Screening Assay Protocols Targeting the Nav1.7 Channel Using Qube High-Throughput Automated Patch-Clamp System.

Abstract

This article describes the basic procedure for setting up the screening protocol and recording data for Nav1.7 on a Qube automated patch-clamp system. Three protocols along with step-by-step details are provided. First, we describe a protocol to estimate Vhalf , the voltage at which half of the channels are inactivated, using traditional steady-state inactivation measurement as well as a new adaptive online estimation. Second, we establish a state-dependent protocol using adaptive online Vhalf measurement to obtain a concentration response curve (CRC) on known reference blockers. Last, we introduce a use-dependent protocol. In our hands, the sample reference demonstrated good state- and use-dependent inhibition of Nav1.7. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Comparing Vhalf measurements using offline calculation and a new adaptive online estimation Basic Protocol 2: Concentration response experiments using a state-dependent protocol Basic Protocol 3: Concentration response experiments using a use-dependent protocol Support Protocol: Cell culture protocol.