David G. McKenna

High Throughput Electrophysiology Using a Fully Automated, Multiplexed Recording System

The drug discovery process centers around finding and optimizing novel compounds active at therapeutic targets. This process involves direct and indirect measures of how compounds affect the behavior of the target in question. The sheer number of compounds that must be tested poses problems for classes of ion channel targets for which direct functional measurements (e.g., traditional patch-clamping) are too cumbersome and indirect measurements (e.g., Ca(2+)-sensitive dyes) lack sufficient sensitivity or require unacceptable compromises. We present an optimized process for obtaining large numbers of direct electrophysiological measurements (two-electrode voltage-clamp) from Xenopus oocytes using a combination of automated oocyte handling, efficient and flexible liquid delivery, parallel operation, and powerful integrated data analysis. These improvements have had a marked impact, increasing the contribution electrophysiology makes in optimizing lead compound series and the discovery of new ones. The design of the system is detailed along with examples of data generated in support of lead optimization and discovery.

Potent desensitization of human P2X3 receptors by diadenosine polyphosphates

In this study, the receptor desensitizing effects of diadenosine polyphosphates at recombinant human P2X3 (hP2X3) receptors were examined. Administration of Ap3A, Ap4A, Ap5A or Ap6A inhibited the hP2X3 receptor-mediated response to a subsequent application of 3 muM alphabeta-methyleneATP (alphabeta-meATP), in a concentration-dependent manner, with IC50 values 2707, 42, 59 and 46 nM, respectively. These agonists did not desensitize alphabeta-meATP responses mediated by the slowly desensitizing heteromeric human P2X2/3 receptor. hP2X3 receptor desensitization was reversible and was not observed following the increase in intracellular Ca2+ levels produced by carbachol. A similar pattern of desensitization evoked by Ap5A was also observed using electrophysiological recordings of Xenopus oocytes expressing hP2X3 receptors. These data demonstrate that diadenosine polyphosphates, found endogenously in the central nervous system, can readily desensitize hP2X3 receptors at nanomolar concentrations that are 10-fold lower than are required to produce agonist-induced receptor activation. Thus, P2X3 receptor desensitization by diadenosine polyphosphates may provide an important modulatory mechanism of P2X3 receptor activation in vivo.

Automated Parallel Oocyte Electrophysiology Test station (POETs): a screening platform for identification of ligand-gated ion channel modulators.

Ligand-gated ion channels (LGICs) play important roles in the regulation of cellular function and signaling and serve as excellent drug targets. However, fast desensitization of most LGICs limits the choice of reliable methods to identify agonists, antagonists, and/or modulators in a high throughput manner. In this study, automated Parallel Oocyte Electrophysiology Test station (POETs) was used to screen a directed compound library against a rapidly desensitizing LGIC and to characterize further the pharmacological properties of the hits. POETs allows up to six two-electrode voltage-clamp experiments to be performed in parallel by automatically loading of the oocytes into flowcells, assessing individual oocyte behavior prior to initiating experiments. Oocytes injected with cRNA were transferred from a chilled 96-well plate into flowcells by the instrument, where they were impaled under software control by two independent electrodes. Expression was tested by measuring current responses to rapid application of agonists. Compounds, prepared in a 96-well format, were tested for effects by coapplication with agonist at a single concentration of 30 microM over 2 s. After compound application, oocytes were washed for a minimum of 30 s, and used repeatedly if the test compounds had no significant effect on the control response. Typical throughput could reach approximately 14 plates/day depending on the protocol. Pilot library screening revealed a hit rate of 0.06%, with active compounds having IC(50) values of 4-40 microM. Hits were also confirmed in native neurons using patch-clamp techniques. We conclude that automated POETs serves as a suitable platform for screening and expedient identification of LGIC modulators.

Epibatidine, a nicotinic acetylcholine receptor agonist, inhibits the capsaicin response in dorsal root ganglion neurons.

In patch-clamp recordings from small-medium diameter dorsal root ganglion neurons in culture, (+/-)-epibatidine (1 microM) was able to inhibit the capsaicin response (IC(50)=0.32 microM) in neurons where there was no detectable direct nicotinic response. Thus, (+/-)-epibatidine may inhibit the vanilloid receptor in a manner that is not dependent upon nicotinic current activation, representing another mechanism by which such ligands could modulate vanilloid receptor signaling.