Regulation of Myometrial Contractility: Defining the Contribution of the MaxiK Potassium Channel and the L- and T-type Calcium Channels. Sarah L. D. Waite B.Sc. (Hons) Submitted to The University of Sheffield for the degree of Doctor of Philosophy Academic Unit of Reproductive and Developmental Medicine Department of Human Metabolism The University of Sheffield 2015 1 2 Table of Contents TABLE OF FIGURES ........................................................................................................................10 TABLE OF TABLES ..........................................................................................................................12 ACKNOWLEDGEMENTS ................................................................................................................14 PUBLICATIONS ARISING FROM THIS THESIS .........................................................................16 PUBLICATIONS ASSOCIATED WITH THIS THESIS ..................................................................16 ABSTRACT ........................................................................................................................................18 ABBREVIATIONS .............................................................................................................................20 CHAPTER 1 INTRODUCTION ........................................................................................................24 1.1 REPRODUCTION ......................................................................................................................... 25 1.1.1 Parturition as a Controlled Inflammatory Event ...................................................................... 25 1.1.2 Sterile Inflammation ................................................................................................................. 26 1.2 REGULATION OF THESE PARTURITION EVENTS ......................................................................... 26 1.2.1 The Role of Surfactant Protein A (SP-A) .................................................................................. 28 1.2.2 Matrix Metalloproteinase (MMP) Expression .......................................................................... 30 1.2.3 Prostaglandin Synthesis ........................................................................................................... 31 1.2.4 Transcriptional Regulation by NFκB ....................................................................................... 33 1.2.5 Progesterone Withdrawal ......................................................................................................... 35 1.2.6 Ion Channel Expression............................................................................................................ 36 1.2.6.1 The MaxiK Channel (KCNMA1) .......................................................................................................37 1.2.6.2 L-Type Calcium Channel (CACNA1C) .............................................................................................37 1.2.7 Summary of Current Theories .................................................................................................. 37 1.3 PRETERM BIRTH ........................................................................................................................ 38 1.3.1 Current Tocolytic Therapies ..................................................................................................... 38 1.3.1.1 β2 Agonists .........................................................................................................................................39 1.3.1.2 Calcium Channel Antagonists ............................................................................................................40 1.3.1.3 Oxytocin Receptor Antagonist ...........................................................................................................41 1.3.1.4 Progesterone .......................................................................................................................................42 1.3.1.5 Summary ............................................................................................................................................42 1.4 SMOOTH MUSCLE STRUCTURE .................................................................................................. 43 1.4.1 Molecular Basis of Smooth Muscle Contraction ...................................................................... 44 1.4.2. Stimulation of Smooth Muscle Contraction ............................................................................. 46 1.4.3 Myometrial Contractility .......................................................................................................... 47 1.4.3.1 Resting Membrane Potential ..............................................................................................................47 1.4.3.2 Generation of Action Potentials .........................................................................................................49 1.4.3.3 Return to Resting Membrane Potential...............................................................................................49 1.4.4 Regulation of Uterine Contractility .......................................................................................... 50 1.5 CALCIUM ACTIVATED POTASSIUM CHANNELS AND VOLTAGE-DEPENDANT CALCIUM CHANNELS WITHIN THE MYOMETRIUM .................................................................................................. 51 1.5.1 The KCNMA1 (BK or slo-1) Channel ....................................................................................... 51 1.5.1.1 Calcium Sensitivity in the KCNMA1 Channel ...................................................................................52 1.5.1.2 Voltage Sensitivity in the KCNMA1 Channel ...................................................................................53 1.5.1.3 Splice Variants in the KCNMA1 Channel ..........................................................................................54 1.5.1.4 KCNMA1 Channel Expression During Gestation ..............................................................................58 1.5.1.5 Regulation of KCNMA1 Expression ..................................................................................................60 1.5.1.6 Regulation of Splice Variant Expression ............................................................................................61 1.5.2 Voltage-Dependant Calcium Channels .................................................................................... 61 1.5.2.1 Calcium Selectivity in the Voltage-Dependant Calcium Channel ......................................................62 1.5.2.2 Voltage Sensitivity in the Voltage-Dependant Calcium Channels .....................................................62 1.5.3 The L-Type Calcium Channel (CACNA1C) .............................................................................. 63 1.5.3.1. Splice Variants in the CACNA1C Calcium Channel ........................................................................63 1.5.3.2 C-Terminal Proteolytic Cleavage of the CACNA1C Calcium Channel .............................................67 1.5.3.3 CACNA1C Calcium Channel Expression During Gestation ..............................................................68 3 1.5.3.4 Regulation of CACNA1C Calcium Channel Expression ................................................................... 70 1.5.4 The T-type Calcium Channel (CACNA1G) ............................................................................... 71 1.5.4.1 Splice Variants in the CACNA1G Calcium Channel .......................................................................... 72 1.5.4.2 CACNA1G Calcium Channel Expression During Gestation ............................................................. 75 1.5.4.3 Regulation of CACNA1G Calcium Channel Expression ................................................................... 76 1.5.5 Accessory Proteins of the Voltage Dependant Calcium Channels ............................................ 77 1.5.5.1 Role of the Beta (β) Subunit (CACNB1-4) ........................................................................................ 77 1.5.5.2 Role of the Alpha 2 Delta (α2δ) Subunit (CACA2D1-4) ................................................................... 78 1.5.6 KCNMA1, CACNA1C and CACNA1G Channel Associations. ................................................. 79 1.5.6.1 Caveola and Caveolins in the Myometrium ....................................................................................... 79 1.5.6.2 The Role Caveolae in Calcium Handling ........................................................................................... 80 1.5.6.3 The Hormonal Control of Caveolae and Caveolins ............................................................................ 80 1.5.6.4 Role of Caveolae and Caveolins During Gestation ............................................................................ 81 1.6 WORK DESCRIBED IN THIS THESIS .............................................................................................83 CHAPTER 2: MATERIALS AND METHODS ................................................................................ 88 2.1 MATERIALS ................................................................................................................................89 2.1.1 General Materials Reagents and Enzymes ...............................................................................