KV7 potassium channels: A focus on human intra-pulmonary arteries A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences 2015 Sean Brennan 1 List of contents Chapter 1 : Introduction ........................................................................................................ 15 1.1 Anatomy and physiology of the lungs .......................................................................... 15 1.2 Pulmonary artery structure ......................................................................................... 15 1.3 Pulmonary hypertension .............................................................................................. 16 1.3.1 Definition .............................................................................................................. 16 1.3.2 Characteristics ....................................................................................................... 17 1.3.3 Classification ......................................................................................................... 17 1.3.4 Treatment ............................................................................................................. 19 1.4 Regulation of the pulmonary vasculature.................................................................... 21 1.4.1 Neuronal regulation .............................................................................................. 21 1.4.2 Humoral regulation ............................................................................................... 22 1.4.3 Respiratory gases and the pulmonary artery ........................................................ 24 1.5 Calcium and contraction .............................................................................................. 25 1.6 Pulmonary artery smooth muscle cells and membrane potential .............................. 28 1.7 Pulmonary artery smooth muscle cell proliferation. ............................................... 29 1.8 Pulmonary artery smooth muscle cell migration. .................................................... 30 1.9 Potassium ion channels in pulmonary artery smooth muscle ..................................... 31 1.10 Potassium channel involvement in regulating the membrane potential of pulmonary artery smooth muscle ........................................................................................................ 36 1.10.1 KATP (KIR6.1) channels ........................................................................................... 36 2+ + 1.10.2 Large-conductance Ca -activated K (BKCa) channels ........................................ 37 + 1.10.3 Voltage-gated K (KV) channels ........................................................................... 38 1.10.4 Two-pore domain channels ................................................................................ 39 1.10.5 Two alternative hypotheses to explain the resting potential of PASMC. ........... 39 1.10.5.1 Hypothesis 1- KV1.5 and KV2.1 set the resting membrane potential in pulmonary artery smooth muscle cells. ..................................................................... 40 2 1.10.5.2 Hypothesis 2: The KV7 sub-family and TASK channels set the resting membrane potential in pulmonary artery smooth muscle cells. .............................. 40 1.11 K+ channels and pulmonary artery smooth muscle cell proliferation, apoptosis and migration. ........................................................................................................................... 42 1.12 Molecular diversity in KV7 channels ........................................................................... 44 1.12.1 KCNQ Splice Variants........................................................................................... 48 1.12.2 KCNE1-5 auxiliary subunits ................................................................................. 48 1.13 Expression and the physiological roles of KV7 channels ............................................ 50 1.13.1 KV7 channels in vascular smooth muscle ............................................................ 51 1.14 KV7 channel Pharmacology .................................................................................... 53 1.14.1 KV7 channel blockers ....................................................................................... 53 1.14.2 KV7 channel activators .................................................................................... 55 1.15 Project aims ............................................................................................................... 59 1.16 Alternative format ..................................................................................................... 60 Chapter 2 : Functional role for KV7 potassium channels in human pulmonary artery .......... 63 2.1 Abstract ........................................................................................................................ 64 2.2 Introduction ................................................................................................................. 64 2.3 Methods ....................................................................................................................... 66 2.3.1 Human Tissue samples .......................................................................................... 66 2.3.2 Rat pulmonary arteries ......................................................................................... 68 2.3.3 Measurement of mRNA Expression ...................................................................... 68 2.3.3.1 RNA isolation .................................................................................................. 68 2.3.3.2 RNA integrity .................................................................................................. 69 2.3.3.3 cDNA synthesis ............................................................................................... 70 2.3.3.4 End-point PCR ................................................................................................ 70 2.3.3.5 Quantitative RT-PCR ....................................................................................... 71 2.3.3.6 Primers ........................................................................................................... 71 3 2.3.3.7 Primer Efficiency ........................................................................................... 73 2.3.3.8 qRT-PCR normalisation .................................................................................. 74 2.3.4 Western blot ......................................................................................................... 76 2.3.5 Myography ............................................................................................................ 77 2.3.5.1 Mounting vessels ........................................................................................... 77 2.3.5.2 Normalisation protocol .................................................................................. 78 2.3.5.3 Vessel validation protocol .............................................................................. 79 2.3.5.4 Endothelium integrity .................................................................................... 79 2.3.5.5 Experimental protocols for KV7 blockers ....................................................... 80 2.3.5.6 Experimental protocols for KV7 activators ..................................................... 80 2.3.6 Drugs ..................................................................................................................... 81 2.3.7 Data analysis ......................................................................................................... 81 2.4 Results .......................................................................................................................... 82 2.4.1 RNA quality ........................................................................................................... 82 2.3.2.8 Primer Efficiency and specificity .................................................................... 82 2.4.2 Housekeeping gene selection ............................................................................... 85 2.4.3 KV7 channel expression in human intra-pulmonary and omental arteries ........... 87 2.4.4 Endothelium integrity ........................................................................................... 91 2.4.5 Effects of KV7 channel blockers on human intra-pulmonary artery tone ............. 93 2+ 2.4.6 Constriction induced by KV7 channel blockers requires voltage-gated Ca influx. ....................................................................................................................................... 95 2.4.7 Dilator effects of KV7 channel activators on human intra-pulmonary and omental arteries. .......................................................................................................................... 97 2.4.8 Retigabine acts via membrane hyperpolarisation and inhibition of voltage-gated Ca2+ influx ....................................................................................................................... 99 2.5 Discussion ................................................................................................................... 102 2.5.1 KV7 channels expressed in human iPAs ............................................................... 102 4 2.5.2 KCNE expression ................................................................................................