Conformational Changes in a Mammalian Voltage-Dependent Potassium Channel Inactivation Peptide †
University of KwaZulu-Natal, Port Natal, KwaZulu-Natal, South Africa Biochemistry
(Impact Factor: 3.02).
03/1998; 37(6):1640-5. DOI: 10.1021/bi972350c
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.
Available from: Tetsu Urano
- " CLSM images show fibrin network (green) formation in the presence of platelets labeled with R-6G (red). TF addition evoked fibrin matrix assembly and concurrent immediate binding of the fibrin/fibrinogen by platelets (white arrows) followed by progressive PS exposure on the platelet surface which was detected by platelet ANX (white) binding. "
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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 02/2013; 8(2):e55466. DOI:10.1371/journal.pone.0055466 · 3.23 Impact Factor
Available from: biochem.wisc.edu
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ABSTRACT: Several methods for determination of the secondary structure of proteins by spectroscopic measurements are reviewed. Circular dichroism (CD) spectroscopy provides rapid determinations of protein secondary structure with dilute solutions and a way to rapidly assess conformational changes resulting from addition of ligands. Both CD and Raman spectroscopies are particularly useful for measurements over a range of temperatures. Infrared (IR) and Raman spectroscopy require only small volumes of protein solution. The frequencies of amide bands are analyzed to determine the distribution of secondary structures in proteins. NMR chemical shifts may also be used to determine the positions of secondary structure within the primary sequence of a protein. However, the chemical shifts must first be assigned to particular residues, making the technique considerably slower than the optical methods. These data, together with sophisticated molecular modeling techniques, allow for refinement of protein structural models as well as rapid assessment of conformational changes resulting from ligand binding or macromolecular interactions. A selected number of examples are given to illustrate the power of the techniques in applications of biological interest.
Analytical Biochemistry 02/2000; 277(2):167-76. DOI:10.1006/abio.1999.4320 · 2.22 Impact Factor
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ABSTRACT: Historically, much attention has focused on the mechanisms of activity-dependent plasticity since the description of long-term potentiation by Bliss and Lomo in the early 1970s, while extrasynaptic changes have received much less interest. However, recent work has concentrated on the role of back-propagating action potentials in hippocampal dendrites in synaptic plasticity. In this review, we focus on the modulation of back-propagating action potentials by K+ currents in the dendrites of hippocampal cells. We described the primary K+-channel subunits and their interacting subunits that most likely contribute to these currents, and how these sites can be regulated by phosphorylation and other mechanisms. In conclusion, we provide a model for an alternative form of coincidence detection through K+ channels in the hippocampus.
Molecular Neurobiology 03/2002; 25(1):51-66. DOI:10.1385/MN:25:1:051 · 5.14 Impact Factor
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