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Endolysosomal Patch Clamping
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Cheng-Chang Chen, Christian Grimm, Christian Wahl-Schott, Martin Biel
Indirect electrophysiological methods have been employed to solve or circumvent the above problems (Pitt et al., 2010; Brailoiu et al., 2010). These are (1) planar lipid bilayer recordings or (2) redirection of the endolysosomal protein to the plasma membrane for conventional patch clamping. In the bilayer recordings, purified ion channel proteins or membrane fractions containing the organelles are reconstituted into synthetic phospholipid bilayers. In the second method, the lysosomal targeting sequences of endolysosomal ion channels are mutated, resulting in lysosomal ion channels to be expressed predominantly on the plasma membrane instead of endolysosomal membranes. The main drawback of both methods is that the new membrane environment is fundamentally different from the endolysosomal membranes. In particular, the specific membrane composition and local membrane proteins are very different. The absence of possible interaction partners and cofactors may also increase the risk of incorrect channel gating phenomena or even change in conformation of the channel structure. Recently, two direct electrophysiological approaches were developed to characterize endolysosomal ion channels directly on individual intact endolysosomes: (a) The solid base electrophysiology technique (Schieder et al., 2010a) and (b) a modified conventional patch clamp technique (Dong et al., 2008; Chen et al., 2017a).
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).
The neurosciences at the Max Planck Institute for Biophysical Chemistry in Göttingen
Published in Journal of the History of the Neurosciences, 2023
Although in the early experiments (Figure 5), the signal/noise ratio of their measurements was not yet ideal, Neher, Sakmann, and their colleagues managed to considerably improve this so-called patch clamp technique, developing it to become the most important instrument in modern neurophysiology (Reyes 2019). The corresponding publication (Hamill et al. 1981) has been cited more than 18,000 times to date.13See https://apps.webofknowledge.com/full_record.do?product=WOS&search_mode=GeneralSearch&qid=1&SID=C6sNCFVQOdB5G9NRJ6j&page=1&doc=1 (accessed May 30, 2020). Neher, Sakmann, and their colleagues initially studied the physiological properties of nAchR and the Na+ channels together, but at some point they began pursuing their own different research interests. Whereas Sakmann concentrated on the postsynaptic neurotransmitter receptors, Neher chose the release of neurotransmitters from the presynaptic side as his research focus. In 1991, they received the Nobel Prize for Physiology or Medicine for their discovery of the function of single membrane channels in nerve cells (Neher 1992b; Sakmann 1992b).
Design, synthesis, and biological evaluation of arylmethylpiperidines as Kv1.5 potassium channel inhibitors
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2022
Lingyue Zhao, Qian Yang, Yiqun Tang, Qidong You, Xiaoke Guo
The whole-cell membrane currents were recorded by the patch-clamp technique, using an EPC-10 double patch-clamp amplifier (HEKA, Pfalz, Germany). Recording pipettes, made from borosilicate glass (1.2 mm, o.d.), pulled with a pipette puller (PIP5, HEKA, Germany), had resistances of between 4 and 6 Ω when filled with the pipette solution. After a giga-seal (>10 GΩ) was obtained, the cell membrane was ruptured by gentle suction to establish the whole-cell configuration. The series resistance was electrically compensated to minimise the capacitive surge on the current recording. Peak current amplitude was determined after baseline correction. Pulse software (HEKA, Pfalz, Germany) was used to generate voltage pulse protocols and to record and analyse data. Compounds were applied at least 5 min after current stabilisation. The data are presented as the mean and standard deviation (mean ± SD). The differences between control levels and the changes caused by the compound application were tested using Student's t-test. A value of p < 0.05 was considered statistically significant. All experiments were performed at 25 °C.
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
Typically, voltage clamping is used to screen the effects of drugs or mutated membrane proteins on a single ion channel or on a single cell, one cell at a time. While such electrophysiological measurements can provide high-resolution information about ion channel performance and functional state on a microsecond timescale, these techniques suffer from being labor-intensive and time-consuming. Indeed, the major tool for ion channel research, the patch-clamp technique, has used the same principle since it was described in 1981 [70]. (This classic paper introducing ‘giga-seal’ has been cited in more than 19,000 publications). The situation changed in 1997, when the first commercial parallel multichannel patch-clamp instrumentation came to the market enabling true high throughput screening (HTS) approach in drug discovery [71]. Relatively large libraries of compounds were screened to develop better agonists or antagonists for epilepsy, major depressive disorder, and neuropathic pain [66,68,72,73].