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An Investigation of the Properties and Functions of Connexins in the Mammalian Inner Ear

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An Investigation of the Properties and Functions of Connexins in the Mammalian Inner Ear An Investigation of the Properties and Functions of Connexins in the Mammalian Inner Ear John Joseph Kelly Ear Institute University College London A thesis submitted for the degree of Doctor of Philosophy September 2011 Declaration I, John Kelly, confirm that the work presented in this thesis is my own. Where information or assistance has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Acknowledgements First and foremost, I would like to thank my supervisors: Dr. Dan Jagger for your continued support, advice and patience throughout my Ph.D., and Prof. Andy Forge for all your help and guidance along the way. It’s been a privilege to have you both as my mentors. I’d also like to thank other members of the group: Regina, for your expertise and friendship and for helping me integrate into the lab with ease; Ruth, for your help and advice over the years; Graham for your expertise and recommendations of areas to explore in the UK (one day….), and Nicole, for your company both inside and outside of the lab, and for the fond memories of our road trip around southern California with Cassy. Lisa, I’ve had the pleasure of working with you in two different labs. Thank you for all your help and advice and for your infectious positivity! I would like to thank Dr. Sally Dawson for introducing me to the world of molecular biology and to both Sally and Dr. Jonathan Gale for the advice you have both given me. Emily, you’ve been a fantastic lab, desk and tea-break buddy. Thank you for all your help and advice and for putting up with all my silly questions. Miriam, thank you for all your help in the lab. Tommy and Valentina, you both made me feel so welcome when I joined and your help in the lab was very much appreciated. It has been a pleasure working with you all. I’d like to thank Deafness Research UK for funding my Ph.D. studies and giving me the opportunity to present my work in the USA. I am indebted to all the friends I have made at the Ear Institute over the past five years. Elena (BT/Mou) we started this together and we’ll finish this together. It’s been a long journey but I couldn’t have asked for a better person to have shared it with. Freeman, you’ve been a constant source of entertainment and laughter. Marc Astick, you nearly ruined my Ph.D. after introducing me to stick cricket, but I forgive you. Zoë, you always made sure we were in the pub by 5.30pm (at the latest) on a Friday night. Bjorn and Lucy, we’ve enjoyed a drink (and a whiskey) or two! Amongst others, Greg, Pavel, Manu, Jason, Ghada, Joey, Joerg, Nico, and all those in the “back-office”; thank you all for your support and friendship over the years, it’s been a pleasure getting to know you all. Adam, thanks for being a fantastic mate and giving me sound advice along the way, and a huge thank you to my fellow Ph.D. buddies Clare (we made it!), Jenny, Mark, Fran, Maz and Andrew for adopting me as their unofficial 7th group member. 3 We’ve had some incredible and unforgettable times. I’d like to thank Ben, Mace, Kate, Steve, Emily, Mattea, Unyime, Phil, John, Tom and Kieran for giving me an escape- route from work and not giving up on me towards the end. I can’t have asked for a better bunch of friends. Last, but certainly not least, I’d like to thank my family. Mum, Dad, Maria, Sarah, Cath and Gel. Your support and encouragement has been amazing, without which this Ph.D. wouldn’t have been possible. John Kelly September 2011 4 Abstract Connexin 26 (Cx26) and Cx30 are the two predominant gap junction constituents expressed in the mammalian cochlea. Mutations in either gene cause hereditary deafness, indicating an essential role for connexins in auditory function. Gap junctions consisting of Cx26 and Cx30 have been implicated in several cochlear processes; however, the precise functions and life-cycle of connexins in the cochlea are poorly defined. Three aspects of inner ear connexin biology were investigated. Most connexins traffic to the plasma membrane (PM) via the conventional secretory pathway. Conflicting data exists for Cx26 trafficking, whereas that of Cx30 had not been previously studied. Trafficking of Cx26 and Cx30 were investigated using stably-transfected HeLa cell lines. Treatment with brefeldin-A (a Golgi-disrupting drug) did not prevent targeting of Cx30 to the PM, whereas Cx26 was strongly inhibited. These data suggest that Cx30 may traffic to the PM via a Golgi-independent pathway, which is in contrast to a Golgi-dependent pathway for Cx26. Gap junctional intercellular communication (GJIC) pathways are hypothesised to support K+ recycling in the cochlea. This study investigated the development of GJIC in the lateral wall (LW) of live rat cochlear slices. Cx26 and Cx30 immunofluorescence revealed a progressive increase of gap junction expression from postnatal day 0 (P0) to P7-P8. Dye-coupling was compartmentalised between P2-P5, but was extensive by P7. These data suggest that GJIC matures several days in advance of hearing onset and provides anatomical evidence of a putative K+-recycling pathway. Finally, Cx30–/– mice are deaf and fail to develop an endocochlear potential (EP). This study investigated the expression of proteins involved in EP generation and found that the potassium channel Kir4.1 was noticeably reduced in the stria vascularis (SV) of Cx30–/– mice. In contrast to a separate study, the SV endothelial barrier was intact. In addition, anatomical analysis was consistent with loss of Cx30 retarding maturation of SV. 5 Table of Contents Acknowledgements ....................................................................................................... 3 Abstract ......................................................................................................................... 5 List of Figures ............................................................................................................. 11 List of Tables............................................................................................................... 14 List of Abbreviations................................................................................................... 15 1 INTRODUCTION .................................................................................................. 18 1.1 Gap Junctions ................................................................................................... 18 1.1.1 Identification of intercellular communication and gap junctions ............. 19 1.1.2 Connexin nomenclature ............................................................................ 22 1.1.3 Structure of gap junctions ......................................................................... 24 1.1.4 Gap junction expression and disease......................................................... 27 1.2 The Mammalian Inner Ear ............................................................................... 29 1.2.1 General structure of the inner ear .............................................................. 29 1.2.2 Sensory epithelium and mechanoelectrical transduction .......................... 31 1.2.3 Ion transporting epithelium ....................................................................... 34 1.3 Gap junctions in the mammalian inner ear ....................................................... 38 1.3.1 Gap junction networks .............................................................................. 38 1.3.2 Connexin expression ................................................................................. 39 1.3.3 Function of inner ear gap junctions ........................................................... 40 1.4 Scope of the thesis ............................................................................................ 43 2 MATERIALS AND METHODS ............................................................................ 44 2.1 Materials ........................................................................................................... 44 2.2 Cell Culture ...................................................................................................... 44 2.2.1 HeLa cell culture ....................................................................................... 44 2.2.2 Connexin expression constructs ................................................................ 44 6 2.2.3 Transient expression of connexin constructs ............................................ 44 2.2.4 Stable expression of connexin constructs ................................................. 45 2.2.5 Cryopreservation of cell lines ................................................................... 45 2.2.6 Drug treatment .......................................................................................... 46 2.2.7 Temperature manipulation ........................................................................ 46 2.3 Animals ............................................................................................................ 46 2.4 Primary Culture of Cochlear Fibrocytes........................................................... 46 2.5 Immunofluorescence labelling and confocal microscopy ................................ 47 2.6 Reverse transcription PCR ............................................................................... 51 2.7 Quantitative real-time PCR .............................................................................. 52 2.8 Connexin 30 transgenic mice ........................................................................... 53 2.8.1 Generation of Cx30 knockout
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