Activation of protein kinase C sensitizes human VR1 to capsaicin and to moderate decreases in pH at physiological temperatures in Xenopus oocytes.

The two-electrode voltage-clamp technique was used to evaluate the effect of protein kinase C (PKC) activation on ion current flow in Xenopus laevis oocytes injected with cRNA coding for the human vanilloid receptor (VR1). In the presence of 30 nM phorbol-12,13-dibutyrate (PDBu), current evoked by an effective concentration (EC(30)) of capsaicin (CAP) was potentiated by 638+/-117% (n=8). PDBu exhibited an EC(50) of about 17+/-3 nM for this effect (n=8). Potentiation was not observed when VR1 expressing oocytes were exposed to both 30 nM PDBu and 1 microM staurosporine. In the presence of 300 nM PDBu, the EC(50) for CAP shifted from 899+/-78 to 139+/-2 1 nM (n=11 and 5, respectively). In the presence of 30 nM PDBu, the maximal current amplitude evoked by application of CAP increased by 86+/-21% (n=10), in a staurosporine sensitive manner. Application of 1 microM PDBu alone elicited a capsazepine sensitive current within 3 min of exposure. This effect was observed in the absence of previous exposure of the oocyte to CAP and was abolished in the presence of 1 microM staurosporine. No current was elicited during a 10 min application of 300 nM PDBu, the longest interval assessed. Prior to 30 nM PDBu exposure, no current was evoked at temperature ramps from room temperature (22-23 degrees C) up to 37 degrees C at pH 6.8, 7.0, or 7.4. Following PDBu treatment, VR1 mediated current was evoked at 26 degrees C at pH 7.0. Likewise, following 30 nM PDBu treatment, current was evoked by application of pH 6.8 alone and a further increase in current amplitude was evoked by heat at 24 degrees C in a staurosporine sensitive manner. These data provide direct evidence that PKC activation can increase VR1 current evoked by candidate physiological activators, pH and heat. This observation provides an empirical foundation for explaining some types of inflammatory pain in terms of PKC activation, small decreases in tissue pH levels, and small increases in skin temperature, all of which can accompany inflammatory conditions.

A high-throughput HERG potassium channel function assay: an old assay with a new look.

In this paper, we describe an assay using radioactive rubidium (86Rb) efflux to screen functional human ether-a go-go-related gene (HERG) K+ channels in a high-throughput screening (HTS) format. This assay offers an alternative way to examine junctional interactions between chemical compounds and HERG K+ channels. Follow-up experiments and discussions were carried out to address a variety of factors that affect potency evaluation within the Rb efflux assay. Factors that can affect the assay results, such as assay time, efflux rate, and compound blocking kinetics, are discussed in detail. Our results provide some explanations for the variances of the assay results and offer some guidelines for using the Rb efflux assay to evaluate compound interactions with HERG K+ channels in the pharmaceutical industry.