Roland Schönherr

MOLECULAR DETERMINANTS FOR ACTIVATION AND INACTIVATION OF HERG, A HUMAN INWARD RECTIFIER POTASSIUM CHANNEL

1. The human eag‐related potassium channel, HERG, gives rise to inwardly rectifying K+ currents when expressed in Xenopus oocytes. 2. The apparent inward rectification is caused by rapid inactivation. In extracellular Cs+ solutions, large outward currents can be recorded having an inactivation time constant at 0 mV of about 50 ms with an e‐fold change every 37 mV. 3. HERG channel inactivation is not caused by an amino‐terminal ball structure, as a deletion of the cytoplasmic amino terminus (HERG delta 2‐373) did not eliminate inactivation. However, channel deactivation was accelerated about 12‐fold at ‐80 mV. 4. Mutation of S631 to A, the homologous residue of eag channels, in the outer mouth of the HERG pore completely abolished channel inactivation. 5. Activity of HERG channels depended on extracellular cations, which are effective for channel activation, in the order Cs+ > K+ > > Li+ > Na+. The point mutation S631A strongly reduced this channel regulation. 6. By analogy to functional aspects of cloned voltage‐gated potassium channels, rectification of HERG, as well as its kinetic properties during the course of an action potential, are presumably governed by a mechanism reminiscent of C‐type inactivation.

The inhibitory effect of the antipsychotic drug haloperidol on HERG potassium channels expressed in Xenopus oocytes

The antipsychotic drug haloperidol can induce a marked QT prolongation and polymorphic ventricular arrhythmias. In this study, we expressed several cloned cardiac K+ channels, including the human ether‐a‐go‐go related gene (HERG) channels, in Xenopus oocytes and tested them for their haloperidol sensitivity. Haloperidol had only little effects on the delayed rectifier channels Kv1.1, Kv1.2, Kv1.5 and IsK, the A‐type channel Kv1.4 and the inward rectifier channel Kir2.1 (inhibition <6% at 3 μm haloperidol). In contrast, haloperidol blocked HERG channels potently with an IC50 value of approximately 1 μm. Reduced haloperidol, the primary metabolite of haloperidol, produced a block with an IC50 value of 2.6 μm. Haloperidol block was use‐ and voltage‐dependent, suggesting that it binds preferentially to either open or inactivated HERG channels. As haloperidol increased the degree and rate of HERG inactivation, binding to inactivated HERG channels is suggested. The channel mutant HERG S631A has been shown to exhibit greatly reduced C‐type inactivation which occurs only at potentials greater than 0 mV. Haloperidol block of HERG S631A at 0 mV was four fold weaker than for HERG wild‐type channels. Haloperidol affinity for HERG S631A was increased four fold at +40 mV compared to 0 mV. In summary, the data suggest that HERG channel blockade is involved in the arrhythmogenic side effects of haloperidol. The mechanism of haloperidol block involves binding to inactivated HERG channels.

Effects of imipramine on ion channels and proliferation of IGR1 melanoma cells

Human IGR1 cells are a model for malignant melanoma. Since progression through the cell cycle is accompanied by transient cell hyperpolarization, we studied the properties of potassium and chloride ion channels and their impact on cell growth. The major potassium current components were mediated by outward rectifying ether à go-go (hEAG) channels and Ca2+-activated channels (KCa) of the IK/SK type. The major chloride channel component was activated by osmotic cell swelling (Clvol). To infer about the contribution of these channels to proliferation, specific inhibitors are required. Since there is no specific blocker for hEAG available, we used the tricyclic antidepressant imipramine, which blocked all channels mentioned, in combination with blockers for KCa (charybdotoxin) and Clvol (DIDS and pamoic acid). Incubation of IGR1 cells for 48 hr in 10–15 mM imipramine reduced DNA synthesis and metabolism without significant effects on apoptosis. hEAG channels were most sensitive to imipramine (IC50: 3.4 mM at +50 mV), followed by KCa (13.8 mM at +50 mV) and Clvol (12 mM at ?100 mV), indicating that hEAG expression may be of importance for proliferation of melanoma cells. The contribution of KCa channels could be excluded, as 500 nM charybdotoxin, which completely blocked KCa, had no effect on proliferation. The impact of Clvol also seems to be minor, because 500 mM pamoic acid, which completely blocked Clvol, did not affect proliferation either.

Inhibition of human ether à go‐go potassium channels by Ca2+/calmodulin

Intracellular Ca2+ inhibits voltage‐gated potassium channels of the ether à go‐go (EAG) family. To identify the underlying molecular mechanism, we expressed the human version hEAG1 in Xenopus oocytes. The channels lost Ca2+ sensitivity when measured in cell‐free membrane patches. However, Ca2+ sensitivity could be restored by application of recombinant calmodulin (CaM). In the presence of CaM, half inhibition of hEAG1 channels was obtained in 100 nM Ca2+. Overlay assays using labelled CaM and glutathione S‐transferase (GST) fusion fragments of hEAG1 demonstrated direct binding of CaM to a C‐terminal domain (hEAG1 amino acids 673–770). Point mutations within this section revealed a novel CaM‐binding domain putatively forming an amphipathic helix with both sides being important for binding. The binding of CaM to hEAG1 is, in contrast to Ca2+‐activated potassium channels, Ca2+ dependent, with an apparent KD of 480 nM. Co‐expression experiments of wild‐type and mutant channels revealed that the binding of one CaM molecule per channel complex is sufficient for channel inhibition.

Multiple PIP2 binding sites in Kir2.1 inwardly rectifying potassium channels

Inwardly rectifying potassium channels require binding of phosphatidylinositol‐4,5‐bisphosphate (PIP2) for channel activity. Three independent sites (aa 175–206, aa 207–246, aa 324–365) were located in the C‐terminal domain of Kir2.1 channels by assaying the binding of overlapping fragments to PIP2 containing liposomes. Mutations in the first site, which abolished channel activity, reduced PIP2 binding of this fragment but not of the complete C‐terminus. Point mutations in the third site also reduced both, channel activity and PIP2 binding of this segment. The relevance of the third PIP2 binding site provides a basis for the understanding of constitutively active Kir2 channels.

Identification of ether à go-go and calcium-activated potassium channels in human melanoma cells.

Abstract. Ion channels and intracellular Ca2+ are thought to be involved in cell proliferation and may play a role in tumor development. We therefore characterized Ca2+-regulated potassium channels in the human melanoma cell lines IGR1, IPC298, and IGR39 using electrophysiological and molecular biological methods. All cell lines expressed outwardly rectifying K+ channels. Rapidly activating delayed rectifier channels were detected in IGR39 cells. The activation kinetics of voltage-gated K+ channels in IRG1 and IPC298 cells displayed characteristics of ether à go-go (eag) channels as they were much slower and depended both on the holding potential and on extracellular Mg2+. In addition, they could be blocked by physiological concentrations of intracellular Ca2+. In accordance with these electrophysiological results, analysis of mRNA revealed the expression of a gene coding for h-eag1 channels in IGR1 and IPC298 cells, but not in IGR39 cells. At elevated Ca2+ concentrations various types of Ca2+-activated K+ channels with single-channel characteristics similar to IK and SK channels were detected in IGR1 cells. The whole-cell Ca2+-activated K+ currents were not voltage dependent, insensitive for 100 nm apamin and 200 μmd-tubocurarine, but were blocked by charybdotoxin (100 nm) and clotrimazole (50 nm). Analysis of mRNA revealed the expression of hSK1, hSK2, and hIK channels in IGR1 cells.

Clinical relevance of ion channels for diagnosis and therapy of cancer

Ion channels have a critical role in cell proliferation and it is well documented that channel blockers can inhibit the growth of cancer cells. The concept of ion channels as therapeutic targets or prognostic biomarkers attracts increasing interest, but the lack of potent and selective channel modulators has hampered a critical verification for many years. Today, the knowledge of human ion channel genes is almost complete and molecular correlates for many native currents have already been identified. This information triggered a wave of experimental results, identifying individual ion channels with relevance for specific cancer types. The current pattern of cancer-related ion channels is not arbitrary, but can be reduced to few members from each ion channel family. This review aims to provide an overview of the molecularly identified ion channels that might be relevant for the most common human cancer types. Possible applications of these candidates for a targeted cancer therapy or for clinical diagnosis are discussed.

Molecular cloning and functional expression of a human peptide methionine sulfoxide reductase (hMsrA)

Oxidation of methionine residues in proteins to methionine sulfoxide can be reversed by the enzyme peptide methionine sulfoxide reductase (MsrA, EC 1.8.4.6). We cloned the gene encoding a human homologue (hMsrA) of the enzyme, which has an 88% amino acid sequence identity to the bovine version (bMsrA). With dot blot analyses based on RNA from human tissues, expression of hMsrA was found in all tissues tested, with highest mRNA levels in adult kidney and cerebellum, followed by liver, heart ventricles, bone marrow and hippocampus. In fetal tissue, expression was highest in the liver. No expression of hmsrA was detected in leukemia and lymphoma cell lines. To test if hMsrA is functional in cells, we assayed its effect on the inactivation time course of the A‐type potassium channel ShC/B since this channel property strongly depends on the oxidative state of a methionine residue in the N‐terminal part of the polypeptide. Co‐expression of ShC/B and hMsrA in Xenopus oocytes significantly accelerated inactivation, showing that the cloned enzyme is functional in an in vivo assay system. Furthermore, the activity of a purified glutathione‐S‐transferase‐hMsrA fusion protein was demonstrated in vitro by measuring the reduction of [3H]N‐acetyl methionine sulfoxide.

Functional role of the slow activation property of ERG K+ channels

ERG (ether‐à‐go‐go‐related gene) K+ channels are crucial in human heart physiology (h‐ERG), but are also found in neuronal cells and are impaired in Drosophila‘seizure’ mutants. Their biophysical properties include the relatively fast kinetics of the inactivation gate and much slower kinetics of the activation gate. In order to elucidate how the complex time‐ and voltage‐dependent activation properties of ERG channels underlies distinct roles in excitability, we investigated different types of ERG channels intrinsically present in cells or heterologously expressed in mammalian cells or Xenopus oocytes. Voltage‐dependent activation curves were highly dependent on the features of the eliciting protocols. Only very long preconditioning times produced true steady‐state relationships, a fact that has been largely neglected in the past, hampering the comparison of published data on ERG channels. Beyond this technical aspect, the slow activation property of ERG can be responsible for unsuspected physiological roles. We found that around the midpoint of the activation curve, the time constant of ERG open–close kinetics is of the order of 10–15 s. During sustained trains of depolarizations, e.g. those produced in neuronal firing, this leads to the use‐dependent accumulation of open‐state ERG channels. Accumulation is not observed in a mutant with a fast activation gate. In conclusion, it is well established that other K+ channels (i.e. Ca2+‐activated and M) control the spike‐frequency adaptation, but our results support the notion that the purely voltage‐dependent activation property of ERG channels would allow a slow inhibitory physiological role in rapid neuronal signalling.

Conformational Switch between Slow and Fast Gating Modes: Allosteric Regulation of Voltage Sensor Mobility in the EAG K+ Channel

Voltage-gated EAG K+ channels switch between fast and slow gating modes in a Mg2+-dependent manner by an unknown mechanism. We analyzed molecular motions in and around the voltage-sensing S4 in bEAG1. Using accessibility and perturbation analyses, we found that activation increases both the charge occupancy and volume of S4 side chains in the gating canal. Fluorescence measurements suggest that mode switching is due to a motion of the S2/S3 side of the gating canal. We propose that when S4 is in the resting state and its thin end is in the gating canal, a conformational rearrangement of S2/S3 narrows the canal around S4, forming the Mg2+ binding site. Binding of Mg2+ is proposed to stabilize this conformation and to slow opening of the gate by impeding S4s voltage-sensing outward motion.