Nav 1.7 and 1.8 Recordings on the IonFlux System
The discovery that mutations in the SCN9A gene, which encodes the alpha subunit of NaV 1.7 Na+ channels, are the almost certain cause of three different human genetic pain disorders has created a great deal of interest towards synthesizing subtype- specific novel analgesics (Drenth & Waxman, 2007; Momin & Wood, 2008). Clinical discoveries such as these, in rare inherited monogenic channelopathies, often point to general bio- molecular targets for therapeutic intervention in a broader spectrum of diseases. The pain disorders therefore highlight NaV 1.7, but also NaV 1.8 since this channel co- expresses with NaV 1.7 in dorsal root ganglia (DRG) and is the molecular effector of hyperexcitability, as drug targets of increased importance (Ogata & Ohishi 2002; Momin & Wood, 2008).
Until recently, screening of chemical entities targeting ion channels faced a bottle-neck caused by the relatively low throughput of patch-clamping. Patching requires specialized equipment and skilled practitioners, and is unwieldy for compound library- based screen- ing in the pharmaceutical industry. The advent of the automated patch clamp (APC) has nonetheless produced gains in ion channel screening throughput, although the available platforms still face difficulties presented by robotic scheduling leading to sub-optimal time resolution for compound applications and washout. In this application note we de- scribe the use of IonFlux, a novel microfluidics- based APC system, for recording Na+ current conducted by hNaV 1.7 and hNaV 1.8 channels expressed in HEK-293 cells, and block of INa by the local anesthetic lidocaine. The IonFlux system employs ensemble recording arrays consisting of twenty or thirty cells voltage clamped in parallel at 16 or 64 recording sites, each exposed to as many as eight different compounds or concentrations in a 96- well or 384-well format. The IonFlux instrument incorporates no internal robotics and offers rapid, microfluidic compound applications. It has a compact “plate reader” footprint suitable for bench-top operation.