[Show abstract][Hide abstract] ABSTRACT: Two different mechanisms leading to increased current have been described for the small-molecule human ether-à-go-go-related gene (herg) activator NS1643 [1,3-bis-(2-hydroxy-5-trifluoromethylphenyl)-urea]. On herg1a channels expressed in Xenopus laevis oocytes, it mainly acts via attenuation of inactivation and for rat (r) erg1b channels expressed in human embryonic kidney (HEK)-293 cells, it strongly shifts the activation curve to the left. We now investigated the NS1643 effects on erg1b channels in more detail and performed comparative experiments with rat and human erg1a in different expression systems. Significant differences were observed between expression systems, but not between the rat and human isoform. In HEK-293 or Chinese hamster ovary (CHO) cells, activation of rat erg1b channels occurred in a dose-dependent manner with a maximum current increase of 300% obtained with 10 μM NS1643. In contrast, the NS1643-induced strong leftward shift in the voltage dependence of activation further increased with higher drug concentration, needed more time to develop, and exhibited use dependence. Coexpression of KCNE1 or KCNE2 did not attenuate this NS1643 effect on erg1 channel activation and did thus not mimic the lower drug potency on this parameter observed in oocytes. NS1643 (10 μM) slowed erg1b channel deactivation and recovery from inactivation without significant changes in activation and inactivation kinetics. With the exception of accelerated activation, NS1643 affected erg1a channels similarly, but the effect was less pronounced than in erg1b or erg1a/1b channels. It is noteworthy that rerg1b and herg1a inactivation estimated from fully activated current voltage relationships were unaltered in the continued presence of 10 μM NS1643 in the mammalian expression systems, indicating qualitative differences from NS1643 effects in X. laevis oocytes.
Preview · Article · Aug 2011 · Molecular pharmacology
[Show abstract][Hide abstract] ABSTRACT: The expression and functional role of ether-à-go-go-related gene (erg) K+ channels were examined in the bovine epididymal duct. Sperm transit through the epididymal duct relies on spontaneous phasic contractions (SC) of the peritubular smooth muscle wall. Isometric tension studies revealed SC-enhancing effects of the erg channel blockers E-4031, dofetilide, cisapride, and haloperidol and SC-suppressing effects of the activator NS-1643. In the corpus epididymidis, EC50 values of 32 nM and 8.3 microM were determined for E-4031 and NS-1643, respectively. E-4031 was also able to elicit contraction in epithelium-denuded corpus segments, which lacked SC. In the cauda region, E-4031 and NS-1643 exerted effects on agonist-induced contraction similar to those observed in the proximal duct. Experiments with nifedipine and thapsigargin suggested that the excitatory effects of E-4031 depended mainly on external calcium influx and not on intracellular calcium release. Western blot and RT-PCR assays revealed the expression of both, erg1a and erg1b, in all duct regions. Because erg1b appears to predominate in the epididymal duct, patch-clamp experiments were performed on heterologously expressed erg1b channels to investigate the sensitivity of this splice variant to NS-1643. In contrast to its effects on erg1a, NS-1643 induced a concentration-dependent current increase mainly due to a marked leftward shift in erg1b channel activation by approximately 30 mV at 10 microM, explaining the inhibitory effect of the drug on epididymal SC. In summary, these data provide strong evidence for a physiological role of erg1 channels in regulating epididymal motility patterns.
No preview · Article · Apr 2008 · AJP Regulatory Integrative and Comparative Physiology