 microM ACh. The delay between ACh application and the activation of K current was inversely related to [ACh] and reached a minimum value of 0.7-1.0 s at high [ACh].

/pdf/muscarinic-activation-of-ionic-currents-measured-by-a-new-xaerj4ehzt.pdf

Muscarinic activation of ionic currents measured by a new whole-cell recording method

The P2Z receptor is responsible for adenosine triphosphate (ATP)-dependent lysis of macrophages through the formation of membrane pores permeable to large molecules. Other ATP-gated channels, the P2X receptors, are permeable only to small cations. Here, an ATP receptor, the P2X7 receptor, was cloned from rat brain and exhibited both these properties. This protein is homologous to other P2X receptors but has a unique carboxyl-terminal domain that was required for the lytic actions of ATP. Thus, the P2X7 (or P2Z) receptor is a bifunctional molecule that could function in both fast synaptic transmission and the ATP-mediated lysis of antigen-presenting cells.

https://science.sciencemag.org/content/sci/272/5262/735.full.pdf

The Cytolytic P2Z Receptor for Extracellular ATP Identified as a P2X Receptor (P2X7)

The classical studies of nicotine by Langley at the turn of the 20th century introduced the concept of a “receptive substance,” from which the idea of a “receptor” came to light. Subsequent studies...

/pdf/mammalian-nicotinic-acetylcholine-receptors-from-structure-1woeprh7qg.pdf

Mammalian nicotinic acetylcholine receptors: from structure to function.

1. Inward currents in chromaffin cells were studied with the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). The intracellular solution contained 120 mM-Cs+ and 20 mM-tetraethylammonium (TEA+). Na+ currents were studied after blockade of Ca2+ channels with 1 mM-Co2+ applied externally. Ca2+ currents were recorded after eliminating Na+ currents with tetrodotoxin (TTX). The current recordings were obtained in cell-attached, outside-out and whole-cell recording configurations (Hamill et al. 1981).

2. Single channel measurements gave an elementary current amplitude of 1 pA at -10 mV for Na+ channels. This amplitude increased with hyperpolarization between -10 and -40 mV, but did not vary significantly between -40 and -70 mV.

3. The mean Na+ channel open time was 1 ms at -30 mV. This open time decreased both with depolarization and hyperpolarization. Its value was close to the time constant of inactivation, τh, above -20 mV.

4. Ensemble fluctuation analysis of Na+ currents gave results consistent with those of single channel measurements. Noise power spectra obtained between -35 mV and 0 mV could be fitted with a single Lorentzian. A range of Na+ channel densities of 1·5-10 channels per μm2 was calculated.

5. Cell-attached single Ca2+ channel recordings were obtained in isotonic BaCl2 solution. The single channel amplitude was 0·9 pA at -5 mV, and it became smaller for positive potential values.

6. At -5 mV, single Ba2+ currents appeared as bursts of 1·9 ms mean duration containing on the average 0·6 short gaps. The burst duration was larger at positive potentials.

7. Ensemble fluctuation analysis of Ca2+ channels was performed on whole-cell recordings in external solutions containing isotonic BaCl2 or external Ca2+ (Cao) concentrations of 1 and 5 mM. The unit amplitude calculated in the former case was similar to that obtained in single channel measurements.

8. Noise power spectra of Ca2+ or Ba2+ currents could be fitted by the sum of two, but not one, Lorentzian components.

9. Tail currents could be fitted by the sum of two exponential components. The corresponding time constants had values close to those obtained with noise analysis.

10. The rising phase of Ca2+ and Ba2+ currents was sigmoid. It could be fitted by the sum of three exponentials. The time constant of the largest amplitude component, τ1, was similar to the time constants of the slow component observed in noise and tail experiments. This time constant also corresponded to the burst duration obtained in single channel measurements.

11. The value of τ1 was larger in 5 mM-Cao and in isotonic Ba2+ than in 5 mM-Bao. Thus, the kinetic properties of Ca2+ channels depend on the nature and concentration of the permeating ion.

12. A simple kinetic scheme is proposed to model the activation pathway of Ca2+ channels.

13. Currents in 1 mM-Cao and 5 mM-Cao showed clear reversals around +53 mV and +64 mV respectively. The outward currents observed above these potentials are most probably due to Cs+ ions flowing through Ca2+ channels.

14. The instantaneous current—voltage relation was obtained from tail current data in the range -70 to +100 mV in 5 mM-Cao. The resulting curve displayed an inflexion point around the reversal potential.

15. Very little inactivation of Ca2+ currents was observed. However, a slow current decline was observed in some cells above +10 mV.

16. Conditioning prepulses to positive potentials had potentiating or depressing effects on Ca2+ currents depending on whether the test pulse lay below or above the maximal current potential. The potentiating effect may be linked to the slowest component of the current rise observed below +10 mV. The depressing effect may be related to the slow decline obtained above +10 mV.

17. Analysis of ensemble variance and of tail current amplitudes suggested that the opening probability of Ca2+ channels was at least 0·9 above +40 mV.

18. A slow rundown of Ca2+ currents was observed in whole-cell recordings. The speed of the rundown was dependent on intracellular Ca2+ concentration. The rundown was apparently due to a progressive elimination of the channels available for activation.

19. The density of Ca2+ channels (before rundown) was estimated at 5-15/μm2.

20. In cell-attached experiments, inward current channels were often seen to follow action potentials. These events did not appear to be the usual Na+ and Ca2+ currents. They were probably due to cation influx of either Na+ or Ba2+, depending on the pipette solution, through Ca2+-dependent channels. Voltage-independent single channel activity observed in whole-cell and outside-out recordings may be due to the same channels.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1982.sp014394

Sodium and calcium channels in bovine chromaffin cells

1. Bovine chromaffin cells were enzymatically isolated and kept in short term tissue culture. Their electrical properties were studied using recent advances of the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). 2. When a patch pipette was sealed tightly to a chromaffin cell ('cell-attached configuration') current wave forms due to intracellular action potentials could be observed. The frequency of the wave forms was altered by changing the pipette potential. When acetylcholine was present in the pipette solution, acetylcholine-induced single channel currents were evident in the patch recording. Action potential wave forms were then often seen to follow acetycholine-induced single channel currents. 3. In the cell-attached configuration, large single channel current events did not resemble square pulses but showed exponential relaxations with time constants of the order of 50 ms. 4. After rupture of the patch of membrane, the pipette--cell seal remained stable ('whole-cell recording', Hamill et al. 1981). Chromaffin cells were found to have a resting potential of -50 to -80 mV, and an input resistance around 5 G omega. The high cell resistance accounts for the relaxing currents evident in the cell-attached configuration. 5. In the best cases, the effective time constant of the voltage clamp in the whole-cell recording mode was 15 microseconds. Exchange of small ions such as Na+ ions between pipette and cell interior solutions was then complete within 15 s. 6. Acetylcholine-induced currents were obtained at various acetylcholine concentrations. Single acetylcholine-induced channels had a slope conductance of 44 pS between -100 and -55 mV, and a mean duration of 27 ms at -80 mV (at room temperature).

A patch-clamp study of bovine chromaffin cells and of their sensitivity to acetylcholine.

Publisher Summary The ability to propagate and transmit signals is conferred to neurons by proteins embedded in the lipid membrane. Knowledge of the molecular properties of these macromolecules would therefore provide a sound basis for the understanding of neuronal functions. A great deal of information on such molecular functions has been provided by two recently developed electrophysiological techniques: noise analysis and single channel recording. Both these techniques allow the study of integral proteins that are assumed to function as so-called ionic channels or membrane pores regulating the flow of ions across the membrane. As such, these proteins must span the whole membrane thickness, and provide a hydrophilic pathway across the membrane. Noise analysis works on the macroscopic fluctuations in membrane current that result from the statistical superposition of many microscopic unit responses. Exploiting some fundamental laws of statistics it deduces from the macroscopic fluctuations the properties of the elementary units that constitute them. Noise analysis is applicable to a wider range of preparations and experimental conditions; however, it depends on certain assumptions, the validity of which, in turn, has to be proven by single channel recording. This chapter provides a summary of some of the molecular features of ionic channels encountered in nerve and nerve-like cells. The chapter focuses on results obtained by single channel recording. Where necessary, it also includes noise results.

Elizabeth M. Fenwick

Papers

Sodium and calcium channels in bovine chromaffin cells

A patch-clamp study of bovine chromaffin cells and of their sensitivity to acetylcholine.

Ionic channels for signal transmission and propagation.