Human ether à go‐go potassium channels (hEAG1) open in response to membrane depolarization and they are inhibited by Ca2+/calmodulin (CaM), presumably binding to the C‐terminal domain of the channel subunits. Deletion of the cytosolic N‐terminal domain resulted in complete abolition of Ca2+/CaM sensitivity suggesting the existence of further CaM binding sites. A peptide array‐based screen of the entire cytosolic protein of hEAG1 identified three putative CaM‐binding domains, two in the C‐terminus (BD‐C1: 674–683, BD‐C2: 711–721) and one in the N‐terminus (BD‐N: 151–165). Binding of GST‐fusion proteins to Ca2+/CaM was assayed with fluorescence correlation spectroscopy, surface plasmon resonance spectroscopy and precipitation assays. In the presence of Ca2+, BD‐N and BD‐C2 provided dissociation constants in the nanomolar range, BD‐C1 bound with lower affinity. Mutations in the binding domains reduced inhibition of the functional channels by Ca2+/CaM. Employment of CaM‐EF‐hand mutants showed that CaM binding to the N‐ and C‐terminus are primarily dependent on EF‐hand motifs 3 and 4. Hence, closure of EAG channels presumably requires the binding of multiple CaM molecules in a manner more complex than previously assumed.

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 microM at +50 mV), followed by KCa (13.8 microM at +50 mV) and Clvol (12 microM 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 microM pamoic acid, which completely blocked Clvol, did not affect proliferation either.

Ion channels and intracellular Ca2+ are thought to be involved in cell proliferation and may play a role in tumor development. We therefore characterized Ca(2+)-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 Ca(2+)-activated K+ channels with single-channel characteristics similar to IK and SK channels were detected in IGR1 cells. The whole-cell Ca(2+)-activated K+ currents were not voltage dependent, insensitive for 100 nm apamin and 200 microm d-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.

The depolarisation activated human ether à go-go (hEAG) potassium channels are primarily expressed in neuronal tissue but their appearance in various tumour entities is also indicative of an oncogenic role. Because upregulation of hEAG channels may yield to an enhanced cell proliferation, interventions increasing hEAG1 currents may serve similar purposes. We therefore investigated the effects of polyunsaturated fatty acids on hEAG1 channels. Arachidonic acid (AA) lowered their activation threshold, accelerated the activation kinetics and increased the open probability with a half-maximal concentration of about 4 microM. This effect correlated with the number of double bonds (db) in the fatty acids, increasing from oleic acid (1 db), linolenic acid (3 db), AA (4 db) to eicosapentaenoic acid (5 db). Unlike other voltage-gated K(+) channels, hEAG1 channels are not blocked by arachidonic acid. Therefore, in particular at typical resting potentials of tumour cells (-30 mV), AA potently activated hEAG1 channels in a reversible manner. Proliferation and metabolic activity of hEAG1-expressing human melanoma cells increased when cells were exposed to AA concentrations of 5 microM and this effect was suppressed in the presence of the hEAG1 blocker LY97241 suggesting that the proliferative effect of AA is in part mediated by activation of hEAG channels.

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