Automated Patch Clamp Analysis of nAChα7 and Na_V1.7 Channels

Abstract

Automated patch clamp devices are now commonly used for studying ion channels. A useful modification of this approach is the replacement of the glass pipet with a thin planar glass layer with a small hole in the middle. Planar patch clamp devices, such as the three described in this unit, are overtaking glass pipets in popularity because they increase throughput, are easier to use, provide for the acquisition of high-quality and information-rich data, and allow for rapid perfusion and temperature control. Covered in this unit are two challenging targets in drug discovery: voltage-gated sodium subtype 1.7 (Na(V)1.7) and nicotinic acetylcholine α7 receptors (nAChα7R). Provided herein are protocols for recording activation and inactivation kinetics of Na(V)1.7, and activation and allosteric modulation of nAChα7R.

Automated Patch Clamp Meets High-Throughput Screening: 384 Cells Recorded in Parallel on a Planar Patch Clamp Module

“…The cell lines used here were cultured as previously described. 5,[19][20][21] In brief, cells were cultured in T75 culture flasks in the media recommended by the supplier and passaged every 2-3 days when they were 50%-80% confluent. The cells were passaged regularly to ensure that the cells were single when passaged and harvested.…”

Identification of Drug–Drug InteractionsIn Vitro: A Case Study Evaluating the Effects of Sofosbuvir and Amiodarone on hiPSC-Derived Cardiomyocytes

“…CT6007; Kv11.1 (hERG) HEK293, no. CT6001, all from Chantest) were cultured as previously described (Becker et al, 2013;Bruggemann et al, 2009;Obergrussberger et al, 2014). In brief, cells were cultured in T75 culture flasks in manufacturer recommended media and passaged at 50-80% confluency (every 2-3 days) to ensure a healthy suspension of completely dissociated cells prior to recording.…”

Electrophysiology-Based Assays to Detect Subtype-Selective Modulation of Human Nicotinic Acetylcholine Receptors

“…Several published studies have shown the utility of this technology in nicotinic receptor research. Characterization of the α7 nAChR subtype has been reported by several groups that used PatchXpress (16 well; Molecular Devices), 18 QPatch (16 and 48 well; Sophion, Ballerup, Denmark), 19 , 20 or SynchroPatch (96 well; Nanion, Munich, Germany) 21 systems. Similar to our results, α7 assays performed on these platforms were shown to be effective in identifying PAMs and to require coapplication of the PAM for accurate measurement of agonist and antagonist potency owing to the rapid desensitization characteristic of α7 receptors.…”