Conformational changes in a mammalian voltage-dependent potassium channel inactivation peptide.
ABSTRACT Fast inactivation is restored in inactivation deletion mutant voltage-gated potassium (Kv) channels by application of synthetic inactivation 'ball' peptide. Using Fourier transform infrared and circular dichroism spectroscopy, we have investigated the structure of synthetic Kv3.4 channel ball peptide, in a range of environments relevant to the function of the ball domain. The ball peptide contains no alpha-helix or beta-sheet in reducing conditions in aqueous solution, but when cosolubilized with anionic lipid or detergent in order to mimic the environment which the ball domain encounters during channel inactivation, the ball peptide adopts a partial beta-sheet structure. Oxidation of the Kv3.4 ball peptide facilitates formation of a disulfide bond between Cys6 and Cys24 and adoption of a partial beta-sheet structure in aqueous solution; the tendency of the oxidized ball peptide to adopt beta-sheet is generally greater than that of the reduced ball peptide in a given environment. THREADER modeling of the Kv3.4 ball peptide structure predicts a beta-hairpin-like conformation which corresponds well to the structure suggested by spectroscopic analysis of the ball peptide in its cyclic arrangement. A V7E mutant Kv3.4 ball peptide analogue of the noninactivating Shaker B L7E mutant ball peptide cannot adopt beta-structure whatever the environment, and regardless of oxidation state. The results suggest that the Kv3.4 ball domain undergoes a conformational change during channel inactivation and may implicate a novel regulatory role for intramolecular disulfide bond formation in the Kv3.4 ball domain in vivo.
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ABSTRACT: Recent studies have explored the utility of Fourier transform infrared spectroscopy (FTIR) in dynamic monitoring of soluble protein-protein interactions. Here, we investigated the applicability of FTIR to detect interaction between synthetic soluble and phospholipid-embedded peptides corresponding to, respectively, a voltage-gated potassium (Kv) channel inactivation domain (ID) and S4-S6 of the Shaker Kv channel (KV1; including the S4-S5 linker "pre-inactivation" ID binding site). KV1 was predominantly α-helical at 30°C when incorporated into dimyristoyl-l-α-phosphatidylcholine (DMPC) bilayers. Cooling to induce a shift in DMPC from liquid crystalline to gel phase reversibly decreased KV1 helicity, and was previously shown to partially extrude a synthetic S4 peptide. While no interaction was detected in liquid crystalline DMPC, upon cooling to induce the DMPC gel phase a reversible amide I peak (1633 cm(-1)) consistent with novel hydrogen bond formation was detected. This spectral shift was not observed for KV1 in the absence of ID (or vice versa), nor when the non-inactivating mutant V7E ID was applied to KV1 under similar conditions. Alteration of salt or redox conditions affected KV1-ID hydrogen bonding in a manner suggesting electrostatic KV1-ID interaction favored by a hairpin conformation for the ID and requiring extrusion of one or more KV1 domains from DMPC, consistent with ID binding to S4-S5. These findings support the utility of FTIR in detecting reversible interactions between soluble and membrane-embedded proteins, with lipid state-sensitivity of the conformation of the latter facilitating control of the interaction.PLoS ONE 01/2012; 7(11):e49070. · 3.73 Impact Factor
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ABSTRACT: Fine-tuned regulation of K(+) channel inactivation enables excitable cells to adjust action potential firing. Fast inactivation present in some K(+) channels is mediated by the distal N-terminal structure (ball) occluding the ion permeation pathway. Here we show that Kv1.4 K(+) channels are potently regulated by intracellular free heme; heme binds to the N-terminal inactivation domain and thereby impairs the inactivation process, thus enhancing the K(+) current with an apparent EC50 value of ∼20 nM. Functional studies on channel mutants and structural investigations on recombinant inactivation ball domain peptides encompassing the first 61 residues of Kv1.4 revealed a heme-responsive binding motif involving Cys13:His16 and a secondary histidine at position 35. Heme binding to the N-terminal inactivation domain induces a conformational constraint that prevents it from reaching its receptor site at the vestibule of the channel pore.Proceedings of the National Academy of Sciences 09/2013; · 9.74 Impact Factor
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ABSTRACT: Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor- and thrombin-treated samples but not in ionomycin-treated samples. FK633, an α(IIb)β(3) antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment.PLoS ONE 01/2013; 8(2):e55466. · 3.73 Impact Factor