Hartmut Suessbrich

Blockade of HERG channels expressed in Xenopus oocytes by the histamine receptor antagonists terfenadine and astemizole.

The widely used histamine receptor antagonists terfenadine and astemizole were shown to prolong the QT interval in electrocardiographic recordings in cases of overdose or inappropriate co‐medications, indicating a possible interaction with cardiac K+ channels. Here, terfenadine and astemizole both inhibited the human ether‐a‐go‐go related gene (HERG) encoded channels expressed in Xenopus oocytes at nanomolar concentrations in a use‐ and voltage‐dependent fashion. In contrast, inhibition of other delayed rectifier (Kvl.1 and IsK) or inward rectifier K+ channels (IRK1) was much weaker and occurred only at high micromolar concentrations. These results suggest that blockade of HERG channels by terfenadine and astemizole might contribute to the cardiac side effects of these compounds.

INHIBITION OF IKS IN GUINEA PIG CARDIAC MYOCYTES AND GUINEA PIG ISK CHANNELS BY THE CHROMANOL 293B

The chromanol derivative 293B was previously shown to inhibit a cAMP regulated K+ conductance in rat colon crypts. Subsequent studies on cloned K+ channels from the rat demonstrated that 293B blocks specifically IsK channels expressed in Xenopus oocytes, but does not affect the delayed and inward rectifier Kv1.1 and Kir2.1, respectively. In the present study, the specificity of 293B for the cardiac K+ conductances IKs and IKr, and for the cloned guinea pig IsK channel and the human HERG channel, which underly IKs and IKr, respectively, was analyzed. 293B inhibited both the slowly activating K+ conductance IKs in cardiac myocytes and guinea pig IsK channels expressed in Xenopus oocytes with a similar IC50 (2-6 μmol/1). In contrast, high concentrations of 293B had only a negligible effect on the more rapid activating IKr. Similarly, 293B exerted no effect on HERG channels expressed in Xenopus oocytes. In summary, 293B appears to be a rather specific inhibitor of IKs and the underlying IsK channels.

Obligatory Amino Acid Exchange via Systems bo,+-like and y+L-like A TERTIARY ACTIVE TRANSPORT MECHANISM FOR RENAL REABSORPTION OF CYSTINE AND DIBASIC AMINO ACIDS

Mutations in the rBAT gene cause type I cystinuria, a common inherited aminoaciduria of cystine and dibasic amino acids due to their defective renal and intestinal reabsorption (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Gallucci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-426; Calonge, M. J., Volipini, V., Bisceglia, L., Rousaud, F., De Sanctis, L., Beccia, E., Zelante, L., Testar, X., Zorzano, A., Estivill, X., Gasparini, P., Nunes, V., and Palacín, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9667-9671). One important question that remains to be clarified is how the apparently non-concentrative system bo,+-like, associated with rBAT expression, participates in the active renal reabsorption of these amino acids. Several studies have demonstrated exchange of amino acids induced by rBAT in Xenopus oocytes. Here we offer evidence that system bo,+-like is an obligatory amino acid exchanger in oocytes and in the “renal proximal tubular” cell line OK. System bo,+-like showed a 1:1 stoichiometry of exchange, and the hetero-exchange dibasic (inward) with neutral (outward) amino acids were favored in oocytes. Obligatory exchange of amino acids via system bo,+-like fully explained the amino acid-induced current in rBAT-injected oocytes. Exchange via system bo,+-like is coupled enough to ensure a specific accumulation of substrates until the complete replacement of the internal oocyte substrates. Due to structural and functional analogies of the cell surface antigen 4F2hc to rBAT, we tested for amino acid exchange via system y+L-like. 4F2hc-injected oocytes accumulated substrates to a level higher than CAT1-injected oocytes (i.e. oocytes expressing system y+) and showed exchange of amino acids with the substrate specificity of system y+L and L-leucine-induced outward currents in the absence of extracellular sodium. In contrast to L-arginine, system y+L-like did not mediate measurable L-leucine efflux from the oocyte. We propose a role of systems bo,+-like and y+L-like in the renal reabsorption of cystine and dibasic amino acids that is based on their active tertiary transport mechanism and on the apical and basolateral localization of rBAT and 4F2hc, respectively, in the epithelial cells of the proximal tubule of the nephron.

Specific block of cloned Herg channels by clofilium and its tertiary analog LY97241

The class III antiarrhythmic drug clofilium is known to block diverse delayed rectifier K+ channels at micromolar concentrations. In the present study we investigated the potency of clofilium and its tertiary analog LY97241 to inhibit K+ channels, encoded by the human ether‐a‐go‐go related gene (HERG). Clofilium blocked HERG channels in a voltage‐dependent fashion with an IC50 of 250 nM and 150 nM at 0 and +40 mV, respectively. LY97241 was almost 10‐fold more potent (IC50 of 19 nM at +40 mV). Other cloned K+ channels which are also expressed in cardiac tissue, Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv4.2, Kir2.1, or K Ks, were not affected by 100‐fold higher concentrations. Block of HERG channels by LY97241 was voltage dependent and the rate of HERG inactivation was increased by LY97241. A rise of [K+]0 decreased both, rate of HERG inactivation and LY97241 affinity. The HERG S631A and S620T mutant channels which have a strongly reduced degree of inactivation were 7‐fold and 33‐fold less sensitive to LY97241 blockade, indicating that LY97241 binding is affected by HERG channel inactivation. In summary, the antiarrhythmic action of clofilium and its analog LY97241 appears to be caused by their potent, but distinct ability for blocking HERG channels.

Specific blockade of slowly activating IsK channels by chromanols — impact on the role of IsK channels in epithelia

Chromanols, which were recently shown to inhibit cAMP‐mediated Cl− secretion in colon crypts via a blockade of a cAMP‐activated K+ conductance, were analyzed for their effects on distinct cloned K+ channels expressed in Xenopus oocytes. The lead chromanol 293B specifically inhibited I sK channels with an IC50 of 7 μmol/l without affecting the delayed rectifier Kv1.1 or the inward rectifier Kir2.1. Moreover, several other chromanols displayed the same rank order of potency for I sK inhibition as demonstrated in colon crypts. Finally, we tested the effects of the previously described I sK blocker azimilide on cAMP mediated Cl− secretion in rat colon crypts. Similar to 293B azimilide inhibited the forskolin induced Cl− secretion. These data suggest that I sK protein induced K+ conductances are the targets for the chromanol 293B and its analogues, and azimilide.

Role of the ISK protein in the IminK channel complex.

The ISK (also called minK) protein, although it is structurally unrelated to any other ion channel subunit, induces slowly activating, voltage-dependent K+ channels (IminK) in Xenopus oocytes or HEK293 cells. The quaternary structure of the IminK channel complex has long remained a mystery, but recent studies suggest an interaction of the ISK protein with a traditional K+ channel subunit, identified in man as KVLQT1. It is unclear at this point what the mechanism of this interaction is, or whether the ISK protein may also interact with other ion channel subunits. However, there is an abundance of information regarding the role and regulation of the ISK protein in the IminK channel complex, discussed in this review by Andreas Busch and Hartmut Suessbrich. The ISK protein is expressed in different tissues, where IminK activation may have distinct net effects on cell function. This fact makes IminK an excellent target for pharmacological agents.

Inhibitory effects of oxidants on n-type K+ channels in T lymphocytes and Xenopus oocytes

Abstract Reactive oxygen species (ROS) appear to be involved in Fas-induced programmed cell death. We have previously demonstrated a tyrosine-kinase-dependent inhibition of the n-type K+ channels (Kn) by Fas stimulation. Thus, the effect of hydrogen peroxide (H2O2) on the function of Kn was examined using the patch-clamp technique. Incubation of Jurkat human T lymphocytes with 100 μM H2O2 resulted in a 46 ± 5% inhibition of the macroscopic whole-cell current. Experiments performed at the single-channel level using the cell-attached configuration revealed that the probability of the channel being open diminished upon incubation in H2O2. The effect was not dependent on src-like kinases, since H2O2 did not trigger tyrosine phosphorylation of the Kn channel protein and herbimycin A did not prevent channel inhibition. Kv1.3 channels underly the Kn of T lymphocytes and were expressed in Xenopus oocytes and subjected to electrophysiological analysis by the two-electrode voltage-clamp technique. Application of 1 mM H2O2 and 500 μM t-BOOH (tert, butylhydroperoxide) resulted in a marked inhibition of the K+ current within 20 min. Both the membrane-permeable thiol-group oxidizing agent DTNP [2,2′-dithiobis-(5-nitropyridine)] and the membrane-impermeable DTNB [5,5′-Dithiobis-(2-nitrobenzoic acid)] (50 μM) inhibited Kv1.3 channels, suggesting that extracellular domains of Kv1.3 are affected. These results point to a direct modulation of Kn by various oxidative agents.

Blockade of epithelial Na+ channels by triamterenes — Underlying mechanisms and molecular basis

The three subunits (α, β, γ) encoding for the rat epithelial Na+ channel (rENaC) were expressed in Xenopus oocytes, and the induced Na+ conductance was tested for its sensitivity to various triamterene derivatives. Triamterene blocked rENaC in a voltage-dependent manner, and was 100-fold less potent than amiloride at pH 7.5. At −90mV and −40mV, the IC50 values were 5 μM and 10 μM, respectively. The blockage by triamterene, which is a weak base with a pKa of 6.2, was dependent on the extracellular pH. The IC50 was 1 μM at pH 6.5 and only 17 μM at pH 8.5, suggesting that the protonated compound is more potent than the unprotonated one. According to a simple kinetic analysis, the apparent inhibition constants at −90mV were 0.74 μM for the charged and 100.6 μM for the uncharged triamterene. The main metabolite of triamterene, p-hydroxytriamterene sulfuric acid ester, inhibited rENaC with an approximately twofold lower affinity. Derivatives of triamterene, in which the p-position of the phenylmoiety was substituted by acidic or basic residues, inhibited rENaC with IQ50 values in the range of 0.1–20 μM. Acidic and basic triamterenes produced a rENaC blockade with a similar voltage and pH dependence as the parent compound, suggesting that the pteridinemoiety of triamterene is responsible for that characteristic. Expression of the rENaC α-subunit-deletion mutant, Δ278–283, which lacks a putative amiloride-binding site, induced a Na+ channel with a greatly reduced affinity for both triamterene and amiloride. In summary, rENaC is a molecular target for triamterene that binds to its binding site within the electrical field, preferably as a positively charged molecule in a voltage-and pH-dependent fashion. We propose that amiloride and triamterene bind to rENaC using very similar mechanisms.