The Pennsylvania State University The Graduate School Intercollege Program of Neuroscience STRUCTURE-FUNCTION RELATIONSHIPS AND BIOCHEMICAL MANIPULATION OF 6-TM SUPERFAMILY ION CHANNELS A Dissertation in Neuroscience by Aditya Pisupati 2018 Aditya Pisupati Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2018 The dissertation of Aditya Pisupati was reviewed and approved* by the following: Timothy Jegla Associate Professor of Biology Dissertation Advisor Chair of Committee Melissa Rolls Professor of Biochemistry and Molecular Biology Bernhard Luscher Professor of Biology, Biochemistry and Molecular Biology, Psychiatry Santhosh Girirajan Associate Professor of Biochemistry and Molecular Biology William Hancock Professor of Bioengineering Kevin Alloway Professor of Neural and Behavioral Sciences Co-Director of Graduate Program in Neuroscience *Signatures are on file in the Graduate School ABSTRACT The six-transmembrane (6-TM) superfamily of ion channels is a diverse group of tetramer-forming ion channels. Members of this superfamily are regulated by a many different physical and chemical stimuli, including (but not limited to): organic compounds, temperature, mechanical stress, and transmembrane potential. 6-TM channels are critical for regulating neuronal excitability, cardiac pacemaking, sensing of thermal and mechanical stimuli, and maintaining water balance in plants among many other functions. In this thesis, I will first present an overview of 6-TM containing channels. This will be followed by three different projects involving channels from three families within the 6-TM superfamily: Shaker, Transient Receptor Potential Vanilloid (TRPV), and Cyclic Nucleotide Binding Domain (CNBD) channels. The project involving Shaker family channels identifies the stoichiometry of an obligatory heteromeric subunit and determines that this stoichiometry is regulated by a mechanism involving the activation gate. In the TRPV project, C. elegans and D. melanogaster TRPV channels are found to be activated by the vitamin B3 metabolite nicotinamide. In the investigations of CNBD containing channels, it is determined that heme appears to be an evolutionarily conserved regulator of CNBD-containing channels in both plant and animal lineages. Finally, I will discuss where these works fit into our current understanding of ion channels and physiology and will speculate on the outcome of new questions posed by the results of these three projects. iv TABLE OF CONTENTS List of Figures .......................................................................................................................... vii List of Tables ........................................................................................................................... x Acknowledgements .................................................................................................................. xi Chapter 1 Background on 6-TM Ion Channels ........................................................................ 1 Ion channel gating ............................................................................................................ 1 Biological significance of 6-TM ion channels ................................................................. 5 Shaker family channels ............................................................................................ 7 TRPV family channels ............................................................................................. 12 CNBD-containing family channels .......................................................................... 14 Preview of Thesis ............................................................................................................. 22 References ........................................................................................................................ 24 Chapter 2 Experimental techniques for probing ion channel structure-function relationships ..................................................................................................................... 32 Xenopus laevis oocytes as a model for studying ion channels ......................................... 32 Preparation of Xenopus oocytes for cRNA injections .............................................. 34 Two-Electrode Voltage Clamp (TEVC) .......................................................................... 35 How to perform Two-Electrode Voltage Clamp ...................................................... 37 Analysis of TEVC Data ............................................................................................ 38 Total Internal Reflectance Fluorescence (TIRF) Microscopy .......................................... 40 Performing TIRF Microscopy Experiments ............................................................. 40 Analysis of TIRF data .............................................................................................. 42 References ........................................................................................................................ 44 Chapter 3 S6 gate divergence in regulatory subunits restricts the stoichiometry of heteromeric Shaker family channels ................................................................................ 45 Abstract ............................................................................................................................ 46 Introduction ...................................................................................................................... 47 Methods ............................................................................................................................ 51 Molecular Cloning.................................................................................................... 51 cRNA synthesis and Xenopus oocyte preparation .................................................... 53 Electrophysiology..................................................................................................... 54 TIRF microscopy...................................................................................................... 55 Sequence Alignments ............................................................................................... 56 Molecular Modeling ................................................................................................. 56 Results .............................................................................................................................. 57 The highly-conserved S6 activation gate sequence degenerates in regulatory subunits ............................................................................................................. 57 TIRF microscopy reveals multiple Kv2.1:Kv6.4 heteromer stoichiometries ........... 61 v The S6 gate of Kv6.4 limits formation of 2:2R heteromers ..................................... 64 The Kv6.4 gate blocks function of 2:2R heteromers ................................................ 67 Cd2+ block of cysteine mutants confirms 2:2R functionality of Kv6.4-PIPIIV- Kv2.1CT ........................................................................................................... 74 Discussion ........................................................................................................................ 77 References ........................................................................................................................ 87 Chapter 4 Activation of invertebrate TRPV heterotetramers by the vitamin B3 metabolite nicotinamide ..................................................................................................................... 91 Introduction ...................................................................................................................... 92 Methods ............................................................................................................................ 93 Molecular cloning and cRNA synthesis ................................................................... 93 Xenopus oocyte recordings ....................................................................................... 93 TIRF Photobleaching ............................................................................................... 93 Sequence Alignment ................................................................................................ 94 Results .............................................................................................................................. 94 Nicotinamide is an OSM9/OCR4 channel agonist ................................................... 94 Nicotinamide-evoked activity is conserved in the Drosophila TRPV homolog formed by Inactive and Nanchung ................................................................... 95 OSM9 and OCR4 form a 2:2 heterotetramer ........................................................... 96 Discussion ........................................................................................................................ 98 Nicotinamide as a potential endogenous ligand for invertebrate TRPV channels ... 99 Phylogenetic analysis of TRPV evolution................................................................ 100 The structural basis of interdependent heteromerization.......................................... 102 References ........................................................................................................................ 105 Chapter 5 Heme modulation