rectifying potassium channels A thesis submitted to the University of Leicester for the deg Doctor of Philosophy 2003 Gayle Martine Passmore BSc (Leicester) Department of Cell Physiology & Pharmacology University of Leicester UMI Number: U4955B9 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U495539 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 For Nan Abstract The patch clamp technique was used to investigate the effect of mutations in the P-region on permeation characteristics and ionic selectivity in members of the Kir2.0 subfamily. Kjr2.2 exhibits electrophysiological characteristics similar to those of Kir2.1 though there are differences in both unitary conductance and the kinetics of Ba2+ blockage. The P-region of these channels is virtually identical with the exception that leucine (L) at position 148 in Kjr2.2 is replaced by phenylalanine (F) in Kir2.1. The effects of mutating L148 to phenylalanine in Kjr2.2 and F147 to leucine in Kir2.1 on unitary conductance and channel sensitivity to Ba2+ were investigated. Neither mutation altered unitary conductance from that seen in the wild-type channel. However, mutation L148F in Kjr2.2 reduced the association rate constant for Ba2+ blockage without affecting affinity. In contrast, mutation F147L in Kjr2.1 increased channel affinity for Ba2+ without affecting the association rate constant. Thus, residues outside the P-region are responsible for the difference in unitary conductance and some of the differences in Ba2+ block in Kjr2.2 and Kir2.1. The effects on ionic selectivity of substituting single amino acid residues at position 143 in the P-region of Kir2.1 were also investigated. Substitution of isoleucine by hydrophobic residues such as valine (I143V) and leucine (I143L) raised the relative Rb+ permeability, whilst substitution of the more hydrophilic residue threonine (I143T) enhanced K+ selectivity. Two further mutants, I143C and I143S, failed to yield currents, but could be rescued by bathing cells in extracellular solution containing lOmM dithiothreitol (DTT). The permeability ratios were then similar to wild-type. The rescue of mutant channels by DTT suggests that the pore of Kjr channels may undergo conformational changes. In vitro translation studies suggest that channel function is rescued through the disruption of an intra­ subunit disulphide bond. Acknowledgements First, and foremost, I would like to express my sincere thanks to my supervisor Professor Peter Stanfield for his advice and assistance throughout the duration of my PhD, and also for his patience during the latter three years. Also, thanks to Dr Noel Davies for his help with Tracan and general problem solving, Dr Ian Ashmole for his help and ideas with the molecular biology and biochemistry, Dr Mike Sutcliffe for his Kn channel modelling and Drs Blair Grubb and Robert Norman for their help with Western blotting. I would also like to thank Drs Chris Abrams, Gareth Thompson, Michael Holland and Christina Skipper who gave me much needed advice in the laboratory on a daily basis. The members of my new lab at UCL have provided me with much encouragement and I would particularly like to thank Professor David Brown and Dr Steve Marsh for their continued support over the last three years. Last, but by no means least, I would like to thank my parents for all of their loving support over the years, without which it would have been impossible to achieve this goal. iv Table of Contents Title Page i Dedication ii Abstract iii Acknowledgements iv Table of Contents V List of Figures xi List of Tables xiii Abbreviations ? Chapter 1 - Introduction 1 1.0. General Introduction 1 1.1. Ion Channels 4 1.1.1. A historical perspective of ion channels 4 1.1.2. Classification of ion channels 7 1.1.3. A role for potassium channels 7 1.2. Potassium channel diversity 9 1.2.1. Early potassium channel cloning studies 11 1.2.2. The three classes of potassium channel 13 1.2.3. Six-transmembrane domain potassium channels 13 1.2.3a. Shaker -related potassium channels 13 Gating 14 Inactivation 15 13-subunits 20 1.2.3b. Calcium-activated potassium channels (KCa) 20 BKca channels 20 SKca channels 22 IKca channels 22 1.2.3c. Eag-like potassium channels 23 1.2.3d. KCNQ potassium channel family 24 1.2.4. Two-transmembrane domain potassium channels 25 1.2.4a. Kir 1.0 subfamily 26 Tissue distribution of Kir1.0 subfamily channels 26 Properties of Kir1.0 subfamily channels 27 Role o f Kirl . 0 subfamily channels 28 1.2.4b. Kir2.0 subfamily 28 Kir2J 28 Kir2.2 30 Kir23 31 Kir2A 32 Kir2.0 channels interact with anchoring proteins 32 Modulation of Kir2.0 subfamily channels 33 1.2.4c. Kir3.0 subfamily 34 Tissue distribution 34 Kaoi is a heteromultimer o f Kir3.1 and Ktr3.4 35 Neuronal G-protein-gated inward rectifier potassium Channels 35 Modulation ofKir3.0 subfamily channels 36 1.2.4d. Kir4.0 subfamily 37 Biophysical properties of KiA.O subfamily channels 37 Distribution and role of Ki/l-0 subfamily channels 38 KiA-0 channels interact with anchoring proteins 39 Heteromeric Ki/l.x channels display distinct biophysical properties 39 1.2.4e. Kir5.0 subfamily 40 Distribution and role o f Kir5.1 /PSD-95 40 Kir5.1 heteromers 40 Distribution and role of heteromeric Ki/l.x-Kir5.1 Channels 41 1.2.4f. Kir6.0 subfamily 41 Role o f K a t p channels 42 Properties ofKi,6.0 channels 42 Sulphonylurea receptors 43 Structure and properties ofKATP channels 44 1.2.4g. Kir7.0 subfamily 44 Biophysical properties ofKir7.1 45 Distribution and role of Kir7.1 45 1.2.4h. Drosophila Kirs 46 1.2.4i. A family of Kir channels in prokaryotes 46 1.2.5. Two-pore domain potassium channels 46 1.3. Assembly and stoichiometry of potassium channels 48 VI 1.3.1. Kv channels assemble as tetramers 48 1.3.2. The T1 domain is required for tetramerization of Kv channels 49 1.3.3. Kir channels assemble as tetramers 50 1.3.4. channels can form heteromers 50 1.3.5. Structural determinants of Kjr channel assembly 51 1.4. Inward rectification 52 1.4.1. Properties of inward rectifier potassium channels 53 1.4.1a. Inward rectification depends on Vm and the external K+ concentration 53 1.4.1b. The conductance is proportional to the square root of [K+]0 55 1.4.1c. Activation 55 1.4.Id. Inactivation 55 1.4. le. Block by external cations 56 1.4. If. Anomalous mole fraction effects 57 1.4.2. Mechanism of inward rectification 57 1.4.2a. The K-activated K-channel model 58 1.4.2b. The blocking particle model 59 1.4.2c. M g 2+block 60 1.4.2d. Polyamine block 60 1.4.2e. Intrinsic gating 63 1.4.2f. Do Kir channels exhibit intrinsic rectification? 66 1.4.3. Structural determinants of inward rectification 67 1.5. Physiological importance of Kir channels 69 1.5.1. K^ channels underlie K+ homeostasis in the kidney 69 1.5.2. K^ channels underlie K+-induced vasodilation 70 1.5.3. K^ channels regulate cardiac action potentials 70 1.5.4. Kir channels help regulate the heartbeat 71 1.5.5. K^ channels regulate blood glucose levels 71 1.5.6. Kir channels are important in development 73 1.6. Selectivity 75 1.6.1. Structural determinants of K+ selectivity in Kv channels 75 1.6.2. Theories of selectivity 77 1.6.3. Molecular basis of selectivity 78 1.7. Aims 81 vii Chapter 2 - Materials & Methods 82 2.1. Molecular biology 82 2.1.1. Cloning and site-directed mutagenesis 82 2.1.2. Purification of plasmid DNA 83 2.2. Cell culture 83 2.2.1. Cell culture reagents and plastic-ware 83 2.2.2. Incubator 83 2.2.3. Cell lines 84 2.2.4. Growth of cells in culture 84 2.2.5. Chinese hamster ovary (CHO) cells 84 2.2.5a. Routine growth and maintenance of CHO cells 85 2.2.5b. Subculture of CHO cells 85 2.2.5c. Preparation of CHO cells for electrophysiological recording 86 2.2.6. Murine erythroleukaemia (MEL) cells 86 2.2.6a. Routine growth and maintenance of MEL cells 87 2.2.6b. Subculture of MEL cells 87 2.2.6c. Induction of MEL cells 87 2.2.6d. Preparation of MEL cells for electrophysiological recording 87 2.2.7. Storage of cell lines 88 2.2.8. Haemocytometer cell counts 89 2.3. Transfection Methods 89 2.3.1. Lipid-mediated transfection 90 2.3.2. Transient transfection of CHO cells using PerFect Lipid™ 90 2.3.3. Transient transfection of CHO cells using FuGENE™ 6 91 2.3.4. Levels of expression 91 2.3.4a.The amount of cDNA used for PerFect Lipid transfections was reduced 92 2.3.4b.The incubation time for Perfect Lipid transfections was reduced 92 2.3.4c. A weaker promoter was used to drive expression 92 2.3.4d.