Min Ru Qiu

Inhibition of the human ether-a-go-go-related gene (HERG) potassium channel by cisapride : affinity for open and inactivated states

Cisapride is a prokinetic agent which has been associated with QT prolongation, torsades de pointes and cardiac arrest. The cellular mechanism for these observations is high affinity blockade of IKr (encoded by HERG). In a chronic transfection model using CHO‐K1 cells, cisapride inhibited HERG tail currents after a step to +25 mV with similar potency at room and physiological temperatures (IC50 16.4 nM at 20–22°C and 23.6 nM at 37°C). Channel inhibition exhibited time‐, voltage‐ and frequency‐dependence. In an envelope of tails test, channel blockade increased from 27±8% after a 120 ms depolarizing step to 50±4% after a 1.0 s step. These findings suggested affinity for open and/or inactivated channel states. Inactivation was significantly accelerated by cisapride in a concentration‐dependent manner and there was a small (−7 mV) shift in the voltage dependence of steady state inactivation. Channel blockade by cisapride was modulated by [K+]o, with a 26% reduction in the potency of channel blockade when [K+]o was increased from 1 to 10 mM. In conclusion, HERG channel inhibition by cisapride exhibits features consistent with open and inactivated state binding and is sensitive to external potassium concentration. These features may have significant clinical implications with regard to the mechanism and treatment of cisapride‐induced proarrhythmia.

The nuclear chloride ion channel NCC27 is involved in regulation of the cell cycle

1 NCC27 is a nuclear chloride ion channel, identified in the PMA‐activated U937 human monocyte cell line. NCC27 mRNA is expressed in virtually all cells and tissues and the gene encoding NCC27 is also highly conserved. Because of these factors, we have examined the hypothesis that NCC27 is involved in cell cycle regulation. 2 Electrophysiological studies in Chinese hamster ovary (CHO‐K1) cells indicated that NCC27 chloride conductance varied according to the stage of the cell cycle, being expressed only on the plasma membrane of cells in G2/M phase. 3 We also demonstrate that Cl− ion channel blockers known to block NCC27 led to arrest of CHO‐K1 cells in the G2/M stage of the cell cycle, the same stage at which this ion channel is selectively expressed on the plasma membrane. 4 These data strongly support the hypothesis that NCC27 is involved, in some as yet undetermined manner, in regulation of the cell cycle.

Inhibition of HERG channels stably expressed in a mammalian cell line by the antianginal agent perhexiline maleate.

Perhexiline has been used as an anti‐anginal agent for over 25 years, and is known to cause QT prolongation and torsades de pointes. We hypothesized that the cellular basis for these effects was blockade of IKr. A stable transfection of HERG into a CHO‐K1 cell line produced a delayed rectifier, potassium channel with similar properties to those reported for transient expression in Xenopus oocytes. Perhexiline caused voltage‐ and frequency‐dependent block of HERG (IC50 7.8 μM). The rate of inactivation was increased and there was a 10 mV hyperpolarizing shift in the voltage‐dependence of steady‐state inactivation, suggestive of binding to the inactivated state. In conclusion, perhexiline potently inhibits transfected HERG channels and this is the probable mechanism for QT prolongation and torsades de pointes. Channel blockade shows greatest affinity for the inactivated state.

Generation and characterization of mice with null mutation of the chloride intracellular channel 1 gene.

CLIC1 belongs to a family of highly conserved and widely expressed intracellular chloride ion channel proteins existing in both soluble and membrane integrated forms. To study the physiological and biological role of CLIC1 in vivo, we undertook conditional gene targeting to engineer Clic1 gene knock‐out mice. This represents creation of the first gene knock‐out of a vertebrate CLIC protein family member. We first generated a Clic1 Knock‐in (Clic1FN) allele, followed by Clic1 knock‐out (Clic1−/−) mice by crossing Clic1FN allele with TNAP‐cre mice, resulting in germline gene deletion through Cre‐mediated recombination. Mice heterozygous or homozygous for these alleles are viable and fertile and appear normal. However, Clic1−/− mice show a mild platelet dysfunction characterized by prolonged bleeding times and decreased platelet activation in response to adenosine diphosphate stimulation linked to P2Y12 receptor signaling. genesis 48:127–136, 2010.

Blockade by N-3 polyunsaturated fatty acid of the Kv4.3 current stably expressed in Chinese hamster ovary cells.

The Kv4.3 gene is believed to encode a large proportion of the transient outward current (Ito), responsible for the early phase of repolarization of the human cardiac action potential. There is evidence that this current is involved in the dispersion of refractoriness which develops during myocardial ischaemia and which predisposes to the development of potentially fatal ventricular tachyarrhythmias. Epidemiological, clinical, animal, and cellular studies indicate that these arrhythmias may be ameliorated in myocardial ischaemia by n‐3 polyunsaturated fatty acids (n‐3 PUFA) present in fish oils. We describe stable transfection of the Kv4.3 gene into a mammalian cell line (Chinese hamster ovary cells), and using patch clamp techniques have shown that the resulting current closely resembles human Ito. The current is rapidly activating and inactivating, with both processes being well fit by double exponential functions (time constants of 3.8±0.2 and 5.3±0.4 ms for activation and 20.0±1.2 and 96.6±6.7 ms for inactivation at +45 mV at 23°C). Activation and steady state inactivation both show voltage dependence (V1/2 of activation=−6.7±2.5 mV, V1/2 of steady state inactivation=−51.3±0.2 mV at 23°C). Current inactivation and recovery from inactivation are faster at physiologic temperature (37°C) compared to room temperature (23°C). The n‐3 PUFA docosahexaenoic acid blocks the Kv4.3 current with an IC50 of 3.6 μmol L−1. Blockade of the transient outward current may be an important mechanism by which n‐3 PUFA provide protection against the development of ventricular fibrillation during myocardial ischaemia.

Macrophage Inhibitory Cytokine-1 (MIC-1/GDF15) Slows Cancer Development but Increases Metastases in TRAMP Prostate Cancer Prone Mice

Macrophage inhibitory cytokine-1 (MIC-1/GDF15), a divergent member of the TGF-β superfamily, is over-expressed by many common cancers including those of the prostate (PCa) and its expression is linked to cancer outcome. We have evaluated the effect of MIC-1/GDF15 overexpression on PCa development and spread in the TRAMP transgenic model of spontaneous prostate cancer. TRAMP mice were crossed with MIC-1/GDF15 overexpressing mice (MIC-1fms) to produce syngeneic TRAMPfmsmic-1 mice. Survival rate, prostate tumor size, histopathological grades and extent of distant organ metastases were compared. Metastasis of TC1-T5, an androgen independent TRAMP cell line that lacks MIC-1/GDF15 expression, was compared by injecting intravenously into MIC-1fms and syngeneic C57BL/6 mice. Whilst TRAMPfmsmic-1 survived on average 7.4 weeks longer, had significantly smaller genitourinary (GU) tumors and lower PCa histopathological grades than TRAMP mice, more of these mice developed distant organ metastases. Additionally, a higher number of TC1-T5 lung tumor colonies were observed in MIC-1fms mice than syngeneic WT C57BL/6 mice. Our studies strongly suggest that MIC-1/GDF15 has complex actions on tumor behavior: it limits local tumor growth but may with advancing disease, promote metastases. As MIC-1/GDF15 is induced by all cancer treatments and metastasis is the major cause of cancer treatment failure and cancer deaths, these results, if applicable to humans, may have a direct impact on patient care.

A potassium ion channel is involved in cytokine production by activated human macrophages

Macrophages play an important role in immune and inflammatory responses, largely through secretion of bioactive molecule such as cytokines. While calcium is known to be an important regulator of this process, less is known about the role of other ions and the ion channels that regulate them. We have previously implicated an outwardly rectifying potassium channel (Kor) in this process and for this reason we have investigated the role of potassium (K+) and K+ channels in the regulation of tumour necrosis factor‐α (TNF‐α)and interleukin (IL)‐8 production by activated human culture‐derived macrophages. The effect of blockade of Kor is to inhibit phorbol myristate acetate (PMA)‐induced cytokine production by translational or post‐translational mechanisms, an effect that is duplicated by increasing extracellular K+. By contrast, the effects of K+ on LPS‐stimulated cells are far more complex and are probably mediated through the change of osmolality and occur largely at the mRNA level. This data directly implicates K+, and its regulation through Kor, in early events following PMA stimulation of these cells.