Coupling stabilizers open KV1-type potassium channels.

The opening and closing of voltage-gated ion channels are regulated by voltage sensors coupled to a gate that controls the ion flux across the cellular membrane. Modulation of any part of gating constitutes an entry point for pharmacologically regulating channel function. Here, we report on the discovery of a large family of warfarin-like compounds that open the two voltage-gated type 1 potassium (KV1) channels KV1.5 and Shaker, but not the related KV2-, KV4-, or KV7-type channels. These negatively charged compounds bind in the open state to positively charged arginines and lysines between the intracellular ends of the voltage-sensor domains and the pore domain. This mechanism of action resembles that of endogenous channel-opening lipids and opens up an avenue for the development of ion-channel modulators.

Polysaccharides from the Edible Mushroom Agaricus bitorquis (Quél.) Sacc. Chaidam Show Anti-hypoxia Activities in Pulmonary Artery Smooth Muscle Cells.

Three kinds of new water-soluble polysaccharides (FA, FB and FC) were isolated from wild mushroom Agaricus bitorquis (Quél.) Sacc. Chaidam by the classical method "water extraction and alcohol precipitation" and purified by column chromatography. The Mw of FA, FB and FC ranged from 5690 Da to 38,340 Da. The three polysaccharide fractions in the fruiting body were mainly composed of 4 kinds of monosaccharides, including glucose, galactose, mannose, and arabinose, among which glucose and galactose were the major monosaccharides. The FTIR and NMR spectroscopy indicated that the skeleton of three fractions composed of a (1→4)-α-D-glycosidic backbone containing α-D-mannopyranose. In vitro anti-hypoxia activity data showed that three polysaccharide fractions possessed a significant effect on inhibiting PASM cells apoptosis under hypoxia. Among them, FC at the concentration of 200 µg/mL revealed a significant anti-hypoxia effect. These results revealed that the intracellular polysaccharides possessed potent anti-hypoxic activity, which might be related to inhibiting LDH and NADPH oxidase expression and promoting the formation of 5-hydroxytryptamine, dopamine, endothelins, acetylcholine. More importantly, FC showed good performance inducing KV1.5 expression and prohibiting KIR6.2 formation at protein level.

Molecular Determinants of Kv1.3 Potassium Channels-induced Proliferation.

Changes in voltage-dependent potassium channels (Kv channels) associate to proliferation in many cell types, including transfected HEK293 cells. In this system Kv1.5 overexpression decreases proliferation, whereas Kv1.3 expression increases it independently of K(+) fluxes. To identify Kv1.3 domains involved in a proliferation-associated signaling mechanism(s), we constructed chimeric Kv1.3-Kv1.5 channels and point-mutant Kv1.3 channels, which were expressed as GFP- or cherry-fusion proteins. We studied their trafficking and functional expression, combining immunocytochemical and electrophysiological methods, and their impact on cell proliferation. We found that the C terminus is necessary for Kv1.3-induced proliferation. We distinguished two residues (Tyr-447 and Ser-459) whose mutation to alanine abolished proliferation. The insertion into Kv1.5 of a sequence comprising these two residues increased proliferation rate. Moreover, Kv1.3 voltage-dependent transitions from closed to open conformation induced MEK-ERK1/2-dependent Tyr-447 phosphorylation. We conclude that the mechanisms for Kv1.3-induced proliferation involve the accessibility of key docking sites at the C terminus. For one of these sites (Tyr-447) we demonstrated the contribution of MEK/ERK-dependent phosphorylation, which is regulated by voltage-induced conformational changes.

Activation of voltage gated K⁺ channel Kv1.5 by ß-catenin.

Voltage-gated Kv1.5 channels are expressed in a wide variety of tissues including cardiac myocytes, smooth muscle and tumor cells. Kv1.5 channel activity is modified by N-cadherin, which in turn binds the multifunctional oncogenic protein ß-catenin. The present experiments explored the effect of ß-catenin on Kv1.5 channel activity. To this end, Kv1.5 was expressed in Xenopus oocytes with or without ß-catenin and the voltage-gated Kv current determined by dual electrode voltage clamp. As a result, expression of ß-catenin significantly increased the voltage-gated Kv current at positive potentials. The stimulating effect of ß-catenin on Kv1.5 was not dependent on the stimulation of transcription since it was observed even in the presence of the transcription inhibitor actinomycin D. Specific antibody binding to surface Kv1.5 in Xenopus oocytes revealed that ß-catenin enhances the membrane abundance of Kv1.5. Further experiments with brefeldin A showed that ß-catenin fosters the insertion of Kv1.5 into rather than delaying the retrieval from the plasma membrane. According to electrophysiological recordings with mutant ß-catenin, the effect on Kv1.5 requires the same protein domains that are required for association of ß-catenin with cadherin. The experiments disclose a completely novel function of ß-catenin, i.e. the regulation of Kv1.5 channel activity.