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The Interaction Between the Voltage-Gated Potassium Channel Hkv1.3 and Verapamil

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The Interaction Between the Voltage-Gated Potassium Channel Hkv1.3 and Verapamil Ulm University Institute of Applied Physiology Prof. Dr. Birgit Liss The interaction between the voltage-gated potassium channel hKv1.3 and verapamil Dissertation to obtain the doctoral degree of Human Biology (Dr. biol. hum.) of the Medical Faculty of Ulm University Ann-Kathrin Diesch Riedlingen 2017 Amtierender Dekan: Prof. Dr. Thomas Wirth 1. Berichterstatter: Prof. Dr. Stephan Grissmer 2. Berichterstatter: PD. Dr. Karl Föhr Tag der Promotion: 26.01.2018 ii Widmung aus Gründen des Datenschutzes entfernt. iii Table of Content Abbreviation ................................................................................................... vi 1 Introduction ..................................................................................................... 1 1.1 Ion channels ............................................................................................... 1 1.2 Potassium channel classes ....................................................................... 2 1.3 Voltage-gated potassium channels ........................................................... 4 1.3.1 Structure of voltage-gated potassium channels .............................. 4 1.3.2 Opening and closing of the channel .................................................. 6 1.3.3 Transduction of voltage sensing to an open conformation .............. 7 1.3.4 Inactivation in potassium channels .................................................. 8 1.3.4.1 N-type inactivation ................................................................ 9 1.3.4.2 C-type inactivation ............................................................... 12 1.3.5 Ion selectivity.................................................................................... 15 1.3.6 Verapamil ........................................................................................ 18 1.4 Aim of the study ....................................................................................... 19 2 Materials and Methods ................................................................................. 20 2.1 Molecular Biology methods ...................................................................... 20 2.1.1 Solutions and reagents ................................................................... 20 2.1.2 DNA clone and amplification .......................................................... 20 2.2 Cell culture ................................................................................................ 21 2.2.1 Solutions and reagents ................................................................... 21 2.2.2 Cell Culture ..................................................................................... 22 2.2.3 Transfection .................................................................................... 22 2.2.4 Cell preparation for electrophysiological measurements .............. 23 2.3 Electrophysiology ..................................................................................... 24 2.3.1 Solutions used for the electrophysiological measurements .......... 24 2.3.2 The patch-clamp set-up .................................................................. 24 2.4 Analysis .................................................................................................... 27 2.4.1 Fitting equations .............................................................................. 27 2.4.2 The kinetic model used for simulations of the current traces ........ 28 iv 2.4.3 The molecular model of docking verapamil in the hKv1.3 mutant channel .............................................................................................. 29 3 Results ........................................................................................................... 30 3.1 Effect of verapamil on current through hKv1.3 wild-type channels ......... 30 3.2 Varying verapamil concentrations and their effect on current through hKv1.3 wild-type channels ........................................................................ 33 3.3 The relationship between the current decay time constant and the on-rate constant ....................................................................................... 36 3.4 On-rate constants of different hKv1.3 channels in the presence of 30 µM verapamil ....................................................................................... 39 3.5 Three-dimensional model of the hKv1.3 mutant channel ....................... 44 4 Discussion ..................................................................................................... 46 4.1 Simulated current traces verify my assumption that off can be neglected ................................................................................................. 46 4.2 The reason why I did not use the reduction of steady-state peak current as a measure of verapamil’s affinity to block current through open hKv1.3 channels ............................................................................... 48 4.3 Effect of verapamil on current through hKv1.3_T419 mutant channels . 49 4.4 The interference of the cysteines at position 417 and 418 in the hKv1.3 channel with verapamil ................................................................. 50 4.5 Does position 346 in the hKv1.3 channel indirectly influence verapamil to reach its blocking site? ......................................................................... 51 4.6 Major effect on the on-rate of verapamil induced by position 420 in the hKv1.3 channel .......................................................................................... 52 5 Summary ........................................................................................................ 53 6 References ..................................................................................................... 55 Acknowledgements Curriculum vitae v Abbreviations A Ampère KF Potassium fluoride Å Angstrom KOH Potassium hydroxide AMP Ampicillin Kv Voltage-gated potassium B Blocked state channel CaCl2 Calcium chloride l Liter Cat - No. Catalog number LB Lysogeny broth C Closed state M Molar C Cysteine MΩ Megaohm °C Degree Celsius MgCl2 Magnesium chloride DMEM Dulbecco’s Modified Eagle m Milli- Medium min Minute DMSO Dimethyl sulfoxide mM Millimolar EDTA Ethylenediaminetetra- ml Milliliter acetic acid ms Millisecond G Glycine MTSEA 2-aminoethyl methane g Gram thiosulfonate GFP Green fluorescent protein mV Millivolt H Histidine n Number of experiments h Rate constant of inactivation Na+ Sodium ion h human nA Nanoampere HEPES 4-(2-hydroxylethyl)- 1 Na-Ri Normal Ringer Piperazineethanesulfonic O Open state acid OB Open-blocked state HP Holding potential Osm Osmolarity I Current amplitude P Pore I Inactivated state PAA Phenylalkylamine K+ Potassium ion PDB Protein Data Bank KCl Potassium chloride s Second KD Dissociation constant S1-S4 Voltage sensing domain vi S1-6 Transmembrane Helix j Time constant of inactivation SF Selectivity filter in Na-Ringer solution Tau TEA+ Tetraethylammonium decay Time constant of current TM Transmembrane decay V Volt n Time constant of activation VP Verapamil wt Wild-type . vii 1 Introduction 1.1 Ion channels All cells are surrounded by an outer plasma membrane consisting of a phospholipid bilayer. This thin membrane forms a stable barrier that isolates the living cell from its outside environment. But it also means that in parts, substrates necessary for the cell but also ionized wasted products, are retained and can neither enter nor leave the cell which is crucial for its survival. The plasma membrane is highly permeable to lipid soluble, nonpolar molecules whereas charged, polar molecules cannot pass through the membrane. Thus to guarantee the exchange of nutrients, different transmembrane proteins are embedded in the membrane. Some of these membrane proteins form ion channels, macromolecular pores providing a water filled diffusion path through the lipid layer (Hille, 2001). Ion channels are allosteric proteins that can be opened and closed by different stimuli either mechanically or chemically or voltage controlled. The macromolecular pores enable for example Cl-, Na+, K+ and Ca2+ ions to pass highly selectively through the membrane. Ion channels play an important role in a broad range of cellular processes including cell growth, regulation of apoptosis, the release of neurotransmitters and hormones and the functioning of excitable cells (Hoshi et al., 1990; Bosma and Hille, 1992; Deutsch and Chen, 1993; Yellen, 2002; Pal et al., 2006). Malfunction of ion channels can cause different severe diseases. Therefore enhanced knowledge about the different structure and function of ion channels might help to determine the cause of the disorder and to develop highly selective agents for the treatment of the diseases. In the following sections I will describe one group of ion channels, the potassium channels because I performed the experiments described in my thesis on potassium channels. - 1 - 1.2 Potassium channel classes Membrane proteins can be associated with the plasma membrane in different ways. Potassium channels belong to the transmembrane proteins representing one possibility to associate with the membrane. Transmembrane proteins extend across the membrane with parts of the channel protein on either side of the cell membrane. Regarding the number of their transmembrane helices (Sokolova, 2004) potassium channels can be divided into four main classes shown in Fig. 1. A B C D Figure 1 Four different classes of potassium channels. A, Two transmembrane helices channel (2TM/P
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