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Roles of the Ion Channel Nalcn in Neuronal Excitability Control University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Fall 2009 ROLES OF THE ION CHANNEL NALCN IN NEURONAL EXCITABILITY CONTROL Boxun Lu University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Cellular and Molecular Physiology Commons, and the Molecular and Cellular Neuroscience Commons Recommended Citation Lu, Boxun, "ROLES OF THE ION CHANNEL NALCN IN NEURONAL EXCITABILITY CONTROL" (2009). Publicly Accessible Penn Dissertations. 245. https://repository.upenn.edu/edissertations/245 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/245 For more information, please contact [email protected]. ROLES OF THE ION CHANNEL NALCN IN NEURONAL EXCITABILITY CONTROL Abstract The resting membrane potential (RMP) of a neuron is set by a complex balance between charged ions, ion channels and transporters. Many of the ion channels have been identified at the molecular level. Missing from the molecular identification has been the voltage-insensitive background sodium ‘‘leak’’ conductance that depolarizes the RMP from the equilibrium potential of potassium and provides a crucial contribution to neuronal excitability One candidate for the molecular identity of this conductance is the protein NALCN. NALCN is a previously uncharacterized orphan member in the sodium/calcium channel family. It is widely expressed in the nervous system. My thesis project was designed to uncover the properties of NALCN and to find its functional roles, especially its contribution to the neuronal excitability as an ion channel. I found that NALCN formed a voltage-insensitive background sodium leak conductance. Such a conductance was detected in hippocampal neurons cultured from the wild-type mice but not the NALCN- /- mutant mice with targted disruption in the NALCN gene. The conductance in the NALCN-/- neurons was restored when NALCN cDNA was transfected. These results suggest that NALCN provides the major contribution to the voltage-insensitive background sodium leak conductance. We also discovered that the NALCN channel could be activated by the neuropeptides substance P (SP) and neurotensin (NT), and by lowering extracellular Ca2+ concentration ([Ca2+]e), both of which elicit excitatory effects on several types of neurons. In addition, we found that NALCN was activated by the two types of stimuli via distinct intracellular signaling pathways and the two had synergistic effects on each other. Finally, application of the neuropeptides or lowering [Ca2+]e did not excite hippocampal neurons cultured from the mutant, suggesting that the NALCN channel complex is a primary target for neuronal excitation control by the neuropeptides and extracellular Ca2+. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Dejian Ren Keywords Neuronal excitability, background leak current, NALCN, Ion channel, Neuropeptides Subject Categories Cellular and Molecular Physiology | Molecular and Cellular Neuroscience This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/245 ROLES OF THE ION CHANNEL NALCN IN NEURONAL EXCITABILITY CONTROL Boxun Lu A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2009 ________________________________________________________________________ Dejian Ren, Ph.D., Supervisor of Dissertation ________________________________________________________________________ Daniel S. Kessler, Ph.D., Graduate Group Chair Dissertation Committee: Dr. J. Kevin Foskett, Professor (Committee Chair) Dr. Jonathan Raper, Professor Dr. Stephen M. Baylor, Professor Dr. Marc F. Schmidt, Assistant Professor i ACKNOWLEDGEMENTS I deeply appreciate the Ph.D. training that I have received from the Cell and Molecular Biology graduate program at the University of Pennsylvania. This could never have been achieved without the tremendous support that I have received from my mentors, colleagues, friends and family. First and foremost, I would like to thank my Ph.D. advisor, Dr. Dejian Ren, who gave me my academic life. I was the fortunate one that became Dejian’s first Ph.D. student and I feel truly privileged to have worked with him. As a very good and rigorous scientist, he taught me in many aspects about the way to do science. For example, Dejian taught me that I should always try to understand and evaluate data in the published papers, instead of skimming through and trying to remember the conclusions. I have been greatly benefited from his suggestion, without which I would have accepted many inaccurate conclusions and missed many chances of making new discoveries in my research. He also greatly improved my scientific presentation skills. I’ve learnt that I should always carefully consider the background and interest of the audience of the presentation so that I can prepare it to be understandable and informative to the specific group of audience. I’ve also learnt that it’s better to admit “I don’t know” than to give inaccurate information. Dejian is also an excellent life coach, who has continuously inspired me to find a meaning in life and to develop my own strategies to get there. He also encouraged me to communicate more with others, which has had large influence on my life and my job search. Second, I’d like to thank my committee members, Dr. J. Kevin Foskett, Dr. Jonathan Raper, Dr. Stephen M. Baylor, and Dr. Marc F. Schmidt. I feel fortunate to ii have such a group of committee members, who have given me many valuable suggestions and kept my work in pace. Kevin has been a mentor during my laboratory rotations, a lecturer of the classes I took, and the chair of my thesis committee. It is hard to describe how much help he has given me during the various stages of my Ph.D study. His telling me of “not taking shortcuts” deeply carved in my mind, and the philosophical wisdom of this instruction will guide my career and my life. Steve has been always interested in my research and he always raised thoughtful questions and discussed with me after every committee meeting, which benefit me greatly. For example, his discussion with me about the “principle of independence” and the old paper from Hodgkin and Huxley helped me understand my own data more clearly. John and Marc are two great neuroscientists who helped me better understand the significance of my work and think about possible future directions of my research. Marc also sent me very encouraging emails after each committee meeting, which I deeply appreciate. Third, I’d like to thank all the previous and current members of the Ren lab for discussion, technical assistance and encouragement. My colleagues and I have built a strong bond with each other, which has made my everyday life in the lab so joyful. Many thanks go to Drs. Yanhua Su, Jingsheng Xia, Jin Liu, Yan Wang, Haikun Wang, Qi Zhang, Yilong Shu, Chunlei Cang and Screenivasulu Gunti, and Janice Harlow. Finally and most importantly, I am very grateful to my family: my parents, my wife and my son, for their love and support in my graduate career, without which I can’t survive. While there are too many words in my mind, “I love you always” is what I most want to say to them. iii ABSTRACT ROLES OF THE ION CHANNEL NALCN IN NEURONAL EXCITABILITY CONTROL Author: Boxun Lu Supervisor: Dr. Dejian Ren The resting membrane potential (RMP) of a neuron is set by a complex balance between charged ions, ion channels and transporters. Many of the ion channels have been identified at the molecular level. Missing from the molecular identification has been the voltage-insensitive background sodium ‘‘leak’’ conductance that depolarizes the RMP from the equilibrium potential of potassium and provides a crucial contribution to neuronal excitability One candidate for the molecular identity of this conductance is the protein NALCN. NALCN is a previously uncharacterized orphan member in the sodium/calcium channel family. It is widely expressed in the nervous system. My thesis project was designed to uncover the properties of NALCN and to find its functional roles, especially its contribution to the neuronal excitability as an ion channel. I found that NALCN formed a voltage-insensitive background sodium leak conductance. Such a conductance was detected in hippocampal neurons cultured from the wild-type mice but not the NALCN-/- mutant mice with targted disruption in the NALCN gene. The conductance in the NALCN-/- neurons was restored when NALCN cDNA was transfected. These results suggest that NALCN provides the major contribution to the voltage-insensitive background sodium leak conductance. iv We also discovered that the NALCN channel could be activated by the 2+ neuropeptides substance P (SP) and neurotensin (NT), and by lowering extracellular Ca 2+ concentration ([Ca ]e), both of which elicit excitatory effects on several types of neurons. In addition, we found that NALCN was activated by the two types of stimuli via distinct intracellular signaling pathways and the two had synergistic effects on each other. 2+ Finally, application of the neuropeptides or lowering [Ca ]e did not excite hippocampal neurons cultured from the mutant, suggesting that the NALCN channel complex is a primary target for neuronal excitation control by the neuropeptides and extracellular Ca2+. v TABLE OF CONTENTS ACKNOWLEDGEMENTS
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