Explore chapters and articles related to this topic
Neuropeptide Receptor-Ion Channel Coupling in the Mammalian Brain
Published in Gerard O’Cuinn, Metabolism of Brain Peptides, 2020
The experimental approaches used to identify the effectors linked to a particular neuropeptide in the mammalian brain are the same as those applied in the more widely studied neurotransmitter candidates such as 5-HT, GABA, acetylcholine acting at muscarinic receptors, and adrenergic receptor-effector systems (see reviews1,2,3). Initially electrophysiological recordings are made to ascertain which, if any, ionic currents are affected by the neuropeptide. For this type of experiment intracellular recording, usually from identified neurones in a brain slice preparation, can be undertaken. When discontinuous single-electrode voltage clamp methods are also applied this technique will yield information on the ionic current(s) involved in the neuropeptide response. A more recent introduction to the field of brain slice electrophysiology is the use of whole-cell recording. This variation of the patch clamp technique is also commonly employed for voltage-clamp studies. It offers the advantage of improved voltage-clamp and the possibility to dialyze, at least in part, the cytoplasmic contents of the neurone. This is advantageous when one wishes to introduce suspected intracellular messengers but can be a problem if the initial whole-cell recording protocol leads to loss of neuropeptide response possibly as a result of loss of endogenous messenger.
Experimental models and measurements to study cardiovascular physiology
Published in Neil Herring, David J. Paterson, Levick's Introduction to Cardiovascular Physiology, 2018
Neil Herring, David J. Paterson
The patch clamp technique can be used to voltage, current or action potential clamp (where the membrane voltage is varied in the same pattern as a recorded native action potential) the entire cell. It can also be used on a small patch of membrane to record the response of a single ion channel (cell-attached patch). If this small area of membrane is then separated from the rest of the cell, an inside-out or outside- out patch configuration can be formed and the ion channel interior or exterior exposed to different ionic concentrations or neurotransmitters (see Figure 19.4).
Beyond Enzyme Kinetics
Published in Clive R. Bagshaw, Biomolecular Kinetics, 2017
Channels allow the selective transport of molecules down their concentration gradient and hence require no additional energy source. Characterization of channel proteins was more challenging because, once solubilized for purification, there is no remaining function, such as ATPase activity, to assay. In the early days, purification often relied on the tight binding of specific ligands, based on naturally occurring toxins. The amount of ligand bound per milligram of protein was used analogously to specific activity in enzyme purification. The situation for ion channels changed dramatically with the advent of patch clamping [261–263], whereby a small section of a native membrane containing one or a few channel proteins is isolated using a micropipette. The technique depends on forming tight seal (resistance >10 Gigaohms) between the glass electrode and membrane, such that the conductance of the pipette is determined by the properties of the channel protein captured within the membrane patch. When an ion channel opens, many hundreds to thousands of ions pass down their concentration gradient, giving a measurable current. This natural amplification allowed the kinetics of single channels to be determined via their stochastic opening and closing and constituted the first single-molecule kinetic assays in the biological field [15,16,263]. Ion channel opening is often modulated by specific ligand binding or the voltage across the membrane. In the patch-clamp technique, an additional electrode is inserted, which enables the membrane potential to be controlled and clamped at a known voltage. The analysis of such records is considered further in Section 9.4.
Automated patch clamp in drug discovery: major breakthroughs and innovation in the last decade
Published in Expert Opinion on Drug Discovery, 2021
Alison Obergrussberger, Søren Friis, Andrea Brüggemann, Niels Fertig
Patch-clamp electrophysiology remains an important technique in studying ion channels; indeed, it is still considered the gold standard since it was first described by Neher and Sakmann in the 1970s [1]. Ion channels are integral membrane proteins which allow ion current flow across the cell membrane. They are involved in almost all physiological processes, and their malfunction underlies many disease states, making them important pharmacological targets. Conventional patch clamp is a very information-rich technique, but it requires skilled personnel to perform experiments, and typically, only one experiment can be performed at a time. In the late 1990s and early 2000s, the field of ion-channel research was revolutionized by the development of the automated patch-clamp (APC) technique. The most successful approach involved replacing the patch-clamp pipette with a planar substrate (for review, see [2]), making the experiments easier to perform and offering the option for recording multiple cells in parallel. In the last two decades, much has changed in the field of ion-channel drug discovery and APC, with increased throughput and enhanced simplicity. We summarize the main changes in the last decade and attempt to look into the future of what’s to come.
Using Xenopus oocytes in neurological disease drug discovery
Published in Expert Opinion on Drug Discovery, 2020
Steven L. Zeng, Leland C. Sudlow, Mikhail Y. Berezin
Once the vitelline membrane is removed, the patch clamp can be performed. Several typical single channel configurations of patch clamp used in oocytes are shown in Figure 4. The difference between the methods lies in the relative orientation of the oocyte membrane and the rim of the glass electrode. Cell-attached configuration forms a good gigaseal with the intact membrane and is often used to study ligand-gated channels. A whole-cell patch clamping where the patch clamping electrode is used to attain direct electrical contact with the interior of the cell allows the entire cell membrane’s voltage to be controlled. This technique is applied more often on smaller cells, such as mammalian cells HEK 293 cells and is not optimal when used with cells the size of Xenopus oocytes [68], resulting in poor space voltage clamp regulation. The loose patch involves a comparatively low resistance seal and is also not very commonly used in oocytes. This would work for cells with high ion current density but would have large electrical noise characteristics.
A major interspecies difference in the ionic selectivity of megakaryocyte Ca2+-activated channels sensitive to the TMEM16F inhibitor CaCCinh-A01
Published in Platelets, 2019
Kirk A. Taylor, Martyn P. Mahaut-Smith
The nature of the ionic permeability of TMEM16F channels is controversial. Studies of native cells and expression systems conclude that human and murine TMEM16F channels are predominantly anion-permeable [4,9–11]. However, multiple reports demonstrate that heterologously expressed TMEM16F displays significant permeability to monovalent cations (PNa/PCl: 0.3 [12] 0.5 [13] and 1.38 [14]). Such studies have principally relied upon the whole-cell patch clamp configuration. Contrastingly, using excised inside-out membrane patch recordings, one group has reported that murine TMEM16F forms non-selective cation channels with a greater permeability to Ca2+ than monovalent cations both endogenously in the native megakaryocyte (MK) and in expression systems [8,15]. This raises the question of whether the properties of TMEM16F are influenced by patch excision or by the environment of a specific cell type. MKs are responsible for generating all proteins within their anuclear product and exhibit functional platelet responses [16,17], thus are frequently used as a surrogate for electrophysiological studies of the tiny, fragile platelet.