Blockade of human IsK channels expressed in Xenopus oocytes by the novel class III antiarrhythmic NE-10064.

cRNA encoding the human IsK protein was injected into Xenopus oocytes and the expressed channels were investigated using the two-microelectrode voltage-clamp method. The novel class III antiarrhythmic NE-10064 (1-[[[5-(4-chlorophenyl)-2-furanyl]methylene]-amino]-3- [4-(4-methyl-1-piperazinyl)-butyl]-2,4-imidazolidinedione dihydrochloride) was tested for its ability to block these channels. The compound displayed potent inhibitory effects with an EC50 of 5.4 microM. The block caused by NE-10064 was use-dependent, i.e. channels had to be activated for the inhibition to occur. Further, the reversal of the inhibition during the wash-out period was use-dependent. Finally, the blockade of human IsK channels by NE-10064 appeared to be voltage-dependent, being more pronounced at depolarized potentials. We conclude that this novel class III antiarrhythmic is a potent inhibitor of human IsK channels and suggest that such effects could be involved in its antiarrhythmic action.

Positive regulation by chloride channel blockers of IsK channels expressed in Xenopus oocytes.

cRNA encoding the human IsK protein was injected into Xenopus oocytes and the induced IsK channels were investigated using the two-microelectrode voltage-clamp method. Niflumic acid, mefenamic acid, flufenamic acid, and 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid, which are commonly used in Xenopus oocytes to suppress endogenous Ca(2+)-activated Cl- channels, were tested for their effects on IsK channels. At low concentrations (10 microM) all compounds increased IsK amplitude and decreased the rate of IsK deactivation. At 100 microM these compounds further decreased the rate of IsK deactivation, resulting in persistent activation of IsK, similar to what has been previously described for the action of organic cross-linkers on IsK. However, at 100 microM niflumic acid and flufenamic acid decreased the time-dependent outward current, whereas 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and mefenamic acid caused an additional increase. When Cl- was completely substituted with gluconate, IsK had somewhat altered activation properties, but niflumic acid produced similar positive regulatory effects on IsK and shifted the voltage needed to evoke half-maximal IsK activation (V1/2) by about -20 mV. In summary, these compounds positively regulate IsK, presumably by stabilizing open IsK channels.

Electrophysiological analysis of Na+/Pi cotransport mediated by a transporter cloned from rat kidney and expressed in Xenopus oocytes.

Phosphate (Pi) reabsorption in renal proximal tubules involves Na+/Pi cotransport across the brush border membrane; its transport rate is influenced by the Na(+)-coupled transport of other solutes as well as by pH. In the present study, we have expressed a cloned rat renal brush border membrane Na+/Pi cotransporter (NaPi-2) in Xenopus laevis oocytes and have analyzed its electrophysiologic properties in voltage- and current-clamp studies. Addition of Pi to Na(+)-containing superfusates resulted in a depolarization of the membrane potential and, in voltage-clamped oocytes, in an inward current (IP). An analysis of the Na+ and/or Pi concentration dependence of IP suggested a Na+/Pi stoichiometry of 3:1. IP was increased by increasing the pH of the superfusate; this phenomenon seems to be mainly related to a lowering of the affinity for Na+ interaction by increasing H+ concentration. The present data suggest that known properties of Pi handling at the tubular/membrane level are "directly" related to specific characteristics of the transport molecule (NaPi-2) involved.

Inhibition of human IsK channels expressed in Xenopus oocytes by calmodulin antagonists.

The calmodulin antagonists, trifluoperazine, chlorpromazine and W7 (10-[3-(4-methyl-1-piperazinyl)-propyl]-2-(trifluomethyl)-10H-phen othiazine , 2-chloro-10-(dimethylaminopropyl)-phenothiazine and N-(6-aminohexyl)-5-chloro-1-naphtalen-sulfonamide, respectively), were tested for their effects on human IsK channels expressed in Xenopus oocytes and their interference with the previously described [Ca2+]i-mediated regulation of IsK. An increase in [Ca2+]i accelerated IsK activation and increased the current amplitude, as has been previously observed. Chlorpromazine, trifluoperazine and W7 inhibited depolarization-activated IsK channels with an EC50 between 70 and 100 microM. None of the calmodulin antagonists abolished the regulation of IsK by A23187 (calcimycin) or hypotonic extracellular fluid, although the inhibitory effects of these compounds were also obvious after enhancement of [Ca2+]i. In conclusion, the calmodulin antagonists inhibit IsK at both physiological and enhanced [Ca2+]i.