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.

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Human ether a © go-go potassium channel 2 (hEAG2) was cloned and its properties were compared with the previously characterized isoform hEAG1. In the Xenopus oocyte expression system the time course of activation was about four times slower and the voltage required for half-maximal subunit activation was about 10 mV greater for hEAG2 channels. However, its voltage dependence was smaller and, therefore, hEAG2 channels start to open at more negative voltages than hEAG1. Coexpression of both isoforms and kinetic analysis of the resulting currents indicated that they can form heteromeric channel complexes in which the slow activation phenotype of hEAG2 is dominant. Upon expression in mammalian cells, quinidine blocked hEAG1 channels (IC 50 1.4 W WM) more potently than hEAG2 channels (IC 50 152 W WM), thus providing a useful tool for the functional distinction between hEAG1 and hEAG2 potassium channels. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Large-conductance voltage-and Ca 2+-activated K + (Slo1 BK) channels serve numerous cellular functions, and their dysregulation is implicated in various diseases. Drugs activating BK channels therefore bear substantial therapeutic potential, but their deployment has been hindered in part because the mode of action remains obscure. Here we provide mechanistic insight into how the dehydroabietic acid derivative Cym04 activates BK channels. As a representative of NS1619-like BK openers, Cym04 reversibly left-shifts the half-activation voltage of Slo1 BK channels. Using an established allosteric BK gating model, the Cym04 effect can be simulated by a shift of the voltage sensor and the ion conduction gate equilibria toward the activated and open state, respectively. BK activation by Cym04 occurs in a splice variant-specific manner; it does not occur in such Slo1 BK channels using an alternative neuronal exon 9, which codes for the linker connecting the transmembrane segment S6 and the cytosolic RCK1 domain-the S6/RCK linker. In addition, Cym04 does not affect Slo1 BK channels with a two-residue deletion within this linker. Mutagenesis and modelbased gating analysis revealed that BK openers, such as Cym04 and NS1619 but not mallotoxin, activate BK channels by functionally interacting with the S6/RCK linker, mimicking site-specific shortening of this purported passive spring, which transmits force from the cytosolic gating ring structure to open the channel's gate.

1 We investigated the inhibition of hEAG1 potassium channels, expressed in mammalian cells and Xenopus oocytes, by several blockers that have previously been reported to be blockers of hERG1 channels. 2 In the whole-cell mode of mammalian cells, LY97241 was shown to be a potent inhibitor of both hEAG1 and hERG1 channels (IC 50 of 4.9 and 2.2 nM, respectively). Clo®lium, E4031, and haloperidol apparently inhibited hEAG1 channels with lower potency than hERG1 channels, but they cannot be considered hERG1-speci®c. 3 The block of hEAG1 channels by LY97241 and clo®lium was time-, use-, and voltage-dependent, best explained by an open-channel block mechanism. 4 Both drugs apparently bind from the intracellular side of the membrane at (a) speci®c site(s) within the central cavity of the channel pore. They can be trapped by closure of the activation gate. 5 In inside-out patches from Xenopus oocytes, hEAG1 block by clo®lium was stronger than by LY97241 (IC 50 of 0.8 and 1.9 nM, respectively). In addition, hEAG1 block by clo®lium was much faster than by LY97241 although there was no di erence in the voltage dependence of the on-rate of block. 6 Physico-chemical di erences of clo®lium and the weak base LY97241 determine the access of the drugs to the binding site and thereby the in¯uence of the recording mode on the apparent block potencies. This phenomenon must be considered when assessing the inhibitory action of drugs on ion channels.

Undesired block of human ERG1 potassium channels is the basis for cardiac side effects of many different types of drugs. Therefore, it is important to know exactly why some drugs particularly bind to these channels with high affinity. Upon expression in mammalian cells and Xenopus laevis oocytes, we investigated the inhibition of the closely related hEAG1 and hEAG2 channels by agents that have previously been reported to block hERG1 channels. Clofilium inhibited hEAG1 and hERG1 with the same potency, whereas hEAG2 was about 150-fold less sensitive to this antiarrhythmic agent. The molecular determinants for this difference are residues Ser436 and Val437 in the inner cavity of the pore and Ala453, which is located in S6 (i.e., remote from the inner cavity). A modeling approach that allowed for partial conformational relaxation of hEAG model structures upon ligand docking suggests that highaffinity block of ether à go-go channels is mediated by an anchoring of the clofilium alkane tail between S6 and the pore helices. In qualitative agreement with experiments, the mutations of hEAG1 residues Ser436 and Val437 to the corresponding larger hEAG2 residues (Thr432, Ile433) resulted in reduced sterical fit between the ligand and the binding cavity. The model is further supported by functional assays involving (ϩ)-N-[1Ј-(6-cyano-1,2,3,4-tetrahydro-2(R)-naphthalenyl)-3,4-dihydro-4(R)-hydroxyspiro(2H-1-benzopyran-2,4Ј-piperidin)-6-yl]methanesulfonamide monohydrochloride (MK-499), terfenadine, quinidine, and tetrabutylammonium that are differentially affected by mutations in the pore pocket.

Although toxic when inhaled in high concentrations, the gas carbon monoxide (CO) is endogenously produced in mammals, and various beneficial effects are reported. For potential medicinal applications and studying the molecular processes underlying the pharmacological action of CO, so-called CO-releasing molecules (CORMs), such as tricabonyldichlororuthenium(II) dimer (CORM-2), have been developed and widely used. Yet, it is not readily discriminated whether an observed effect of a CORM is caused by the released CO gas, the CORM itself, or any of its intermediate or final breakdown products. Focusing on Ca2+- and voltage-dependent K+ channels (KCa1.1) and voltage-gated K+ channels (Kv1.5, Kv11.1) relevant for cardiac safety pharmacology, we demonstrate that, in most cases, the functional impacts of CORM-2 on these channels are not mediated by CO. Instead, when dissolved in aqueous solutions, CORM-2 has the propensity of forming Ru(CO)2 adducts, preferentially to histidine residues, as demonstrated with synthetic peptides using mass-spectrometry analysis. For KCa1.1 channels we show that H365 and H394 in the cytosolic gating ring structure are affected by CORM-2. For Kv11.1 channels (hERG1) the extracellularly accessible histidines H578 and H587 are CORM-2 targets. The strong CO-independent action of CORM-2 on Kv11.1 and Kv1.5 channels can be completely abolished when CORM-2 is applied in the presence of an excess of free histidine or human serum albumin; cysteine and methionine are further potential targets. Off-site effects similar to those reported here for CORM-2 are found for CORM-3, another ruthenium-based CORM, but are diminished when using iron-based CORM-S1 and absent for manganese-based CORM-EDE1.