The Pennsylvania State University The Graduate School Eberly College of Science EVOLUTIONARY ORIGINS AND PIP2 MODULATION OF VOLTAGE-GATED K+ CHANNELS A Dissertation in Molecular, Cellular, and Integrative Biosciences by Xiaofan Li © 2015 Xiaofan Li Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2015 ii The dissertation of Xiaofan Li was reviewed and approved* by the following: Timothy Jegla Assistant Professor of Biology Dissertation Advisor Chair of Dissertation Committee Bernhard Lüscher Professor of Biology Professor of Biochemistry and Molecular Biology Melissa Rolls Associate Professor of Biochemistry and Molecular Biology Chair of the Molecular, Cellular and Integrative Biosciences Graduate Program David Vandenbergh Associate Professor of Biobehavioral Health Associate Director of the Penn State Institute of the Neurosciences *Signatures are on file in the Graduate School iii Abstract Voltage-gated K+ channels are important regulators of neuronal excitability. Bilaterians have eight functionally distinct Voltage-gated K+ channel subfamilies: Shaker, Shab, Shaw, Shal, KCNQ, Eag, Erg and Elk. These subfamilies are defined by sequence conservation, functional properties as well as subfamily-specific assembly. Genome searches revealed metazoan-specificity of these gene families and the presence of prototypic voltage-gated K+ channels in a common ancestor of ctenophores (comb jellies) and parahoxozoans (bilaterians, cnidarians and placozoans). Establishment of the gene subfamilies, however happened later in a parahoxozoan ancestor. Analysis of voltage- gated K+ channels in a cnidarians species Nematostella vectensis (sea anemone) unveiled conservation in functional properties with bilaterian homologs. Phosphoinositide (most notably PIP2) regulation of ion channels is universal in eukaryotes. PIP2 modulates Shaker, KCNQ and Erg channels in distinct manners, while PIP2 regulation of Elk channels has not been reported. I showed that PIP2 modulates the human Elk1 channel in a bimodal fashion: It inhibits voltage activation but also enhances open state stabilization through mode shift. Mutational analysis and structural modeling localized three basic residues on the N-terminal cap domain, S4-5 linker and S6 terminal as putative binding residues for PIP2. Comparison with Shaker and HCN channels hints at a common mechanism by which PIP2 modulates voltage gating in channels. iv TABLE OF CONTENTS List of Figures……………………………………………………………………….…..viii List of Tables………………………………………………………………...………..….xi Acknowledgements………………………………………………………………………xii Chapter 1 Background and Introduction………………………………………………….1 1.1 Voltage-gated K+ channels: structure, function and evolution……………….2 1.1.1 Ion channels are essential for cellular function …………………….2 1.1.2 Structure, origin and function of voltage-gated K+ channels…….…3 1.1.3 Evolution of the nervous system and voltage-gated ion channels….9 1.1.4 Co-evolution of ion channels and the plasma membrane…………14 1. 2 Phosphoinositide regulation of voltage-gated K+ channels………………...16 1.2.1 Introduction to phosphoinositides…………………………………16 1.2.2 PIP2 modulation of ion channel activities…………………………18 + 1.2.3 PIP2 modulation of voltage-gated K channels ……………….......21 1.2.4 Using Elk1 to study PIP2 modulation of EAG family voltage-gated K+ channels………………………………………………25 1. 3 Summary.……………………………………………………………….…..27 1. 4 References…………………………………………………………………..28 Chapter 2 Ether-a-go-go family Voltage-Gated K+ Channels evolved in an ancestral metazoan and functionally diversified in a Cnidarian/Bilaterian Ancestor……………...36 2.1 Abstract………………………………………………………………….…..37 2.2 Introduction……………………………………………………………….…38 2.3 Materials and Methods………………………………………………………41 2.3.1 Identification and cloning of EAG family genes………………….41 v 2.3.2 Phylogenetic analysis……………………………………………...41 2.3.3 In Situ Hybridization………………………………………………42 2.3.4 Functional Expression in Xenopus Oocytes……………………….42 2.4 Results……………………………………………………………………….44 2.4.1 Phylogenetic analysis of EAG family……………………………..44 2.4.2 Analyzing mRNA expression patterns for EAG genes in Nematostella vectensis………………………………………….47 2.4.3 Functional analysis of NvEag and NvElk…………………………51 2.5 Discussion…………………………………………………………………...64 2.6 References………………………………………………………….………..70 Chapter 3 Major diversification of voltage-gated K+ channels occurred in ancestral parahoxozoans……………………………………………………………………………74 3.1 Abstract……………………………………………………………………...75 3.2 Introduction………………………………………………………………….76 3.3 Materials and Methods………………………………………………………79 3.3.1 Gene Identification, cloning and phylogenetic analysis…………..79 3.3.2 Electrophysiology…………………………………………………80 3.4 Results………………………………………………………………………82 3.4.1 Phylogenetic analysis of the Shaker family……………………….82 3.4.2 Functional analysis of Nematostella Shaker channels…………….86 3.4.3 Functional analysis of Mnemiopsis Shaker channels……………...94 3.4.4 Phylogenetic analysis of the KCNQ family……………………….98 3.4.5 Functional analysis of Nematostella KCNQ channel……………101 3.5 Discussion………………………………………………………………….105 3.6 Supplemental Figures and Legends………………………………………..108 vi 3.7 References………………………………………………………………….116 Chapter 4 Bimodal regulation of an Elk subfamily K+ channel by Phosphatidylinositol 4,5-bisphosphate……………………………...………………….120 4.1 Abstract…………………………………………………………………….121 4.2 Introduction………………………………………………………………...122 4.3 Materials and Methods……………………………………………………..126 4.3.1 Molecular Biology……………………………………………….126 4.3.2 Xenopus Oocyte Preparation, Injection and Culture…………….126 4.3.3 Patch Clamp Recordings…………………………………………127 4.3.4 Data analysis……………………………………………………..128 4.3.5 Structural modeling………………………………………………128 4.4 Results……………………………………………………………………...132 4.4.1 Patch Excision alters voltage-dependent gating of Elk1…………132 4.4.2 Elk1 voltage-gating show a robust mode shift that is lost upon patch excision……………………………………………………..134 4.4.3 PIP2 slows activation of Elk1 in excised patches and partially restores mode shift………………………………………..136 4.4.4 Depletion of native PIP2 with CiVSP modulates Elk1 voltage gating…………………………………………………...………139 4.4.5 PIP2 and PIP3 regulate Elk1……………………………………...141 4.4.6 The role of the eag domain in mode shift…………………...…...143 4.4.7 Basic residues in the S45L and post-S6 are required for PIP2-dependent inhibition of Elk1…………………………………..147 4.4.8 Modeling the PIP2 binding site…………………………………..154 4.5 Discussion………………………………………………………………….158 4.6 References……………………………………………………………….…162 vii Chapter 5 Concluding remarks and future research questions………………………….178 5.1 Evolution of voltage-gated K+ channels and the nervous system………….179 5.1.1 Controversy on the origin(s) of the nervous system……………..179 5.1.2 Diversity of Kv channels in the nervous system…………………182 5.1.3 Driving force for ion channel evolution?.......................................184 5.1.4 Unanswered questions and future directions…………………….185 5.2 Phosphoinositide regulation of ion channel activities……………………..187 5.2.1 Ion channel sensitivity to phosphoinositides….………….……...187 5.2.2 Mechanism of phosphoinositide interaction with ion channels….189 5.2.3 Possible functions of phosphoinositide regulation………………190 5.3 References………………………………………………………….193 viii List of Figures Figure 1-1. Anatomy of an action potential……………………………………………….4 Figure 1-2. Structural diagram and evolutionary history of voltage-gated cation channels ……………………………………………………………………………..……6 Figure 1-3. A simplified phylogeny of animal evolution………………………………..10 Figure 1-4. Phosphoinositide structure and PIP2 metabolism……………………………17 Figure 1-5. PIP2 binding site in Kir3.2 (GIRK2) crystal structure………………...…….20 Figure 1-6. PIP2 affects voltage-dependent gating of Cav2.1, Shaker and HCN channels in a similar fashion……………………………………………………….…….22 Figure 2-1. Phylogeny of the metazoan EAG K+ channel family………………………..46 Figure 2-2. In situ hybridization analysis of Nematostella EAG family channel expression patterns……………………………………………………………………....48 Figure 2-3. Comparison of NvEag and MmEag1 voltage activation properties…………51 Figure 2-4. A distinct pattern of acidic side chains confers divalent cation sensitivity to the EAG superfamily voltage sensors……………………………………..54 Figure 2-5. Mg2+ sensitivity in voltage activation is greatly reduced in NvEag…………56 Figure 2-6. NvErg1 exhibits reduced divalent sensitivity……………………………….60 Figure 2-7. Comparison of NvElk and mammalian Elk currents………………………..62 Figure 2-8. Summary of EAG K+ channel family evolution…………………………….63 Figure 3-1. Bayesian inference phylogeny of the metazoan Shaker K+ channel family……………………………………………………………………………84 Figure 3-2. NvShab encodes a typical Shab family delayed rectifier…………………....87 Figure 3-3. Functional properties of homomeric and heteromeric Nematostella Shaw channels…………………………………………………………………………...90 Figure 3-4. Functional properties of homomeric and heteromeric Nematostella Shal channels…………………………………………………………………………….93 Figure 3-5. Ctenophore Shaker family subunits co-assemble with Nematostella and mouse Shaker subfamily subunits………………………………………………...…95 ix Figure 3-6. Ctenophore Shaker family subunits do not co-assemble with Nematostella Shab, Shal or Shaw channels…………………………………………………………….97 Figure 3-7. Bayesian inference phylogeny of the metazoan KCNQ voltage-gated K+ channel family………………………………………………………………………..99 Figure 3-8. Nematostella KCNQ encodes a sub-threshold PIP2-sensitive voltage-gated K+ channel…………………………………………………………….…102 Figure S3-1. Schematic metazoan phylogenies