Guido Gessner

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.n

Dicarbonyl-bis(cysteamine)iron(II): a light induced carbon monoxide releasing molecule based on iron (CORM-S1).

Carbon monoxide releasing molecules (CORMs) deliver controlled amounts of CO to biological targets and organs. The reaction of cysteamine with triirondodecacarbonyl yields dicarbonyl bis(aminoethylthiolato)iron(II) that represents an iron-based CORM with biogenic ligands. X-ray diffraction studies at a single crystal show a cis-arrangement of the carbonyl ligands in trans-position to the amino groups with average Fe-C and C-O distances of 176.8 and 114.8 pm. The CO release is mediated by irradiation with visible light (λ>400 nm). Physiological tests using ion channels sensitive to CO revealed the light- and time-dependent decomposition of CORM-S1 without obvious adverse effects on the cellular level. CORM-S1 is thus suitable for selective CO release and possesses a high potential for therapeutic application.

Functional distinction of human EAG1 and EAG2 potassium channels

Human ether à 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 (IC50 1.4 μM) more potently than hEAG2 channels (IC50 152 μM), thus providing a useful tool for the functional distinction between hEAG1 and hEAG2 potassium channels.

Molecular mechanism of pharmacological activation of BK channels

Large-conductance voltage- and Ca2+-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 model-based 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 channels gate.

Cysteine 723 in the C-linker segment confers oxidative inhibition of hERG1 potassium channels

Excess reactive oxygen species (ROS) play a crucial role under pathophysiological conditions, such as ischaemia/reperfusion and diabetes, potentially contributing to cardiac arrhythmia. hERG1 (KCNH2) potassium channels terminate the cardiac action potential and malfunction can lead to long‐QT syndrome and fatal arrhythmia. To investigate the molecular mechanisms of hERG1 channel alteration by ROS, hERG1 and mutants thereof were expressed in HEK293 cells and studied with the whole‐cell patch‐clamp method. Even mild ROS stress induced by hyperglycaemia markedly decreased channel current. Intracellular H2O2 or cysteine‐specific modifiers also strongly inhibited channel activity and accelerated deactivation kinetics. Mutagenesis revealed that cysteine 723 (C723), a conserved residue in a structural element linking the C‐terminal domain to the channels gate, is critical for oxidative functional modification. Moreover, kinetics of channel closure strongly influences ROS‐induced modification, where rapid channel deactivation diminishes ROS sensitivity. Because of its fast deactivation kinetics, the N‐terminally truncated splice variant hERG1b possesses greater resistance to oxidative modification.

Inhibition of hEAG1 and hERG1 potassium channels by clofilium and its tertiary analogue LY97241

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. In the whole‐cell mode of mammalian cells, LY97241 was shown to be a potent inhibitor of both hEAG1 and hERG1 channels (IC50 of 4.9 and 2.2 nM, respectively). Clofilium, E4031, and haloperidol apparently inhibited hEAG1 channels with lower potency than hERG1 channels, but they cannot be considered hERG1‐specific. The block of hEAG1 channels by LY97241 and clofilium was time‐, use‐, and voltage‐dependent, best explained by an open‐channel block mechanism. Both drugs apparently bind from the intracellular side of the membrane at (a) specific site(s) within the central cavity of the channel pore. They can be trapped by closure of the activation gate. In inside‐out patches from Xenopus oocytes, hEAG1 block by clofilium was stronger than by LY97241 (IC50 of 0.8 and 1.9 nM, respectively). In addition, hEAG1 block by clofilium was much faster than by LY97241 although there was no difference in the voltage dependence of the on‐rate of block. Physico‐chemical differences of clofilium and the weak base LY97241 determine the access of the drugs to the binding site and thereby the influence of the recording mode on the apparent block potencies. This phenomenon must be considered when assessing the inhibitory action of drugs on ion channels.

BKCa channels activating at resting potential without calcium in LNCaP prostate cancer cells

Large-conductance Ca2+-dependent K+ (BKCa) channels are activated by intracellular Ca2+ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BKCa-type K+ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BKL. K+ selectivity, high conductance, activation by Mg2+ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BKCa channels. However, unlike conventional BKCa channels, BKL channels activated in the absence of free cytosolic Ca2+ at physiological membrane potentials; the half-maximal activation voltage was shifted by about −100 mV compared with BKCa channels. Half-maximal Ca2+-dependent activation was observed at 0.4 μM for BKL (at −20 mV) and at 4.1 μM for BKCa channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BKL conductance. Expression of hSlo-β1 in LNCaP cells shifted voltage-dependent activation to values between that of BKL and BKCa channels and reduced the slope of the Popen (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BKCa subunit, which is responsible for the BKL phenotype in a dominant manner. BKL-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BKL in LNCaP cells is regulated by serum-derived factors, however not by androgens.

A mutually exclusive alternative exon of slo1 codes for a neuronal BK channel with altered function.

Large-conductance Ca2+- and voltage-activated K+ (BK) channels are comprised of four pore-forming α-subunits (Slo1), whose mRNA is alternatively spliced in a cell-specific manner. Here we report the first case of a correctly spliced mutually exclusive exon in a mammalian (human and mouse) BK channel; an exon coding for the region from S6 to the RCK1 domain is exchanged for an alternative exon of the same length. The slo1 transcript with this novel exon is present in native brain tissues and inclusion of the alternative exon profoundly alters the channels gating characteristics: faster activation at low Ca2+ concentrations and greater open probability at resting membrane potential at high Ca2+ concentrations. The novel gating features conferred by the alternative exon are dominant over those of the commonly described Slo1 variant when coexpressed. The evolutionarily-preserved splicing of the Slo1 S6-RCK1 linker segment possess great potential to fine-tune neuronal excitability.

CORM-EDE1: A Highly Water-Soluble and Nontoxic Manganese-Based photoCORM with a Biogenic Ligand Sphere

[Mn(CO)5Br] reacts with cysteamine and 4-amino-thiophenyl with a ratio of 2:3 in refluxing tetrahydrofuran to the complexes of the type [{(OC)3Mn}2(μ-SCH2CH2NH3)3]Br2 (1, CORM-EDE1) and [{(OC)3Mn}2(μ-SC6H4-4-NH3)3]Br2 (2, CORM-EDE2). Compound 2 precipitates during refluxing of the tetrahydrofuran solution as a yellow solid whereas 1 forms a red oil that slowly solidifies. Recrystallization of 2 from water yields the HBr-free complex [{(OC)3Mn}2(μ-S-C6H4-4-NH2)2(μ-SC6H4-4-NH3)] (3). The n-propylthiolate ligand (which is isoelectronic to the bridging thiolate of 1) leads to the formation of the di- and tetranuclear complexes [(OC)4Mn(μ-S-nPr)2]2 and [(OC)3Mn(μ-S-nPr)]4. CORM-EDE1 possesses ideal properties to administer carbon monoxide to biological and medicinal tissues upon irradiation (photoCORM). Isolated crystalline CORM-EDE1 can be handled at ambient and aerobic conditions. This complex is nontoxic, highly soluble in water, and indefinitely stable therein in the absence of air and phosphate buffer. CORM-EDE1 is stable as frozen stock in aqueous solution without any limitations, and these stock solutions maintain their CO release properties. The reducing dithionite does not interact with CORM-EDE1, and therefore, the myoglobin assay represents a valuable tool to study the release kinetics of this photoCORM. After CO liberation, the formation of MnHPO4 in aqueous buffer solution can be verified.

The amiodarone derivative KB130015 activates hERG1 potassium channels via a novel mechanism.

Human ether à go-go related gene (hERG1) potassium channels underlie the repolarizing I(Kr) current in the heart. Since they are targets of various drugs with cardiac side effects we tested whether the amiodarone derivative 2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran (KB130015) blocks hERG1 channels like its parent compound. Using patch-clamp and two-electrode voltage-clamp techniques we found that KB130015 blocks native and recombinant hERG1 channels at high voltages, but it activates them at low voltages. The activating effect has an apparent EC(50) value of 12microM and is brought about by an about 4-fold acceleration of activation kinetics and a shift in voltage-dependent activation by -16mV. Channel activation was not use-dependent and was independent of inactivation gating. KB130015 presumably binds to the hERG1 pore from the cytosolic side and functionally competes with hERG1 block by amiodarone, E4031 (N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl] carbonyl] phenyl] methanesulfonamide dihydrochloride), and sertindole. Vice versa, amiodarone attenuates hERG1 activation by KB130015. Based on synergic channel activation by mallotoxin and KB130015 we conclude that the hERG1 pore contains at least two sites for activators that are functionally coupled among each other and to the cavity-blocker site. KB130015 and amiodarone may serve as lead structures for the identification of hERG1 pore-interacting drugs favoring channel activation vs. block.