Ngcam Targeting Regulates Axon and Dendrite Bundling

Function and Mechanism of Polarized Targeting of Neuronal

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Joshua A. Barry, B.S.

Graduate Program in Molecular, Cellular and Developmental Biology

The Ohio State University

2013

Committee:

Chen Gu, Advisor Anthony Brown Tsonwin Hai Peter Mohler

Joshua A. Barry

2013

ABSTRACT

A neuron’s ability to survive and function properly requires many factors including a multitude of proteins. Membrane proteins are a specific class that are expressed within the plasma membrane and function in a variety of roles from action potential firing to neurotransmitter reuptake. To perform this variety of functions requires proper localization of these proteins, however, the exact functional relevance and underlying mechanisms that regulates this targeting is still an area of intense research. In this thesis I examined the functional role that NgCAM had on inducing bundling of axons or dendrites via regulation by domain deletion or phosphorylation. I also examined how the polarized targeting of the splice variants of Kv3.1 could affect the maximal spiking frequency of neurons. Then I explored how Kv3.1 could induce clustering and activation of its motor kinesin 1/KIF5. Finally, I looked at the role that metal binding sites, specifically zinc, plays on the localization and activity of Kv3.1. My various areas of research are all linked by the shared idea that the proper localization of membrane proteins can regulate a neuron’s function.

DEDICATION

This work is dedicated to my mom and dad, and my two sisters Jillian and Emma. – thanks for supporting me and keeping me going

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ACKNOWLEDGEMENTS

The time spent in graduate school has been a life changing experience for me.

When I was in high school I knew I wanted to be a scientist. Then I went to college and earned a B.S. in biology, but it wasn’t until I started graduate school that I learned what a true scientist is. Scientists don’t just sit at the bench and discover answers to questions they have. Every day is a learning experience, with the successes and failures of experiments that molded me into the competent scientist I am today. But I did not do all this work by myself. I would like to thank the following people for all their support.

First, I must say thank you to Dr. Chen Gu. He has stood behind me one-hundred percent as I stumbled my way through learning how to become a better scientist. When I started in his lab I had a basic textbook understanding about molecular biology, but he gave me the tools to build a foundation of molecular biology techniques that I will always carry with me and help me pursue my career as a scientist.

I would also like to thank the enormous help of the past and present members of the Gu lab. Dr. Mingxuan Xu taught me all I know about molecular biology. Dr.

Yuanzheng Gu taught me about electrophysiology and how to generate the hippocampal neuron culture that I used almost every week. Peter Jukkola, the other graduate student in Dr. Gu’s lab, taught me how the molecules I break apart and study at the molecular

iv level function in the whole brain. These three people are not only coworkers but also friends and I will miss them.

I would like to thank our collaborators: Dr. Robert McDougel and Dr. David

Terman for their help in generating the computer simulations of fast spiking neurons. I would like to thank Andrew Dangel for his help in using the Surface Plasmon Resonance machine to study protein-protein high affinity binding. And finally Dr. Chandra Shrestha for her help on the competition assay between KIF5 and Kv3.1 and other laboratory work.

Also, I would like to thank my committee members Dr. Tsonwin Hai, Dr.

Anthony Brown and Dr. Peter Mohler for being on my committee and helping me on this journey to receive my PhD.

Vita

October 10, 1981 ...... Born – Madison, Wisconsin

June 2004 ...... B.S., Biology Mount Union College

2004-2013 ...... Graduate Research Associate The Ohio State University

Publications

1. Barry, J., Xu, M., Gu, Y., Dangel, A., Shrestha, C., and Gu, C. (2013) Activation of Conventional Kinesin Motors in Clusters by Shaw Voltage-Gated Potassium Channels. Journal of Cell Science 126, 2027-2041 Published online March 2013.

2. Gu, Y., Barry, J., and Gu, C. (2013) Kv3 channel assembly, trafficking and activity are regulated by zinc through different binding sites. Journal of Physiology 591, 2491-2507 Published online Feb. 18th, 2013.

3. Barry, J., and Gu, C. (2012) Coupling Mechanical Forces to Electrical Signaling: Molecular Motors and the Intracellular Transport of Ion Channels. The Neuroscientist 19, 145-159 Published online Aug. 24th, 2012.

4. Gu, Y.Z., Barry, J., McDougel, R., Terman, D., and Gu, C. (2012) Alternative splicing regulates Kv3.1 polarized targeting to adjust the maximal spiking frequency. The Journal of Biological Chemistry 287, 1755-1769.

5. Gu, C., and Barry, J. (2011) Function and mechanism of axonal targeting of voltage-sensitive potassium channels. Progress in Neurobiology (review article) 94, 115-132.

6. Xu, M., Gu, Y.Z., Barry, J., and Gu, C. (2010) Kinesin I transports tetramerized Kv3 channels through the axon initial segment via direct binding. Journal of Neuroscience 30, 15987-16001.

7. Barry, J., Gu, Y., and Gu, C. (2010) Polarized targeting of L1-CAM regulates axonal and dendritic bundling in vitro. European Journal of Neuroscience 32, 1618-1631.

vi 8. Baumann, A., Barry, J., Wang, S., Fujiwara Y., Wilson, T.G. (2010) Paralogous genes involved in juvenile hormone action in Drosophila melanogaster. Genetics 185, 1327-1336.

9. Barry, J., Wang, S., Wilson, T.G. (2008) Overexpression of Methoprene-tolerant, a Drosophila melanogaster gene that is critical for juvenile hormone action and insecticide resistance. Insect Biochemistry and Molecular Biology 38, 346-353.

Fields of Study

Major Field: Molecular, Cellular and Developmental Biology Program

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TABLE OF CONTENTS

2013 ...... II ABSTRACT ...... II DEDICATION ...... III ACKNOWLEDGEMENTS ...... IV VITA ...... VI LIST OF TABELS ...... X LIST OF FIGURES ...... XI CHAPTER 1 ...... 1 INTRODUCTION ...... 1 NEURON POLARITY ...... 1 MOTOR PROTEINS: KINESINS ...... 2 CELL ADHESION MOLECULES ...... 3 ION CHANNELS ...... 4 Potassium Channels ...... 6 TRANSPORT OF ION CHANNELS BY ADAPTOR PROTEINS ...... 14 AMPA receptors ...... 14 NMDA receptors ...... 16 GABAA Receptors...... 21 Voltage-gated potassium channel (Kv3) and Kinesin 1 ...... 24 CLC-5 (Chloride/Proton antiporter) and Kinesin 2 ...... 25 REGULATION OF ION CHANNEL AND KINESIN INTERACTION ...... 27 ADAPTOR PROTEIN WITH MULTIPLE BINDING PARTNERS ...... 28 CHANNEL OLIGOMERIZATION AND KINESIN TRANSPORT...... 30 CHAPTER 2 NGCAM TARGETING REGULATES AXON AND DENDRITE BUNDLING ...... 31 SUMMARY ...... 31 * ALL EXPERIMENTS PERFORMED IN THIS CHAPTER BY JOSHUA BARRY WITH HELP FROM DR. CHEN GU. . 31 INTRODUCTION ...... 32 RESULTS ...... 33 METHODS ...... 53 CHAPTER 3 KV3 POLARIZED TARGETING REGULATES NEURONAL ACTIVITY ...... 59 SUMMARY ...... 59 * JOSHUA BARRY PERFORMED ALL IMMUNOSTAINING AND QUANTIFICATION. DR. YUANZHENG GU PERFORMED ALL ELECTROPHYSIOLOGY RECORDINGS. DR. ROBERT MCDOUGEL AND DR. DAVID TERMAN (DEPT OF MATHEMATICS, OSU) PERFORMED COMPUTER MODELING WITH HELP FROM DR. YUANZHENG GU AND DR. CHEN GU...... 59 viii INTRODUCTION ...... 60 RESULTS ...... 61 DISCUSSION...... 76 METHODS ...... 78 CHAPTER 4 KV3 CLUSTERS AND ACTIVATES KIF5 ...... 85 SUMMARY ...... 85 INTRODUCTION ...... 86 RESULTS ...... 87 METHODS ...... 112 CHAPTER 5 ZN2+ REGULATES KV3 CHANNEL ASSEMBLY, TRAFFICKING AND ACTIVITY...... 119 SUMMARY ...... 119 INTRODUCTION ...... 120 RESULTS ...... 121 METHODS ...... 139 CHAPTER 6 CONCLUSIONS ...... 142 NGCAM POLARIZED TARGETING AND BUNDLING OF AXONS OR DENDRITES ...... 142 MAXIMAL SPIKING FREQUENCY REGULATED BY KV3 POLARIZED TARGETING...... 143 CLUSTERING AND ACTIVATION OF KIF5 BY KV3 ...... 144 ZINC’S ROLE IN KV3 ACTIVITY AND TRAFFICKING ...... 145 FUTURE DIRECTIONS ...... 146 REFERENCES ...... 147

LIST OF TABELS

Table Page

Table 1.1. Kinase inihibitors and their concentrations used...... 43

LIST OF FIGURES

Figure Page

Figure 1.1. Major binding proteins in the intracellular transport of AMPA receptors. .... 18

Figure 1.2. Adaptor and motor proteins in the intracellular transport of NMDA receptors...... 20

Figure 1.3. Different adaptor proteins mediate the interaction between GABAA receptors and KIF5 motor...... 23

Figure 1.4. Direct binding between ion channel/transport and kinesin...... 26

Figure 1.5. Regulation of ion channel protein loading to kinesin motors...... 29

Figure 2.1. Expression of NgCAM fusion proteins induced robust axonal bundling in cultured hippocampal neuron...... 35

Figure 2.2. Roles of Ig and C-terminal domains of NgCAM in axonal bundling...... 37

Figure 2.3. Deleting the fibronectin repeats decreased axonal bundling but induced dendritic bundling...... 40

Figure 2.4. Axon-dendrite targeting of NgCAM-GFP and its deletion mutants...... 42

Figure 2.5. Protein phosphorylation regulated axonal targeting of NgCAM and hence neurite bundling...... 45

Figure 2.6. Mouse L1-CAM (mL1CAM) axonal targeting is regulated by protein phosphorylation and induces axonal bundling...... 48

Figure 3.1. Hippocampal neurons in culture spikes at different frequencies in response to prolonged depolarizing inputs...... 64

Figure 3.2. Expression of Kv3.1b effectively converts slow-spiking neurons to fast- spiking ones...... 66

xi Figure 3.3. Regulation of axonal targeting and action potential firing by Kv3.1b truncations...... 70

Figure 3.4. Axonal targeting of Kv3.1b is critical for inducing the maximal AP firing frequency...... 73

Figure 3.5. Modeling and simulations of the effect of Kv3.1 axonal targeting on spiking frequency...... 75

Figure 4.1. Clustering of endogenous KIF5 motors in cultured hippocampal neuron. .... 88

Figure 4.2. Direct binding between the Kv3.1 T1 and KIF5 tail domains...... 91

Figure 4.3. Binding affinity and stoichiometry of Kv3.1 T1 and KIF5B tail domain...... 94

Figure 4.4. Kv3.1 T1 competes with microtubules but not KLC1 for binding to KIF5B tail...... 97

Figure 4.5. KIF5-binding proteins differentially regulate KIF5B tail localization...... 100

Figure 4.6. Mutating the three charged residues or the presence of Kv3.1 clusters KIF5B- YFP...... 103

Figure 4.7. KIF5 clusters reduced in cerebellar neurons of Kv3.1 knockout mice...... 106

Figure 4.8. Summary of the mechanism and consequence of the Kv3-KIF5 binding.. .. 108

Figure 5.1. Extracellular application of zinc reversibly reduces spiking frequency of cultured neurons...... 123

Figure 5.2. A novel binding site for divalent heavy metal ions in the Kv3.1 C-terminus...... 126

Figure 5.3. Three H residues in Kv3.1a C-terminus are required for binding to Co2+ or Zn2+...... 128

Figure 5.4. H459, but not H480 and H481, in the C-terminal domain, is required in Kv3.1b axonal targeting...... 130

Figure 5.5. Mutagenesis studies in searching for potential zinc-binding sites in the membrane portion of Kv3.1b...... 131

Figure 5.6. Constructing a fast-spiking neuron with relative resistance to the zinc inhibition ...... 134

xii Figure 5.7. Distinct binding sites for divalent heavy metal ions in the tetrameric Kv3.1 channel complex...... 136

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CHAPTER 1

INTRODUCTION

This dissertation presents research that examines the underlying functions and mechanisms that regulate and are regulated by the polarized targeting of membrane proteins, specifically NgCAM and Kv3.1. This introduction will give an overview of the background required to understand the thrust of this thesis.

Neurons and glia are the two major cell types in the brain. Glia are the supporting cells for the neurons. The main function of a neuron is the transmission of information.

A neuron can be broken into three major compartments: dendrites which receive multiple synaptic inputs, the soma or cell body which contains the nucleus and protein production machinery, and the axon which sends out the information via action potentials (APs).

This transmission of information requires proper localization of many different membrane proteins such as cell adhesion molecules and ion channels. Many of these proteins must be targeted to a specific region of the neuron, whether that is the dendrites, soma or axon, this is known as polarity or polarized targeting.

Neuron polarity

Neuronal polarity begins at a very young age for neurons. When examining cultured hippocampal neurons as they develop they form many short processes, but after

1 several hours one process grows very rapidly, becoming the axon, while the other process grow much slower and form the dendrites (Dotti et al., 1988). Inside the neuron microtubules (MTs) are the tracks that motor proteins use when transporting their cargo.

MTs themselves are oriented based on polarity. MTs are tubular structures made up of dimers of the globular proteins α- and β-tubulin. MTs grow by addition of αβ tubulin dimers at the + end, while they degrade from the - end. In neuron culture all MTs in the axon are oriented with their + ends facing away from the cell body, while in the dendrites they are randomly oriented (Baas et al., 1988). This polarity of MTs is very important for the directionality of the motor proteins.

The neuron maintains a filter or ‘gate’ located in the axon initial segment that separates the axon from the somatodendritic region. This gate allows axonal cargoes to pass through while blocking other cargoes (Winckler et al., 1999; Song et al., 2009;

Rasband, 2010). This filter requires ankyrin G, a large cytoskeletal scaffolding protein.

If ankyrin G is removed from the axon initial segment then axons begin to take on the molecular and structural features of dendrites, including spines (Zhou et al., 1998;

Sobotzik et al., 2009).

Motor proteins: Kinesins

Proper membrane protein localization requires active transport by molecular motors. There are three groups of motors, kinesin, dynein and myosin (Hirokawa et al.,

2009; Kardon and Vale, 2009; Hammer and Sellers, 2012). In the neuron, kinesin primarily transports its cargo away (anterograde) form the cell body, whereas dynein transports its cargo towards (retrograde) the cell body. Both use the MTs as tracks for

2 trafficking of cargoes. The 45 members of the kinesin family can be divided into three groups based on the location of the motor domain: N-terminal, middle and C-terminal

(Hirokawa and Noda, 2008). Kinesins are a heterotetramer made of two heavy chains

(KHCs) and two light chains (KLCs) (Diefenbach et al., 1998). The N-terminal kinesin contains a motor domain, a coiled-coil domain used for dimerization, and a tail domain that binds cargos and the KLCs which can act as adaptor proteins between KHC and cargoes.

Cell adhesion molecules

As neurons develop within the brain they interact with one another, the most well known of these interactions is the axon-dendrite interaction known as the synapse, but axon-axon and dendrite-dendrite interactions can also occur. These interactions require the membrane proteins known as cell adhesion molecules (CAMs). The CAMs function as an adhesive, causing axons or dendrites to bundle.

CAMs can be broken into four large superfamilies: immunoglobulin (Ig), integrins, cadherins and selectins. Within the Ig superfamily the L1-CAM family, which is made of L1, close homolog of L1, neurofascin and NrCAM, is important in axon guidance and fasciculation, synaptic plasticity and neuronal migration. KIF4 was shown to be involved in the anterograde transport of L1, but whether via direct linkage to KIF4 or through adaptor proteins is not known (Peretti et al., 2000).

NgCAM (neuron-glia cell adhesion molecule) is the chicken homolog of L1. The molecule contains six immunoglobulin (Ig) like domains, four to five fibronectin (FN) type III repeats in the extracellular region, a single transmembrane region and a highly

3 conserved cytoplasmic tail. It has been used as a model molecule to understand the mechanisms regulating polarized membrane protein sorting and targeting in hippocampal neurons. Using a transcytotic pathway NgCAM is expressed first on the somatodendritic surface before being endocytosed and transported to the axon, there is a specific axonal targeting sequence within the cytoplasmic tail that is required for this pathway (Wisco et al., 2003; Yap et al., 2008b). There is also a secondary axonal targeting signal located in the FN repeats that improves axonal targeting (Sampo et al., 2003).

The adhesion ability of L1-CAM is due to the heterophilic and homophilic interactions between L1-CAMs by their Ig domains (Maness and Schachner, 2007). L1-

CAM intracellular trafficking usually mediated by the C-terminus, which contains a conserved FIGQ/AY sequence that binds to ankyrin proteins, which links L1 to the actin cytoskeleton (Bennett and Chen, 2001). Phosphorylation of the tyrosine in the conserved sequence regulates the L1-ankyrin binding (Garver et al., 1997).

Knockout mice carrying L1-CAM mutations showed abnormalities in retino- collicular, corticospinal, thalamocortical and callosal axons, and also in dendritic bundles of cortical pyramidal neurons (Dahme et al., 1997; Cohen et al., 1998; Demyanenko and

Maness, 2003; Demyanenko et al., 2004; Wiencken-Barger et al., 2004; Maness and

Schachner, 2007). This led to the main question of my first chapter: Could the polarized targeting of a CAM affect both axonal and dendritic bundling?

Ion channels

As stated earlier the proper function of a neuron requires the polarized targeting of many membrane proteins, but one of the most critical are ion channels. The three major

4 + + 2+ ion channels required for APs are voltage gated Na (Nav), K (Kv) and Ca channels. A wide variety of ion channels can be produced through alternative splicing and heteromeric oligomerization that occurs within a neuron. How these various channels are trafficked to very specific regions within the neuron is a question that is still being researched today. How a variety of ion channels are transported by so few kinesin motors is a very interesting question. Most believed an adaptor protein was needed to link the ion channel to the kinesin motor, while more recent studies have begun to show some channels interact directly with their motor.

The electrical and chemical signaling that occurs between neurons requires the proper localization of both ligand- and voltage-gated ion channels. These channels must be contained within very specific regions of the neuronal membrane and some have a polarized targeting pattern where they can be enriched either in the dendrites or the axon.

There is a wide variety of ion channels that can be produced through alternative splicing and heteromeric oligomerization that occurs within a neuron. How these various constructs are trafficked to very specific regions within the neuron is a question that is still being answered by researchers everyday.

Kv3 channels are involved in fast spiking neurons because of their unique biophysical properties: high activation threshold (roughly -20 mV) and rapid deactivation kinetics (Rudy and McBain, 2001; Bean, 2007). The Kv3 subfamily has four members

(Kv3.1 to Kv3.4). Kv3.1 has two splice variants (Kv3.1a and Kv3.1b) due to alternative splicing in the C-terminal. These two variants also have different polarized axon- dendrite targeting patterns (Xu et al., 2007). This polarized targeting is due to an axonal

5 targeting motif in the C-terminal that binds both the N-terminal T1 domain and ankyrin G at the axon initial segment (Xu et al., 2007)

Kv3 mediated fast spiking is critical for stabilizing images on the retina via the vestibule-ocular reflex circuit and sound location by the auditory circuit (Parameshwaran et al., 2001; von Hehn et al., 2004; Gittis et al., 2010). Regulating the Kv3 protein levels and phosphorylation of Kv3 is a proposed mechanism in adjusting the spiking frequency of neurons in auditory circuits and the suprachiasmatic nucleus following different acoustic environments and circadian rhythm, respectively (Itri et al., 2005; Song et al.,

2005).

Potassium Channels

Potassium (K+) channels play very important physiological and pathological roles in different organs including the brain, heart and muscle. They are one of the most diverse families of ion channels. There are approximately 80 different genes that encode the pore-forming subunits. This large superfamily can be split into three major families based on the secondary structure of the channel subunits: 1) voltage-sensitive, 2) inwardly rectifying and 3) two-pore domain K+ channels. Voltage-sensitive K+ channels

α subunits contain six to seven transmembrane (TM) domains, and one pore-forming loop located between the last two domains. Each functional channel contains four of these α subunits and possibly is associated with β subunits as well. In this family there are channels that are activated by both membrane potential and intracellular chemical signals.

Inwardly rectifying K+ channels conduct current more efficiently in the inward direction.

They have the simplest structure, two TM domains with a pore-forming loop between

6 them. They also form tetramers and can co-assemble with other membrane proteins.

Two-pore domain K+ channels subunits contain four TM domains and two pore-forming loops. Only two subunits are required to form a functional channel. Most members of this family form leak K+ channels due to have little voltage or time dependence. In the following sections I will give a general overview of the three different families of K+ channels, their function, tissue distribution, and diseases associated with them.

Voltage-sensitive K+ channels

Voltage-sensitive potassium (Kv) channels are a very large family in its own right. It can be divided into four subfamilies based on their homologues in fruit flies

(Drosophila): Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal). Since the cloning of these original four subfamilies, eight other subfamilies (Kv5 to Kv12) have been discovered (Chandy, 1991; Gutman et al., 2005). Only Kv5, Kv6, Kv8 and Kv9 α subunits do not form functional channels, but they can modulate other Kv channels

(Gutman et al., 2005; Vacher et al., 2008).

Kv1, Kv2, Kv3 and Kv4 are most well known for being extensively expressed in neurons where they function in regulating neuronal action potential firing. For example

Kv3 channels mediate the fast spiking neurons in stabilizing images on the retina by the vestibule-ocular reflex circuit, and sound localization by the auditory circuit

(Parameshwaran et al., 2001; von Hehn et al., 2004; Gittis et al., 2010).

Other members of these four channels also have roles outside of central nervous system. For example Kv1.3 is important in T lymphocyte membrane potential (Chandy

7 et al., 1984; DeCoursey et al., 1984; Lewis and Cahalan, 1995). Kv1.5 is expressed in the atrium of the heart (Fedida et al., 2003; Gaborit et al., 2007). Kv1.7 is expressed strongly in the heart and skeletal muscle (Finol-Urdaneta et al., 2006), and in the heart it is important in regulating the frequency, shape and duration of cardiac action potentials

(Brown, 1997; Nerbonne, 2000). Kv1.4, Kv1.6 and Kv2.1 were shown to be highly expressed in rat islets and insulinoma cells of the pancreas (MacDonald et al., 2001).

Kv1.8 has been shown to be expressed in the kidney, specifically proximal tubular cells, glomerular and vascular endothelial cells, and also vascular smooth muscle cells. It is believed to be important in sodium absorption and stabilizing the voltage of cell membranes of the proximal tubular cells and may also regulate vascular tone (Yao et al.,

2002). Kv3.4 is abundant in skeletal muscle and sympathetic neurons, but weakly expressed in only a few neuronal types in the brain, usually where other Kv3 channels are expressed (Rudy et al., 1999).

The Kv7 (KCNQ) subfamily was more recently identified and has a variety of functions. When Kv7.1 (KCNQ1) is associated with its β subunit KCNE1 (minK) it can repolarize action potentials in the heart (Barhanin et al., 1996; Sanguinetti et al., 1996), and can provide a pathway for transepithelial potassium secretion in the inner ear (Vetter et al., 1996; Neyroud et al., 1997). Kv7.2 and Kv7.3 (KCNQ2 and KCNQ3) are important as heterotetramers, where they were observed to be a molecular correlate of the

M-current, which is involved in determining subthreshold excitability of neurons and their responsiveness to synaptic input (Brown and Adams, 1980; Wang et al., 1998a).

Kv7.4 (KCNQ4) and Kv7.5 (KCNQ5) also form heterotetramers with KCNQ3, but

KCNQ4 is much more restricted in its expression, it is primarily expressed in sensory

8 hair cells of the inner ear and certain tracts and nuclei of the central auditory pathway

(Kubisch et al., 1999; Kharkovets et al., 2000; Lerche et al., 2000; Schroeder et al.,

2000). Kv10-Kv12 are grouped into a subfamily known as the KCNH. This subfamily is expressed in the CNS where it plays important roles in regulating neuronal excitability of the nervous system.

Two other groups of K+ channels can be categorized in this family: 1) large conductance Ca2+-regulated, “big” K+ (BK) channels, and 2) small- and intermediate- conductance calcium-regulated K+ channels. The BK channels are among the largest and most complex of the K+ channel superfamily. It is gated by both voltage and intracellular

Ca2+. It contains seven TM domains and one pore-forming loop, with a large conserved

C-terminus that allows channel gating to be altered in response to directly sensing several different intracellular ions and other second messenger systems (Salkoff et al., 2006).

The four members of this family Slo1, Slo 2.1 (Slick), Slo 2.2 (Slack) and Slo3 all differ in their gating properties and Ca2+ regulation (Salkoff et al., 2006). The small- and intermediate-conductance calcium-regulated K+ channels contain six TM domains and one pore-forming loop just like Kv channels, but their activation is regulated by intracellular Ca2+ and is are insensitive to the membrane potential (Weatherall et al.,

2010).

Mutations in Kv1.1 causes episodic ataxia type 1, an autosomal dominant disease

(Browne et al., 1994). Some of these mutants fail to produce functional homomeric channels, and reduced the K+ current when they formed heteromeric channels with wildtype subunits(Adelman et al., 1995). The mouse model of the Kv1.1 knockout

9 shows heterozygous mice appear normal, but homozygous knockouts have frequent generalized epileptic seizures (Smart et al., 1998).

Kv3.4 is upregulated in the early stages of Alzheimer’s disease, which may cause altered synaptic activity and underlie the neurodegeneration observed in Alzheimer’s disease (Angulo et al., 2004). Kv4.1 is expressed in breast cancer cell lines and plays a role in their proliferation (Kim et al., 2010).

Mutations of Kv7.1 contribute to long QT syndrome which causes cardiac arrhythmia and sudden death (Sanguinetti et al., 1996; Wang et al., 1996). Mutations of

Kv7.2 and Kv7.3 cause a rare epilepsy disorder called benign familial neonatal convulsions, which is inherited in an autosomal-dominant method (Schroeder et al., 1998;

Goldberg-Stern et al., 2009; Volkers et al., 2009). Mutations in Kv7.4 produce an inherited syndrome of deafness (Kubisch et al., 1999).

Expression of Kv10.1 ectopically leads to uncontrolled proliferation, while inhibition of Kv101 reduces proliferation. Specific antibodies against Kv10.1 recognized an epitope in over 80% of human tumors of diverse origins tested (Pardo et al., 2005).

Mutations in Kv11.1 (human Ether-a-go-go-related gene hERG) can lead to a potentially fatal disorder called long QT syndrome, which is an inherited cardiac arrhythmia

(Sanguinetti et al., 1995; Moss et al., 2002).

Inward-rectifying K+ channels

Inward-rectifying potassium channels (Kir) allow K+ ions to flow into rather than out of the cell. Each Kir subunit contains two TM domains and one pore-forming loop, and four subunits make up a functional channel. Fifteen Kir subunit genes have been

10 identified and split into seven subfamilies (Kir1.x to Kir7.x). These families can be further categorized into four functional groups: 1) classical Kir channels (Kir2.x); 2) G protein-gated Kir channels (Kir3.x); 3) ATP-sensitive K+ (KATP channels (Kir6.x); 4)

K+-transport channels (Kir1.x, Kir4.x, Kir5.x and Kir7.x) (Hibino et al., 2010). Kir channels are only active when associated with phosphatidylinositol 4,5-bisphosphate

(PIP) (Baukrowitz and Fakler, 2000).

Kir2.1-Kir2.3 are all expressed in cardiomyocytes (Liu et al., 2001), with Kir2.1 expressed 10-fold higher than Kir2.2 and Kir2.3 (Wang et al., 1998b). Kir3 is important in regulating excitability in the heart and brain (Stanfield et al., 2002). Kir4.1 and Kir5.1 are expressed in the brain in astrocytes and retinal Muller cells, and in the kidney in proximal and distal convoluted tubule and cortical collecting duct (Poopalasundaram et al., 2000; Tanemoto et al., 2000; Stanfield et al., 2002; Ishii et al., 2003; Konstas et al.,

2003; Hibino et al., 2004; Tanemoto et al., 2004).

Kir6.2 forms KATP channels in the heart when combined with sulfonylurea receptor 2A (SUR2A) subunits, while other KATP channels are formed of Kir6.1 and

SUR2B subunits in smooth muscle cells (Seino, 1999; Wu et al., 2007). Kir7.1 is expressed in the kidney and the epithelial cells of the choroid plexus, and is proposed to contribute to tubular K+ recycling and secretion (Doring et al., 1998; Ookata et al., 2000;

Derst et al., 2001).

As stated earlier Kir channels are only active when associated with phosphatidylinositol 4,5-bisphosphate (PIP) (Baukrowitz and Fakler, 2000). Mutations of Kir1.1 which impairs PIP2 interaction can lead to disease states like Bartter’s syndrome, which is characterized by low potassium levels (Lopes et al., 2002). Mutation

11 in Kir3.2 lead to a disorder in the mouse called the weaver mutant. Weaver mice have spontaneous seizures, extensive cerebellar granule cell death and progressive loss of substantia nigra neurons (Patil et al., 1995; Navarro et al., 1996).

Two-pore domain K+ channels

Leak K+ channels contribute to resting membrane potential, and are involved in regulating neuronal excitability. Each subunit contains four TM domains with two pore- forming loops, and only two subunits are needed to make a functional channel. The first channel TWIK (tandem of pore domains in a weak inward rectifying K+ channel, now called TWIK-1) was identified in 1996 (Lesage et al., 1996). There are currently fifteen members of the KCNK family in the human genome, but KCNK8, KCNK11 and

KCNK14 do not exist. This family is grouped into six subfamilies, TWIK, TREK,

TASK, TALK, THIK and TRESK.

TWIK-1 has been detected in the kidney, brain and lung (Lesage et al., 1996;

Arrighi et al., 1998). But TWIK K+ current still needs to be analyzed directly in these cell types. TWIK-related K+ (TREK) channel subfamily is made up of TREK-1, TREK-

2 and TRAAK. TREK-1 overexpression altered the morphology of cultured hippocampal neurons with filipodia like structures formed in the axons and dendrites which were enriched with actin and ezrin (Lauritzen et al., 2005). Cultured striatal neurons of TREK-

1 knockout mice developed significantly less growth cones and looked more condensed than wildtype animals, suggesting TREK-1 is necessary for normal morphogenesis

(Lauritzen et al., 2005). Many different two-pore potassium channels are expressed at the

12 mRNA level in the rat heart including TREK-1, TREK-2 and TRAAK (Liu and Saint,

2004). Gastrointestinal, bladder and uterine smooth muscles expressed TREK-1 where it may cause the cells to relax (Koh et al., 2001).

TASK (TWIK-related acid-sensitive K+ channel) subfamily contains TASK-1,

TASK-3 and TASK-5. In type I (glomus) cells of carotid bodies expression of several

TASK channels has been detected, including TASK-1, TASK-2 and TASK-3 (Buckler and Vaughan-Jones, 1994; Yamamoto et al., 2002; Yamamoto and Taniguchi, 2006).

TASK-1/TASK-3 heterodimers found to be responsible for major part of oxygen- sensitive TASK-like background K+ conductance (Kim et al., 2009). In cerebellar granule cells background K+ conductance is regulated by TASK-1/TASK-3 heterodimers in vivo, while heterodimers along with TASK-3 homodimers constituted the current in cultured cells (Kang et al., 2004; Aller et al., 2005).

TALK (TWIK-related alkaline pH-activated K+ channel) subfamily includes

TALK-1, TALK-2 and TASK-2. Because of its sensitivity to extracellular pH TASK-2 was originally named TASK-2, but later it was reassigned to the TALK subfamily due to sequence similarity. TASK-2 is expressed in the kidney in the proximal tubule cells and is indirectly engaged in bicarbonate reabsorption and the accompanying Na+ and water movements (Warth et al., 2004).

TREK-1 was highly expressed in prostate cancer samples and malignant cell lines

(Voloshyna et al., 2008). A dominant negative mutation of TASK-3 was implicated in a maternally inherited disease with mental retardation and characteristic dysmorphism

(Barel et al., 2008). TASK-3 may be involved in carcinogenesis and tumor progression.

TASK-3 was significantly overexpressed in 44% of breast and 35% of lung cancer cases,

13 and overexpression of TASK-3 accelerated tumor formation from implanted oncogenically transformed (C8) fibroblasts (Mu et al., 2003).

Transport of ion channels by adaptor proteins

AMPA receptors

AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptors are ligand-gated glutamate receptor ion channels. These channels are nonselective and allow sodium, potassium and calcium ion influx to occur. AMPA receptors are usually heterotetrameric with a ‘dimer of dimers’ made up of various combinations of the four subunits GluR1 to GluR4. GluR subunits have an extracellular N-terminal, 4 transmembrane domains, with a pore loop between TM2 and TM3, and an intracellular

C-terminal domain (Fig. 1.1A). If the channel contains the GluR2 subunit then it is no longer permeable to calcium (Hollmann et al., 1991). AMPA receptor trafficking has been well studied, with multiple kinesins and adaptors being identified.

GRIP-1

Glutamate receptor interacting protein-1 (GRIP-1) was identified to bind to the C- terminus of GluR2 (Dong et al., 1997). The GRIP family (GRIP-1 and GRIP-2) contain six or seven PDZ domains (Fig. 1.1A). PDZ stands for the first three proteins discovered that contain this domain (PSD-95, Discs-large and ZO-1) (Kennedy, 1995). The fifth

PDZ domain was discovered to bind the C-terminal of GluR2/3 subunits (Dong et al.,

1997; Srivastava et al., 1998; Wyszynski et al., 1999). GRIP-1 was later shown to interact with KIF5 C-terminal tail via a region between PDZ 6 and PDZ 7 (Setou et al.,

14 2002). This interaction caused KIF5 to be directed to the dendrites, where it functions as an AMPA receptor transporter (Setou et al., 2002).

Liprin-α

Liprin-α is another adaptor protein that was found to link GluR2/3 to a different kinesin, KIF1A. Liprin-α is a four member family (α1 to α4) that binds the LAR family of receptor protein tyrosine phosphatases (Pulido et al., 1995; Serra-Pages et al., 1995;

Serra-Pages et al., 1998). Liprin-α has an N-terminal coiled-coil domain and a C- terminal sterile α motifs (Fig. 1.1A). It was the Liprin-α C-terminal that was shown to bind the GRIP-1 PDZ6 domain (Fig. 1.1A) (Wyszynski et al., 2002). This binding site is right next to the GluR2 binding site, which may cause the two binding sites to interact.

This might be true, because interrupting the Liprin-α/GRIP-1 interaction caused a reduction in GluR2 clustering along dendrites and a reduction in AMPA receptor surface expression (Wyszynski et al., 2002). The N-terminal of Liprin-α was shown to bind the

KIF1A C-terminus (Fig. 1.1A) (Shin et al., 2003).

LIN-10

LIN-10 is another PDZ domain protein originally discovered in Caenorhabditis elegans, where it plays a role in trafficking GLR-1 (the C. elegans homolog of mammalian GluR1) (Kaech et al., 1998; Rongo et al., 1998). mLin-10 contains a phosphotyrosine binding domain and two PDZ domains at its C-terminal (Fig. 1.1B)

(Okamoto and Sudhof, 1997; Kaech et al., 1998). The first PDZ domain was shown to interact with the C-terminal of GluR1 and possibly the tail of GluR2 also (Fig. 1.1B)

15 (Stricker and Huganir, 2003). The motor that transports the AMPA receptor via mLin-10 remains unknown, but may be KIF17, the same motor that transports NMDA receptors.

NMDA receptors

NMDA (N-methyl-d-aspartate) receptors are non-selective ligand-gated ionotropic glutamate receptors with a high permeability to calcium. These receptors are important is synaptic plasticity via calcium signaling and are key in learning and memory

(Tsien et al., 1996; Tang et al., 1999). The channel is made of NR1, NR2

Figure 1.1. Major binding proteins in the intracellular transport of AMPA receptors (A) The GluR2/3 subunits of AMPA receptors interact with glutamate receptor interacting protein-1 (GRIP-1), which binds to either KIF5 or KIF1. The diagram of a tetrameric AMPA receptor/channel is on the upper right. The diagram of a single GluR subunit is on the upper left, which contains four TM segments (gray bars), extracellular N-terminal domain, and intracellular C-terminal domain. The GluR2/3 C-termini bind to the PDZ5 domain (aa. 441–658) of GRIP-1. The residue number and the last five residues (-ESVKI*) are from GluR2. The cyan boxes numbered from 1 to 7 indicate seven PDZ domains. The region between PDZ6 and PDZ7 of GRIP-1 (aa. 753–987) interacts with the KIF5 C-terminal cargo binding region (aa. 807–934 from KIF5B). The PDZ6 domain (aa. 658–753) of GRIP-1 interacts with the C-terminal region of Liprin-α, which contains sterile α motifs (red boxes). The C-terminal half of the coiled-coil domain (the dark blue box) in the Liprin-α N-terminus (aa. 351–673) binds to the C-terminal region of the KIF1A motor (aa. 657–1105). Blue balls: N-terminal motor domains of kinesins. “N,” the N-terminus of the protein; “C,” the C-terminus of the protein; the number near the C- terminus; the total residue number. The residue numbers of protein binding sites are given whenever they are available. (B) The GluR1 subunit of AMPA receptors interacts with KIF17 through mLin-10. The PDZ1 domain of mLin-10 (aa. 650–840) can interact with both the GluR1 C-terminus and the KIF17 C-terminal region (aa. 957–1038). Yellow box: phosphotyrosine binding domain.

Figure 1.1. Major binding proteins in the intracellular transport of AMPA receptors.

18 and sometimes NR3 subunits (Laube et al., 1998; Dingledine et al., 1999; Carroll and

Zukin, 2002). Each subunit contains a similar structure of four TM domains with a pore loop between TM2 and TM3 (Fig. 1.2). NMDA receptors are unique because they are gated by both a ligand (both glycine and glutamate are required) and voltage (Kleckner and Dingledine, 1988). These channels are also special because extracellular Mg2+ blocks their voltage-sensitivity (Nowak et al., 1984).

LIN10

As shown above LIN-10 may play a role in AMPA receptor trafficking. But it definitely is important in NMDA trafficking of receptors that contain the NR2B subunit

(Fig. 1.2). A complex of LIN proteins (LIN-7/LIN-2/LIN-10) links the C-terminal of

NR2B with the C-terminal of KIF17 (Fig. 1.2) (Kaech et al., 1998; Jo et al., 1999; Setou et al., 2000). Disruption of mouse KIF17 inhibited NR2B transport, but not NR2A transport, suggesting the NR2A subunit is transported by a different motor (Yin et al.,

2011). Also in KIF17 knockout mice there were reduced levels of NR2 receptors, which resulted in attenuation of NMDA-receptor mediated synaptic current, long term depression and early/late long term potentiation (Yin et al., 2011). Therefore the link between ionotropic glutamate receptors (both AMPA and NMDA) and kinesins appears to rely on various proteins containing PDZ domains (Fig. 1.1 and 1.2).

Figure 1.2. Adaptor and motor proteins in the intracellular transport of NMDA receptors. During the intracellular transport of NMDA receptors, KIF17 interacts with the NR2B subunit through three PDZ-domain proteins. The NR2B C-terminus binds to the only PDZ domain of mLin-7 (aa. 127–197). The last residue number (1484) and the last four residues (-ESDV*) of NR2B are indicated. A small region right before the PDZ domain of LIN-7 (aa. 109–126) interacts with a region in the middle of LIN-2 (aa. 428– 524). An N-terminal region of LIN-2 (aa. 1–247) interacts with the N-terminal region of LIN-10 (aa. 1–581). The PDZ1 of mLin-10 (aa. 650-840) binds to the C-terminal tail of KIF17 (aa. 957–1038). In the PDZ domain proteins, cyan boxes, PDZ domains; yellow box in LIN-10/mLin-10, phosphotyrosine binding domain; orange box in LIN-2, N- terminal CaM kinase domain; green box in LIN-2, SH3 domain; red box in LIN-2, guanylate kinase domain.

20 GABAA Receptors

GABAA (γ-aminobutyric acid type A) receptors are ligand-gated chloride channels, which are important inhibitory neurotransmitter receptors in the central nervous system (Whiting, 1999). The channels are pentamers of a combination of α, β, γ, δ, ε, θ, and π subunits (Bonnert et al., 1999) with the most common structure being 2α, 2β and 1γ

(Tretter et al., 1997). Each subunit has four TM with extracellular N- and C-termini (Fig.

1.2B).

GRIF-1

The β2 subunit of GABAA receptors is the most common subunit in the adult brain (Li and De Blas, 1997; Sur et al., 2001). GRIF-1 (GABAA receptor interacting factor 1) was discovered to interact with the β2 subunit (Beck et al., 2002). GRIF-1 contains two coiled-coil domains in the N-terminal region (Fig. 1.3). The first coiled-coil domain was shown to interact with both the intracellular loop of the β2 subunit (Beck et al., 2002) and the kinesin motor KIF5C (Brickley et al., 2005) (Fig. 1.3). The GRIF-

1/KIF5 interaction was further narrowed to the non-motor domain of KIF5C (Pozo and

Stephenson, 2006) then specifically to the cargo binding domain (Fig. 1.3, (Smith et al.,

2006).

HAP-1

Huntingtin-associated protein 1 (HAP1), another adaptor protein, was shown to regulate the trafficking of GABAA to the synapse (Twelvetrees et al., 2010). HAP1 contains three coiled-coil domains in the N-terminal (Fig. 1.3). HAP1 was shown to

21 directly interact with GABAA receptors via the β3 subunit (Fig. 1.3) (Kittler et al., 2004).

The coiled-coil domain of HAP1 was then shown to directly interact with the C-terminal of KIF5B (Fig. 1.3) (Twelvetrees et al., 2010). Interference of the KIF5-HAP1 interaction by dominant negative KIF5-HAP1 binding domain or HAP1 RNAi reduced the surface expression of GABAA receptors (Twelvetrees et al., 2010).

Figure 1.3. Different adaptor proteins mediate the interaction between GABAA receptors and KIF5 motors. Pentameric structural diagram of the GABAA receptor is shown on the top. The first coiled-coil domain of GRIF-1 (aa. 124–283) can bind to either the second intracellular loop of GABAA β2 (aa. 301–426) or the C-terminal tail region of KIF5B (aa. 827–957). The coiled-coil domains of huntingtin-associated protein- 1 (HAP-1; aa. 153-320) interact with C-terminal of KIF5B (aa. 814–963). Whether and how HAP-1 interacts with GABAA β3 directly remains unknown at this time. Blue boxes: coiled-coil domains.

23 Trafficking via direct binding of channels and motors

Voltage-gated potassium channel (Kv3) and Kinesin 1

As listed above most channel-motor interactions require an adaptor protein, but recent studies have shown some channels bind directly to their motors. Kv channels play roles in neuronal excitability and synaptic transmission. Members of the Kv channel superfamily have a wide variety of expression patterns and biophysical and pharmacological properties (Gu and Barry, 2011). Kv channels contain four voltage- sensing and pore-forming subunits. Each subunit is made of six TM domains with intracellular N- and C-termini (Fig. 1.4A). The T1 domain within the N-terminal is responsible for the tetramerization of the Kv channel subunits within each Kv subfamily

(Li et al., 1992; Xu et al., 1995; Choe et al., 2002). Zn2+ is required for the T1 tetramerization for Kv2, Kv3 and Kv4 but not Kv1 channels (Bixby et al., 1999; Choe et al., 2002; Jahng et al., 2002).

Kv3 channels are involved in fast spiking neurons because of their unique biophysical properties. The Kv3 subfamily has four members (Kv3.1 to Kv3.4). Kv3.1 has two splice variants (Kv3.1a and Kv3.1b) due to alternative splicing in the C-terminal.

These two variants also have different polarized axon-dendrite targeting patterns (Xu et al., 2007). This polarized targeting is due to an axonal targeting motif in the C-terminal that binds both the N-terminal T1 domain and ankyrin G at the axon initial segment (Xu et al., 2007).

Kv3 channel transport is mediated by kinesin 1/KIF5, which binds to the T1 domain (Xu et al., 2010). The specific binding site of the T1 domain was mapped to a

24 region within the tail domain of KIF5 (Fig. 1.4A), and required the proper T1 tetramerization of Kv3 channels (Xu et al., 2010). This study was important in being the first to describe direct binding between an ion channel and kinesin motor with no adaptor required.

CLC-5 (Chloride/Proton antiporter) and Kinesin 2

CLC-5 is a member of the CLC family of voltage-gated chloride channels and chloride/proton antiporters. CLC-5 directly binds to KIF3B/kinesin 2 (Reed et al., 2010).

The C-terminal of CLC-5 interacts with the coiled-coil and cargo binding domain but not the motor domain of KIF3B (Fig. 1.4B) (Reed et al., 2010). siRNA knockdown of

KIF3B caused a reduction in the surface expression of CLC-5. These two examples of channels being transported through direct interaction with their motor is just the beginning of the discovery of what is likely a major mechanism underlying the transport and localization of channels in the membrane.

Figure 1.4. Direct binding between ion channel/transport and kinesins. (A) Structural diagrams of Kv3.1b subunit (left) and tetrameric channel complex of Kv3 (right). T1 domain of Kv3.1 (aa. 1–110) interacts with the C-terminal region of KIF5B (aa. 865–934). Gray bars, transmembrane domains; black ovals, T1 domains. Importantly, only properly tetramerized T1 domains, but not T1 monomers, bind to KIF5 tail domains. In the diagram for a channel tetramer complex (upper right), for clarification, only the C- terminus of one subunit is shown in green, containing the ATM (axonal targeting motif). (B) Diagrams of CLC-5 subunit structure and KIF3. CLC-5 C-terminal domain interacts with the non-motor region (coiled-coil and cargo binding domains) of KIF3. Gray bars, transmembrane domains in CLC-5; purple bars, extra-/intracellular domains; green ovals, CBS (cystathionine beta synthase) domain. KAP3 = kinesin-associated protein 3.

26 Kinesin transport of ion channels by unknown adaptors

The above research has shown that ion channels can interact with their motors with or without adaptor proteins. However, there are some interactions between a channel and its motor that are not known whether they require an adaptor protein or not.

Unlike Kv3 channels Kv1 channels require an auxiliary subunit, Kvβ2, which binds to the Kv1 T1 domain, to possibly mediate axonal targeting (Gu et al., 2003).

Kvβ2 is also known to bind to KIF3A/kinesin 2, which is required for axonal targeting of

Kv1.2/Kvβ2 (Gu et al., 2006). But it is not known whether Kvβ2 interacts directly or indirectly with KIF3, therefore whether it is an adaptor for the Kv1/KIF3 channel/motor complex is still unclear.

Another Kv channel, Kv4.2, is a dendritic A-type Kv channel (Rivera et al., 2003).

Using a dominant-negative screening process it was determined only KIF17 had an effect on the targeting of Kv4.2. However, a recent study using KIF17 knockout mice showed the level of Kv4.2 remained unchanged in these mice, suggesting other motor proteins may be involved in Kv4.2 trafficking (Yin et al., 2011).

Cyclic nucleotide-gated (CNG) ion channels are located to non-motile cilia on olfactory sensory neurons. The CNG channels are tetramers made of subunits CNGA2,

CNGA4, and CNGB1b (Bonigk et al., 2009). KIF17 dominant negative construct was able to block YFP-tagged CNG channels from entering the cilia (Jenkins et al., 2006).

Regulation of Ion Channel and Kinesin Interaction

Proper localization of ion channels requires regulation of the ion channel/kinesin interaction. An ion channel must be able to associate and disassociate with its motor at

27 the right time in the right place. Three major types of regulation are adaptor proteins regulated by binding proteins, posttranscriptional modification (phosphorylation) and oligomerization of channel subunits.

Adaptor protein with multiple binding partners

Adaptor proteins may have other binding partners besides ion channels. For example GRIP-1 binds to many other proteins through different domains including

GRASP-1, a Ras guanine-nucleotide exchange factor (Ye et al., 2000), EphB2 and

EphA7 receptor tyrosine kinases and EphrinB ligands (Torres et al., 1998; Bruckner et al.,

1999; Lin et al., 1999) (Fig. 1.5A). Protein Interacting with C-kinase 1 (PICK1) interacts with the linker II region of GRIP-1 to regulate AMPA receptor trafficking (Lu and Ziff,

2005), while Phosphoinositide 3-kinase enhancer binds to the 4th PDZ domain (Chan et al., 2011) and neuronal early endosomal protein, NEEP21, binds to the C-terminus

(Steiner et al., 2005) (Fig. 1.5A). These binding proteins may interfere with the interaction of GRIP with AMAP receptors and/or KIF5, which may alter the transport of the channels.

Figure 1.5. Regulation of ion channel protein loading to kinesin motors. (A) Multiple binding proteins of GRIP-1. Cyan boxes, PDZ domains. (B) Phosphorylation of KIF17 at aa. Ser1029 by CaM kinase II causes disassociation from mLin-10. (C) Kv3.1 tetramerization is required for the interaction with KIF5 motor [Source. Adapted from Xu and others (2010)].

29 Posttranslational modification

The channel-kinesin or channel-adaptor-kinesin interaction can be regulated by posttranslational modification, especially phosphorylation. For example the phosphorylation of S1029, by CaM kinase II, in the C-terminus of KIF17 disrupts the

KIF17/mLin-10 interaction (Guillaud et al., 2008). This disruption causes a release of mLin-10 from KIF17 (Fig. 1.5B).

Another example is the phosphorylation of GRIP-1 at S917, by an unknown kinase, which regulates the surface expression of the AMPA receptor subunit GluR2

(Kulangara et al., 2007). PICK1 is a protein kinase C α adaptor protein that interacts with

GRIP-1. When this interaction is disrupted the phosphorylation of S880 of GluR2 is impaired, which causes a reduced surface expression of GluR2 (Lu and Ziff, 2005).

Channel oligomerization and kinesin transport

The major example of channel assembly (oligomerization) that regulates the channel-kinesin complex is the tetramerization of Kv3 channels. Only the fully tetramerized T1 domains of Kv3.1 interact with KIF5 motors, but not monomers of the

T1 domain (Fig. 1.5C) (Xu et al., 2010). However heterooligomerization of channel proteins may also be important in channels interacting with their motors. For example

GluR2/3 binds to KIF5 by using GRIP-1, while GluR1 binds to KIF17 via mLin-10. This may lead to a ‘tug of war’ between the two adaptor-kinesin constructs for the same channel.

CHAPTER 2

NGCAM TARGETING REGULATES AXON AND DENDRITE BUNDLING

SUMMARY

Whether a single cell adhesion molecule (CAM) can induce both axonal and dendritic bundling due to polarized targeting is an interesting question. Different L1-

CAM proteins have different expression and targeting patterns, which may play different roles in axonal and dendritic morphogenesis. In this chapter I used NgCAM to examine the basic molecular mechanisms underlying the sorting and targeting of this polarized membrane protein. My lab* generated a new in vitro transfection method which allowed expression of different constructs in different neurons in the same culture. This allowed for the study of the interaction of the neurites of transfected neurons. Polarized targeting of NgCAM to the axon induced axonal bundling due to the extracellular Ig domains.

Surprisingly the polarized targeting of NgCAM could be switched to the dendrites due to either blocking protein kinase activity or mutagenesis. Therefore these results suggest the polarized targeting of L1-CAM can regulate the axonal or dendritic bundling.

 All experiments performed in this chapter by Joshua Barry with help from Dr.

Chen Gu.

31 INTRODUCTION

When normally thinking of neuronal interactions most people immediately think of the synapse, the interaction between an axon and a dendrite. But there are other neuronal interactions that can occur between axons or dendrites alone. In the case of axons this term is known as axon fasciculation, while the more general term bundling can be applied to both axons and dendrites.

Establishment of proper neuronal connections over long distances requires selective axonal bundling (Bastiani et al., 1984; Tosney and Landmesser, 1985; Lin et al.,

1994; Yu et al., 2000; Hanson et al., 2008). CAM mediated axon bundling can be regulated by neuronal activity (Walsh and Doherty, 1997; Van Vactor, 1998; Yu et al.,

2000; Hanson et al., 2008; Imai et al., 2009). Dendritic bundling may be important for receiving common excitatory inputs (He and Masland, 1998). A recent study showed that synchronization of shared synaptic inputs may be due to dendritic bundling of gonadotropin-releasing hormone neurons (Campbell et al., 2009).

This bundling occurs due to the interactions between CAMs expressed on the membrane surface of neurons. A large family of CAMs, called the L1-CAM family, has a common structure made up of six immunoglobulin (Ig)-like domains and four to five fibronectin (FN) type III repeats in the extracellular region. This is followed by a single transmembrane domain and a highly conserved cytoplasmic tail. The adhesion ability of

L1-CAM is due to the heterophilic and homophilic interactions between L1-CAMs by their Ig domains (Maness and Schachner, 2007). L1-CAM intracellular trafficking usually mediated by the C-terminus, which contains a conserved FIGQ/AY sequence that binds to ankyrin proteins, which links L1 to the actin cytoskeleton (Bennett and Chen, 32 2001). Phosphorylation of the tyrosine in the conserved sequence regulates the L1- ankyrin binding (Garver et al., 1997).

Knockout mice carrying L1-CAM mutations showed abnormalities in both axons and dendritic bundles (Dahme et al., 1997; Cohen et al., 1998; Demyanenko and Maness,

2003; Demyanenko et al., 2004; Wiencken-Barger et al., 2004; Maness and Schachner,

2007).

NgCAM (neuron-glia cell adhesion molecule) is the chicken homolog of L1-

CAM that has been used as a model CAM molecule in polarized targeting in hippocampal neurons (Sampo et al., 2003; Wisco et al., 2003; Yap et al., 2008a; Yap et al., 2008b). My lab generated a new in-vitro system to assess the hypothesis that the axon-dendritic targeting of a single L1-CAM protein can regulate the axon and dendritic bundling. The new system was used to transfect hippocampal neurons with two different

GFP- or mCherry-tagged NgCAM constructs. The standard Lipofectamine 2000 transfection protocol was modified so each cDNA/lipofectamine complex contains only one cDNA plasmid. Using this method, transfection of neurons with the two complexes resulted in one neuron expressing one protein.

RESULTS

NgCAM induces bundling of axons in hippocampal neuron culture.

To start examining the hypothesis that NgCAM targeting can affect axonal or dendritic bundling I began by tagging NgCAM with GFP or mCherry by fusing the fluorophores to the C-terminus of NgCAM. At 5 DIV hippocampal neurons were

33 transfected and fixed 3 days later. NgCAM-GFP and NgCAM-mCherry expressing neurons were shown to cause highly bundled axons (Fig. 2.1A). As a negative control

GFP and mCherry transfected by themselves induced almost no axonal bundling despite their extensive axonal crossings (Fig. 2.1B).

NgCAM domains play different roles in bundling.

I then deleted the extracellular Ig domains to generate NgCAM-ΔIg-GFP or

NgCAM-ΔIg-mCherry. When these constructs were transfected into neurons the axonal bundling was completely eliminated even though there was still extensive crossing between the NgCAM-ΔIg-GFP and NgCAM-ΔIg-mCherry axons (Fig. 2.2A, D).

Deletion of the C-terminal domain (NgCAM-ΔCt-GFP or NgCAM-ΔCt-mCherry) caused a reduction in axonal bundling (Fig. 2.2B, D).

To quantify axonal bundling I calculated the axonal bundling index (ABI) which is defined as the total length of bundled axon segments divided by the total number of axonal crossings (Fig 2.2C, D).

Figure 2.1. Expression of NgCAM fusion proteins induced robust axonal bundling in cultured hippocampal neurons. GFP and mCherry, were fused to the C-terminus of NgCAM. NgCAM-GFP and NgCAM-mCherry were transfected into different neurons in culture. (A) Axonal bundling was induced between the axons of NgCAM-GFP and NgCAM-mCherry-expressing neurons. NgCAM constructs structural diagrams are provided. Black circles, the Ig domains; blue rectangles, fibronectin repeats; black squares, the membrane-spanning segment; green and red circles, GFP and mCherry; “N” and “C”, the extracellular N- and intracellular C-terminus. Arrows, bundled axonal segments. (B) Little axonal bundling of the axons of GFP- and mCherry-expressing neurons. Asterisks, soma of transfected neurons. Scale bars, 100 μm in (A) and upper panels of (B), 10 μm in lower panels of (B). (All experiments performed by Joshua Barry).

Figure 2.2. Roles of Ig and C-terminal domains of NgCAM in axonal bundling. (A) Deleting the Ig domains (NgCAM-ΔIg) eliminated NgCAM-induced axonal bundling. (B) Deleting the intracellular C-terminal domain of NgCAM (NgCAM-ΔCt) moderately reduced axonal bundling. Axonal bundles were still visible in culture. (C) Diagram for calculating the axonal bundling index (ABI). A pair of neurons expressing NgCAM-GFP (green) and NgCAM-mCherry (red) had bundled axonal segments. Upper right panel is the camera lucida drawing. Thick black lines show segments of axonal bundles (lower left) and asterisks show green and red axon crossings (lower right). The ABI (104μm/crossing) of this pair of neurons equals to the total length of axonal bundles (624 μm) divided by the total number of crossings (6). (D) Summary of ABIs of NgCAM truncations. Control, GFP and mCherry expressing neurons; NgCAM, NgCAM-GFP and NgCAM-mCherry expressing neurons; ΔCt, NgCAM-ΔCt-GFP and NgCAM-ΔCt-mCh expressing neurons; ΔIg, NgCAM-ΔIg-GFP and NgCAM-ΔIg-mCh expressing neurons; ΔFN, NgCAM-ΔFN-GFP and NgCAM-ΔFN-mCh expressing neurons. Asterisks, soma of transfected neurons. Scale bars, 100 μm. One-way ANOVA followed by Dunnett’s test was used for the comparison to the control group; ** P < 0.01. Additional One-way ANOVA followed by Dunnett’s test was performed for comparing the truncations to the wild type NgCAM shown on the top; ** P < 0.01. (All experiments performed by Joshua Barry.)

Figure 2.2. Roles of Ig and C-terminal domains of NgCAM in axonal bundling.

37 NgCAM targeting to dendrites induced dendritic bundling.

The most interesting construct was the deletion of the FN domains. Expression of the FN deletion constructs (NgCAM-ΔFN-GFP or NgCAM-ΔFN-mCherry) was able to induce dendritic bundling (Fig. 2.3A-C) due to the reversal of the targeting from the axon to the dendrite. To confirm this dendritic bundling was unique to the NgCAM FN deletion constructs I used an Ig-domain containing CAM (ICAM5 (telencephalin)) which is expressed in the dendrites as well. ICAM5, however, did not induce dendritic bundling

(Fig. 2.3D) even though it contains nine Ig domains. I confirmed the axon-dendrite targeting of the NgCAM-ΔFN construct by staining for microtubule associated protein 2

(MAP2) a dendritic marker (Fig. 2.3D).

Next, I examined the surface expression targeting of most NgCAM constructs using anti-NgCAM (8D9, which is against the extracellular Ig domains) under nonpermeabilized conditions. I could not test the NgCAM-ΔIg due to the lack of the Ig domains. NgCAM-GFP was enriched on the axonal surface (Fig. 2.4A, D, and F).

NgCAM-ΔCt-GFP was still enriched on the axonal membrane, similar to NgCAM-GFP

(Fig. 2.4B, D, and F). The polarity of NgCAM-ΔFN-GFP was reversed, and this construct was enriched on the dendritic surface (Fig. 4C, D, and F), similar to the

ICAM5-GFP targeting pattern (Fig. 2.4F). All GFP-tagged NgCAM constructs were expressed at the correct size (Fig. 2.4E).

Figure 2.3. Deleting the fibronectin repeats decreased axonal bundling but induced dendritic bundling. (A) The dendrites of a pair of neurons expressing NgCAM-ΔFN- GFP (green) and NgCAM-ΔFN-mCh (red) formed bundles in both parallel (blue arrowhead) and anti-parallel (red arrowhead) manners. The anti-MAP2 staining (blue in merged) indicated the dendrites and dendritic bundles. (B) Anti-parallel dendritic bundling. Dendrites of a pair of neurons expressing NgCAM-ΔFN-GFP (green) and NgCAM-ΔFN-mCh (red) grew towards each other and formed two bundles, indicated by white arrowheads. A high magnification image is provided on the right. (C) Parallel dendritic bundling. A pair of transfected neurons close to each other had an axonal bundle indicated by a white arrow and a parallel dendritic bundle indicated by a white arrowhead. (D) ICAM5 (telencephalin) constructs (ICAM5-GFP and ICAM5-mCh) were mainly localized in dendrites but did not induce dendritic bundling. Yellow circles, Ig domains; Yellow square, the membrane spanning segment. Scale bars, 100 μm. (All experiments performed by Joshua Barry.)

Figure 2.3. Deleting the fibronectin repeats decreased axonal bundling but induced dendritic bundling.

Figure 2.4. Axon-dendrite targeting of NgCAM-GFP and its deletion mutants. Neurons transfected with NgCAM-GFP or its truncations were stained with anti-NgCAM antibody under non-permeabilized conditions, and then stained for MAP2 under permeabilized conditions. Signals are inverted in unmerged images. In merged images (right), GFP fluorescence is in green, anti-NgCAM staining in red, and anti-MAP2 staining in blue. (A) NgCAM-GFP was mainly localized on axonal membranes. (B) NgCAM-ΔCt-GFP was mainly localized on axonal membranes similar to the wildtype. (C) Deletion of the fibronectin repeat domain resulted in dendritic targeting of NgCAM- ΔFN-GFP. NgCAM-ΔFN-GFP was mainly enriched on dendritic membranes. Arrows, axons. Arrowheads, dendrites. Scale bars, 100 μm. (D) Surface levels of the three NgCAM constructs along the axon (blue) and two main dendrites (red and dark red). (E) Western blots of GFP fusion constructs expressed in HEK293 cells. Mouse monoclonal anti-GFP antibody was used. (F) Summary of polarized targeting of NgCAM and ICAM5 constructs. For NgCAM constructs the surface levels were measured, but for ICAM5- GFP the GFP fluorescence intensity reflecting the total protein level was measured. One- way ANOVA followed by Dunnett’s test was used for the comparison among three NgCAM constructs; * P < 0.001. (All experiments performed by Joshua Barry.)

Figure 2.4. Axon-dendrite targeting of NgCAM-GFP and its deletion mutants.

42 NgCAM targeting regulated by protein phosphorylation.

I then wanted to identify the signaling pathway involved in regulating the axonal and dendritic bundling. Since neuronal firing patterns have been shown to regulate axon fasciculation I examined whether blocking voltage-gated sodium channels (100 μM tetrodotoxin), voltage-gated potassium channels (100 μM 4-aminopyridine) or voltage- gated calcium channels (50 nM Cd2+) had any effect on NgCAM induced bundling

(Hanson and Landmesser, 2004, 2006). They did not (Fig. 2.5E).

Then I examined the effect carbachol, an acetylcholine receptor agonist, had on bundling. It significantly increased axonal bundling (Fig. 2.5A, E). Carbachol activates both ligand-gated nicotinic and G protein coupled muscarinic receptors. While activation of nicotinic receptors increases neuronal excitability, muscarinic receptor activation initiates different protein kinase pathways through G proteins, which may also change the neuronal firing patterns. I tested the following list of kinase inhibitors (Table 1.1).

Name Kinase inhibited Concentration Chelerythrine (Chele) Protein kinase C 5 μM

Rp-cAMPS Protein kinase A 10 μM KN-93 Ca2+/calmodulin-dependent 2 μM kinase ML-7 Myosin light chain kinase 2 μM KT5823 Protein kinase G 1 μM GSK3 GSK3 10 nM Kinase Inhibitors Kinase Genistein Tyrosine kinase 20 μM Staurosporine (Stau) Broad spectrum 100 nM PD98059 Extracellular signal-regulated 12 μM kinases JNK JNK kinase 100 nM p38 p38 MAP kinases 1 nM

Inhibitors U0126 Extracellular signal-regulated 10 μM Map Kinase Kinase Map kinases Table 1.1 Kinase inhibitors and their concentrations used.

Figure 2.5. Protein phosphorylation regulated axonal targeting of NgCAM and hence neurite bundling. Hippocampal neurons transfected with either NgCAM-GFP or NgCAM-mCherry at 5 DIV were incubated with various drugs for 2 to 3 days, and then fixed and stained for quantification of polarized targeting and axonal bundling. (A) The carbachol treatment enhanced axonal bundling. White arrows, bundled axons. (B) Inhibiting protein kinase activities by the Stau treatment markedly decreased axonal bundling but increased dendritic bundling. Black arrowheads, dendrites. (C) The Stau treatment resulted in dendritic targeting of NgCAM. (D) Summary of the effects of different drugs on NgCAM-induced axonal bundling. Four panels of drug treatment experiments included the effects of ion channel blockers, AChR agonist, kinase inhibitors, and MAP kinase inhibitors, from left to right. The same control group was used in the four panels. One-way ANOVA followed by Dunnett’s test for comparing three or more groups, * P < 0.05. **P < 0.01. Two-tailed Student’s t-test for comparing two groups, * P < 0.05. (All experiments performed by Joshua Barry.)

Figure 2.5. Protein phosphorylation regulated axonal targeting of NgCAM and hence neurite bundling.

45 Only staurosporine had a significant effect of decreasing axonal bundling (Fig.

2.5B and D) and increasing dendritic bundling. To determine how staurosporine caused a switch from axonal to dendritic bundling I examined the membrane targeting of NgCAM-

GFP with staurosporine treatment. After staurosporine treatment NgCAM targeting was switched from the axon to the dendrites (Fig. 2.5C). I further examined specific kinase inhibitors that block three different pathways of MAP kinases (Table 1.1). Only the JNK inhibitor significantly reduced axonal bundling (Fig. 2.5D).

To determine whether my results with NgCAM were applicable to L1-CAM I fused GFP to the C-terminus of mouse L1-CAM (mL1-CAM-GF) and examined mL1-

CAM-GFP targeting and its effect on neurite bundling. mL1-CAM-GFP was mainly targeted to axons (Fig. 2.6A and C) and treatment with Stau induced a reversal of mL1-

CAM-GFP axon-dendrite polarity (Fig. 2.6B). Also, mL1CAM-GFP expressing neurons induced bundling of axons with neurons that expressed NgCAM-mCherry (Fig. 2.6D).

This suggests my data on NgCAM can represent the bundling and targeting effects of rodent L1-CAM.

Figure 2.6. Mouse L1-CAM (mL1CAM) axonal targeting is regulated by protein phosphorylation and induces axonal bundling. GFP was fused to the C-terminus of mL1CAM (mL1CAM-GFP). (A) When expressed in cultured hippocampal neurons, mL1CAM-GFP (green) mainly localized in axons. Dendrites were revealed with the MAP2 staining (red). (B) After treated with Stau, mL1CAM-GFP was mainly localized in dendrites but not axons. (C) GFP fluorescence levels of control (A) and Stau-treated neurons (B) along the axon (blue) and two main dendrites (red and dark red). Background value, 230, was subtracted. (D) mL1CAM-GFP-expressing axons formed bundles with NgCAM-mCherry-expressing axons. Arrows, axons. Arrowheads, dendrites. Scale bars, 100 μm. (All experiments performed by Joshua Barry).

Figure 2.6. Mouse L1-CAM (mL1CAM) axonal targeting is regulated by protein phosphorylation and induces axonal bundling.

48 DISCUSSION

In this study I show that axonal and dendritic bundling of cultured hippocampal neurons can be regulated by polarized targeting of NgCAM. Axonal NgCAM induced axonal bundling by the trans-homophilic interaction (Fig. 2.1 and 2.2). Reversal of the polarity of NgCAM by mutagenesis or kinase inhibitors induced dendritic bundling (Fig.

2.3-2.5). Also, the effects of expressed NgCAM were similar to mouse L1-CAM (Fig.

2.6).

Mechanistic insights provided from this in-vitro study included: 1. Both homophilic and axonal targeting of L1-CAM is required for axonal bundling. 2.

Dendritic bundling may be regulated by dendritic targeting of L1-CAM via induced activity change or transient targeting during development. 3. Axon-dendrite targeting of

L1-CAM due to activity dependent regulation of protein phosphorylation.

These results suggest that different L1-CAM domains are important for proper axonal bundling. In this system the Ig domains, which mediate trans-homophilic interaction of NgCAM, were required for axonal bundling (Fig. 2.2). These results were consistent that Ig domains of L1-CAM mediate cell-cell interaction (Kunz et al., 1998;

Haspel and Grumet, 2003; Itoh et al., 2004; Maness and Schachner, 2007). The Ig domains of L1 also have additional binding partners including integrins, neurocan and neuropilin. Deletion of the sixth Ig domain of L1 inhibited L1-L1 homophilic interaction and L1 interaction with RGD-dependent integrins, but had not effect on the neurocan or neuropilin interactions (Itoh et al., 2004). Deletion of the cytoplasmic tail reduced axonal bundling (Fig 2.2) which indicates it may play a role in regulating the bundling. L1 proteins maintain their adhesive property even in the absence of the cytoplasmic domain

49 (Hortsch et al., 1995; Wong et al., 1995). In my study the FN domain is required for axonal targeting of NgCAM (Fig. 2.4) consistent with a previous study (Sampo et al.,

2003). Loss of the FN domain failed to induce axonal bundling and actually induced dendritic bundling (Fig. 2.2D).

The targeting of overexpressed NgCAM is not affected by endogenous L1 proteins (Sampo et al., 2003; Wisco et al., 2003). Overexpressed L1-GFP is approximately five times stronger than endogenous L1 levels (Dequidt et al., 2007). The hippocampal neurons we used were at such an early stage that their endogenous levels of

CAMs did not lead to formation of axonal bundles. This study may suggest that both overexpressed and endogenous L1-CAM proteins can be used to induce axonal bundling, it requires a note of caution that the high levels of overexpressed NgCAM may cause non-physiological effects.

These results still left the question of what signaling pathways were involved in the axonal and/or dendritic bundling. Since I already knew that axon-axon interaction are regulated by neuronal activity (Walsh et al., 1997; Van Vactor, 1998; Yu et al., 2000;

Hanson et al., 2008; Imai et al., 2009) I wanted to delve deeper into these signaling pathways. I found that staurosporine, the broad spectrum Ser/Thr kinase inhibitor, not only decreased axonal bundling but induced dendritic bundling (Fig. 2.5B, D), similar to the deletion of the FN domain (Fig. 2.3A-C). Staurosporine treatment also reversed the polarity of NgCAM-GFP (Fig. 2.5C). JNK, a MAP kinase, may partially mediate the effect of staurosporine treatment because it significantly reduced axonal bundling as well

(Fig. 2.5D). At the concentration used the JNK inhibitor II actually blocks the activity of

50 all three JNK genes, JNK 1-3. Even though the other kinase inhibitors I examined had no significant effect I cannot rule out the possibility of multiple protein kinase involvement.

As stated in the introduction axonal targeting of NgCAM occurs by NgCAM being expressed first on the somatodendritic surface before being endocytosed and transported to the axon. A specific axonal targeting sequence within the cytoplasmic tail is required for this pathway (Wisco et al., 2003; Yap et al., 2008b). There was also another secondary axonal targeting signal discovered to be located with the FN domain that improves axonal targeting (Sampo et al., 2003).

Unlike the Sampo group who showed when they deleted the FN domains of overexpressed NgCAM they abolished axonal polarization, and it was diffused throughout the axon, soma and dendrites (Sampo et al., 2003), when I overexpressed the

NgCAM-ΔFN-GFP or NgCAM-ΔFN-mCh constructs I saw an increase in the somatodendritic expression of NgCAM, and a reduction in the axonal expression as shown by anti-MAP2 staining (Fig. 2.3A). This could be due to the fact that they used an untagged NgCAM molecule and examined only cell surface NgCAM by immunostaining. My experiment used NgCAM tagged at the C-terminal with GFP or mCh, this allowed me to examine all the NgCAM expressed within the cell, not just that on the cell surface. It is a possibility that the large 25 kD fluorophore may interfere with targeting of the NgCAM with the FN deletion, but that does not explain why the wildtype and other deletion constructs expression patterns were unaffected (Fig. 4.A). Also, their transfection method was slightly different than our own. Both my lab and the Sampo group used E18 hippocampal neuron culture, but we transfected at 5 DIV and fixed at 7

51 DIV, the Sampo group transfected at 8-11 DIV and fixed one day after transfection. Our younger neurons might behave differently than the older neurons the Sampo group used.

Phosphorylation of the tyrosine residue in the conserved FIGQ/AY sequence in the cytoplasmic tail of L1-CAM is known to regulate the L1-ankrying binding (Garver et al., 1997). However from my data only staurosporine, the pan kinase inhibitor, caused a switch in the polarized targeting of NgCAM from axonal to somatodendritic. This could be due to the pan kinase inhibitor affecting a single or multiple other pathways leading to a change in the targeting of NgCAM. Also this polarized targeting was tested on only overexpressed NgCAM, to examine if a single CAM targeting is altered endogenously it would required further experiments of treating older neurons, that do not form bundles with their neighbors, with staurosporine and then examining the endogenous targeting of

CAMs, specifically L1-CAM. This would confirm my results during overexpression of

NgCAM.

Finally the development of the new assay system offers advantages for studies of neurite bundling. My new system allows different constructs to be easily expressed in different neurons and different developmental stages. I also developed a new way to quantitatively reflect the degree of bundling (ABI value). Overall this study suggests that the targeting of L1-CAM is critical for establishing proper subcellular contacts in neural circuit formation.

52 METHODS cDNA constructs and antibodies

The NgCAM-GFP (green fluorescent protein) was a kind gift from Dr Gary

Banker (Oregon Health & Science University, Portland). In this construct, the stop codon of NgCAM is eliminated. In the linker region between NgCAM and GFP, there is an

EcoRI site. There is another EcoRI site after the GFP region. Thus, the GFP coding region can be easily cut out by an EcoRI digestion. NgCAM-mCherry (mCh) was constructed by inserting the cDNA fragment produced by polymerase chain reaction from the mCherry (a kind gift from Dr Roger Tsien, University of California San Diego, San

Diego) coding region into the EcoRI sites. The construct with mCherry in the right orientation has red fluorescence when expressed in neurons. NgCAM-ΔIg-GFP and

NgCAM-ΔIg-mCh were constructed by deleting the Ig domains between residues Q35 and F539, where two XhoI sites in the same reading frame were engineered by

Quickchange. Two rounds of the Quickchange mutagenesis were used to generate the

NgCAM construct with two engineered XhoI sites. The construct was cut with XhoI and religated without the insert. Using the same strategy, NgCAM-ΔFN-GFP and NgCAM-

ΔFN-mCh were made by deleting the FN repeat domain between residues I609 and

F1140. NgCAM-ΔCt-GFP and NgCAM-ΔCt-mCh were made by deleting the intracellular C-terminal (Ct) region between Y1174 and D1280. Intercellular adhesion molecule 5 (ICAM5)-GFP and mL1-CAM-GFP were made by inserting the coding sequences produced by polymerase chain reaction from ICAM5 and mouse L1-CAM

53 (OpenBiosystem, Huntsville, AL, USA) into the pEGFP-N1 vector (Clontech, Mountain

View, CA, USA), respectively. All of the constructs were confirmed with sequencing.

The following antibodies were used: rabbit polyclonal anti-microtubule- associated protein 2 (MAP2) (Chemicon, Temecula, CA, USA), rabbit polyclonal anti-

Tau1 (Abcam, Cambridge, MA, USA), mouse monoclonal anti-NgCAM antibody (8D9;

Developmental Studies Hybridoma Bank, Iowa City, IA, USA), mouse monoclonal anti-

GFP antibody (Antibodies Inc., Davis, CA, USA) and Cy5- conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA).

Hippocampal neuron culture and transfection

The primary dissociated hippocampal neuron culture was prepared from pregnant

Sprague-Dawley rats at embryonic day 18 under sterile conditions as previously described (Gu et al., 2006; Xu et al., 2007), in accordance with ethical guidelines stipulated by the Ohio State University animal ethics committee. The rats were euthanized by CO2 exposure. Hippocampi were dissected from embryonic day 18 rat embryos. Embryonic hippocampal neurons were disassociated in the dissecting medium

[containing (in mm): 82 Na2SO4, 30 K2SO4, 10 HEPES (pH 7.4), 10 glucose, 6 MgCl2 and 3 mg⁄mL protease 23 (Sigma, St Louis, MO, USA)], resuspended in the plating medium [Minimum Essential Medium Earle’s salts, 1 mm sodium pyruvate, 25 lm l- glutamine, 0.45% glucose, 10% fetal bovine serum and 1 x Pen ⁄ Strep (Invitrogen,

Carlsbad, CA, USA)] and plated onto glass coverslips coated with poly-d-lysine (Sigma) and collagen (Roche, Mannheim, Germany). At 2–4 h after plating, when neurons attached to coverslips, the plating medium was replaced by the maintenance medium

[Neurobasal medium, 1 x B27 supplement (Invitrogen), 0.5 mM l-glutamine and 1 x Pen ⁄

54 Strep]. At 2 days after neuron plating, 1 μm cytosine arabinose (Sigma) was added to the maintenance medium to inhibit glial growth for the subsequent 2 days, and then replaced with the normal maintenance medium. The maintenance medium was replenished twice a week by replacing a half volume. The transfection procedure was modified for studying neuron interactions. Cultured neurons at 5 days in vitro (DIV) were incubated in Opti-

MEM containing 0.4 μg cDNA plasmid of the first construct mixed with 0.75 μL lipofectamine 2000 (Invitrogen) and 0.4 μg cDNA plasmid of the second construct mixed with 0.75 μL lipofectamine 2000 for 20 min at 37° C. Thus, each cDNA⁄cationic lipid complex contained only one type of cDNA plasmid so that each neuron probably expressed only one protein construct. With this transfection method, over 95% of transfected neurons expressed only one construct.

Drug treatment

The following chemicals were used: chelerythrine chloride (a protein kinase C inhibitor), p38 mitogen-activated protein (MAP) kinase inhibitor III, staurosporine (Stau)

(a broad-spectrum Ser ⁄ Thr kinase inhibitor), c-Jun N-terminal kinase (JNK) inhibitor II,

KT5823 (a protein kinase G inhibitor), Rp-cAMPS (a protein kinase A inhibitor), ML-7 hydrochloride (a myosin light chain kinase inhibitor), KN-93 (a selective

Ca2+⁄calmodulin-dependent protein kinase II inhibitor), PD98059 (a MEK1 inhibitor), genistein (a tyrosine kinase inhibitor), glycogen synthase kinase 3 (GSK3) inhibitor IX, and U0126 (a MEK1/2 inhibitor) were purchased from Calbiochem (Gibbstown, NJ,

USA). 4-Aminopyridine, tetrodotoxin, ethanol and carbachol chloride were purchased from Sigma. Drugs were added at 4–6 h after transfection in the proper concentration.

Neurons were usually treated for 48 h before fixation and staining. Within this time

55 window and under the drug concentration used, no detrimental effect on neuron survival was observed.

Immunofluorescence staining

The procedure for immunocytochemistry and fluorescence microscopy was described previously (Gu et al., 2006; Xu et al., 2007). In brief, the neurons expressing

NgCAM or mL1-CAM constructs were fixed and stained under permeabilized conditions

(in the presence of 0.2% Triton) for the endogenous dendritic marker MAP2 or axonal marker Tau1. To reveal the surface level of NgCAM constructs, the transfected neurons were first stained with the 8D9 antibody under non-permeabilized conditions, permeabilized with 0.2% Triton after washing, and then stained with the anti-MAP2 antibody.

Fluorescence imaging and quantification

Fluorescence images were captured with a Spot CCD camera RT slider (Diagnostic

Instruments, Sterling Heights, MI, USA) mounted on a Zeiss (Oberkochen, Germany) upright microscope (Axiophot) using Plan Apo objectives (20 x ⁄0.75 and 100 x ⁄1.4 oil), saved as 16-bit TIFF files, and analyzed with NIH ImageJ and Sigmaplot 10.0 for fluorescence intensity quantification. Exposure times were controlled so that the pixel intensities in dendrites and axons were below saturation, but the same exposure time was used within each group of an experiment. The quantification procedure for the axon ⁄ dendrite targeting ratio (Faxon ⁄ Fdendrite) was described previously (Gu et al., 2006; Xu et al., 2007). Only transfected neurons with clearly separated dendrites and axons, and isolated from other transfected neurons, were chosen for analysis. The co-staining with axonal marker Tau1 and/or dendritic marker MAP2 helped to distinguish axons from

56 dendrites. The targeting ratio (Faxon ⁄ Fdendrite) was determined by the average fluorescence intensity in axons divided by the average fluorescence intensity in dendrites.

To measure the fluorescence intensity, lines were laid along the major processes to acquire the fluorescence intensity profiles (in arbitrary units) of these processes excluding segments fasciculated with other neurites, regions with crossings, proximal dendritic segments within 20 μm of the soma, and fine terminal branches.

The procedure for the quantification of the axonal bundling index (ABI) was as follows. Measurements of the RGB image of a pair of neurons with green and red fluorescence were performed with NIH Image J. The total length of bundled axonal segments between a green and red neuron (Ltotal in μm) was measured. The number of crossings between the green and red axons (Ncross) was manually counted. Each bundled segment was counted as one crossing. The ABI for this pair of neurons was defined as the average length of bundled axons per crossing (ABI in lm⁄ crossing), ABI = Ltotal ⁄

Ncross. Thus, I obtained the ABI for each pair of neurons. An example is provided in Fig.

2C. For each condition, more than 10 pairs of neurons from at least three independent transfections were quantified and their ABIs are given with means and SEs.

Fluorescence recovery after photobleaching imaging

To better visualize the moving carriers containing NgCAM-GFP, Yuanzheng adopted the strategy of fluorescence recovery after photobleaching imaging. First, the

GFP fluorescence of NgCAM-GFP in an axonal segment (~50–150 μm) was bleached by maximal excitation lights for 5–10 min. Then Yuanzheng performed time-lapse imaging using weaker excitation lights (~12.5% of the maximal) with the 8 x neutral density filter

57 to visualize NgCAM-GFP-containing carriers moving from the unbleached regions through the bleached segment.

Statistical analysis

All data were analyzed with pasw⁄spss Statistics 18 (SPSS Inc., Chicago, IL,

USA) and are presented as the mean ± SEM. Two-tailed Student’s t-test was used for comparisons between two groups. Oneway anova followed by Dunnett’s test was used for comparing three or more groups. P < 0.05 was considered statistically significant.

CHAPTER 3

KV3 POLARIZED TARGETING REGULATES NEURONAL ACTIVITY

SUMMARY

The neuron’s main function of integrating synaptic inputs and generating proper outputs, APs, requires the proper localization and function of both ligand- and voltage- gated ion channels. These ion channels regulate the spiking frequency of a neuron, and the fast spiking abilities of neurons is attributed to Kv3 channel presence. We* discovered that expression of Kv3.1b, the longer splice variant of Kv3.1, converted slow- spiking young neurons into fast spiking ones. Kv3.1a, the shorter splice variant of Kv3.1, is somato-dendritically located and was less effective at increasing maximal spiking frequency. This suggests that spiking frequency may be regulated by the polarized targeting of Kv3.1. I discovered an electrostatic repulsion between the N- and C-termini of Kv3.1b which unmasks an axonal targeting motif. This motif is required for the polarized axonal targeting of the Kv3.1b channel.

* Joshua Barry performed all immunostaining and quantification. Dr. Yuanzheng Gu performed all electrophysiology recordings. Dr. Robert McDougel and Dr. David Terman (Dept of Mathematics, OSU) performed computer modeling with help from Dr. Yuanzheng Gu and Dr. Chen Gu.

59 INTRODUCTION

Ligand- and voltage-gated ion channel localization in neurons is important for a neuron’s ability to integrate synaptic inputs and generate action potentials. Neurons are highly polarized cells with multiple dendrites and a single long axon. Various voltage- gated potassium (Kv) channels are differentially localized to dendrites and/or the axon.

In the mammalian brain Kv1 is located primarily in the axon while Kv2 and Kv4 channel family members are mainly localized in dendrites (Trimmer, 1991; Sheng et al., 1992;

Hwang et al., 1993; Wang et al., 1993; Veh et al., 1995; Cooper et al., 1998; Du et al.,

1998; Rasband et al., 1998; Monaghan et al., 2001; Trimmer and Rhodes, 2004). Kv3 channels have complex targeting patterns due to the isoform, alternative splicing and neuronal time which localizes them to either axons or dendrites (Chow et al., 1999;

Ozaita et al., 2002; Ishikawa et al., 2003; Martina et al., 2003; Brooke et al., 2004;

Goldberg et al., 2005; Chang et al., 2007; Xu et al., 2007; Johnston et al., 2010).

Excitatory and inhibitory synaptic potentials are summed and converted into one or more action potentials (APs) at the axon initial segment (AIS) of most neurons. The AIS is where voltage-gated sodium (Nav) channels are highly concentrated. The AIS is also important as a ‘gate’ for the entry of axonal proteins to the axon (Winckler et al., 1999;

Song et al., 2009). Regulation of this input-output relationship is still poorly understood.

The widest range of spiking frequencies is usually accredited to Kv3 channels being present. These channels have high activation threshold (roughly -10 mV) and rapid deactivation kinetics (Rudy and McBain, 2001; Bean, 2007). It is still unknown whether expression of Kv3 channels is sufficient for fast spiking because not all Kv3 channel expressing neurons are able to spike rapidly (Chow et al., 1999). This lead to the main

60 question of this chapter: is spiking frequency regulated by the polarized targeting of Kv3 channels?

We used hippocampal neuron culture as a model to examine how neuronal input- output relationship is affected by Kv3 polarized targeting. Our results showed that slow spiking neurons could be converted to fast spiking neurons by axonal expression of

Kv3.1b. Also my mutagenesis study uncovered new insight into the mechanism that regulates the polarized targeting of Kv3.1.

RESULTS

Spiking frequency of culture hippocampal neurons and endogenous Kv3

Yuanzheng Gu, one of our postdocs, performed whole cell current-clamp recordings on hippocampal neurons in a hippocampal neuron culture (from 16 to 28 DIV)

(Fig. 3.1A). To induce AP firing either 2 μM glutamate was puffed on dendrite or soma or square-wave current pulses were injected into the soma. The induction of AP firing showed two different populations of neurons in culture, slow-spiking neurons (Fig. 3.1B) or fast-spiking neurons (Fig. 3.1C). This is consistent with the fact that hippocampal cultures contain different types of neurons with different firing properties. 1mM TEA, which blocks Kv3 channels, was used to examine the role of Kv3 channels in regulating spiking frequency. Fast-spiking neurons were reversibly suppressed by 1mM TEA (Fig.

3.1D).

He also examined the role of Kv3.1 channels by combining current-clamp recording and post-recording immunostaining. Yuanzheng performed the recordings and

61 captured an image of the recorded neuron, then I performed immunostaining and we discovered that slow spiking neurons often did not express Kv3.1b (Fig. 3.1E), whereas fast-spiking neurons expressed endogenous Kv3.1b in the soma and proximal axon (Fig.

3.1F).

Figure 3.1. Hippocampal neurons in culture spikes at different frequencies in response to prolonged depolarizing inputs. (A) whole cell current-clamp recording on cultured hippocampal neurons. In the experimental diagram of the whole cell recording of a cultured hippocampal neuron (top), a recording electrode (green) records membrane potentials and injects currents to induce APs. A drug perfusion pipette (gray) is used to apply either glutamate (Glu) or TEA to the neuron. Current injection and Glu puffing mimic presynaptic excitatory inputs to induce APs initiated at the AIS (red). APs propagate anterogradely along axons toward axonal terminals, and retrogradely back to soma picked up by the recording electrode. The lower panel shows a transmitted light image of the recoding of cultured hippocampal neurons at 14 DIV. (B) a slow-spiking neuron firing APs induced by either a puffing (0.5–1 s) of 2 μM Glu (upper) or 1-s injection of 130 pA currents (lower). Recording of APs for 20 s is shown on the top, below which is the trace within the first 1 s of drug application. Neurons around 15 to 17 DIV from the P8 culture were used. (C) a fast-spiking neuron firing APs induced by either Glu puffing (upper) or current injection (lower). Black arrowheads indicate the Glu puffing. (D) fast spiking of cultured hippocampal neurons was reversibly blocked by perfusing 1 mM TEA. The first (left) and fifth (right) APs are shown. Summary of the result is at the bottom. Normalized frequency equals to AP frequency divided by the frequency under the control condition. Paired t test, **, p 0.01. (E) AP firing induced by 1-s current injection (left) and axonal levels ofendogenousKv3.1b (right) in a slow- spiking neuron. After recording, the neuron was fixed post hoc, and co-stained with anti- Kv3.1b (green) and anti-pan-NaV (red) antibodies. The anti-Kv3.1b staining is shown in reversed signal (right top). (F) AP firing induced by 1-s current injection (left) and axonal levels of endogenous Kv3.1b (right) in a neuron firing more rapidly. White arrowheads indicate proximal axons. Scale bars, 100 μmin A and 50 μmin E and F. (All recordings performed by Yuanzheng Gu, immunostaining in E and F performed by Joshua Barry).

Figure 3.1. Hippocampal neurons in culture spikes at different frequencies in response to prolonged depolarizing inputs.

64 Kv3.1 expression converts young neurons from slow-spiking to fast-spiking neurons

Yuanzheng then used Kv3 expression to convert slow spiking to fast spiking neurons. Kv3.1aHA and Kv3.1bHA increased the maximal firing frequency (Fig. 3.2A).

YFP-Kv1.2, which is concentrated on axonal membranes, also increased maximal spiking frequency, but not as much as the Kv3 constructs (Fig. 3.2A). YFP-Kv4.2, which is concentrated on somatodendritic membranes, did not increase spiking frequency at all

(Fig. 3.2A). None of the constructs significantly affected the resting membrane potential

(Fig. 3.2B). Closer analysis of the input-output relationship showed Kv3.1bHA significantly increased maximal spiking frequency compared to Kv3.1aHA. Yuanzheng then examined the biophysical properties of Kv3.1aHA and Kv3.1bHA, using YFP-

Kv1.2 and YFP-Kv4.2 as controls. He discovered that the two Kv3.1 splice variants had identical channel biophysical properties (data not shown).

Figure 3.2. Expression of Kv3.1b effectively converts slow-spiking neurons to fast- spiking ones. (A) overexpression of Kv3.1bHA or Kv3.1aHA significantly increased firing frequencies of APs induced by long-pulse current injections. Hippocampal neurons transfected with YFP (as control), Kv3.1aHA + YFP (indicator for transfection), Kv3.1bHA + YFP, YFP-Kv1.2, or YFP-Kv4.2 were examined with whole cell recording. Currents with 1000-ms duration and 10-pA increments from 5 to 145 pA were injected to the neuronal soma through the recording electrode. An example trace of APs induced by 105 pA current is shown for each condition (B) relationship of injected current and AP firing frequency in neurons expressing different Kv channels, between Kv3.1aHA and the Kv3.1bHA t test: **, p 0.01; *, p 0.05. The p values were adjusted by the Holm- Bonferroni method from repeat measures of Kv3.1bHA. The “n” numbers are provided in parentheses. (All data in Figure 3.2 was collected by Yuanzheng Gu).

66 Underlying mechanism of Kv3.1 axonal targeting and its impact on spiking frequency

After Yuanzheng determined slow spiking neurons could be converted to fast spiking neurons due to Kv3.1 expression we wondered if the polarized targeting of Kv3.1 channel plays an important role in regulating the spiking frequency. The two splice variants of Kv3.1, Kv3.1a and Kv3.1b, differ only in their C-termini, with Kv3.1a having a very short tail, while Kv3.1b is much longer. The polarized axon-dendrite targeting of these two splice variants is different, with Kv3.1a more somato-dendritic, while Kv3.1b is more axonal (Xu et al., 2010). Our previous work showed that the splice domain of

Kv3.1b regulates the N/C-terminal interaction to expose an axonal targeting motif which is located in the shared region of Kv3.1a and Kv3.1b (Xu et al., 2007).

I made deletion constructs of the splice domain of Kv3.1b and examined their axon-dendrite targeting (Fig. 3.3A). I found making the deletions of Kv3.1bHA1-554 or

Kv3.1bHA1-530 had no effect on the axonal targeting and was similar to Kv3.1bHA.

Whereas the deletion constructs Kv3.1bHA1-502 and Kv3.1bHA1-513 were contained in the somatodendritic region, similar to Kv3.1aHA (Fig. 3.3A-E). Therefore I hypothesized the regulatory element of the splice domain is somewhere between residues 513 and 530

(Fig. 3.3A).

Yuanzheng then examined the biophysical properties of these channel deletion constructs using voltage-clamp recording on transfect HEK293 cells. All deletion constructs except Kv3.1bHA1-513 had channel properties similar to Kv3.1bHA (data not shown). To examine the deletion constructs effects on spiking frequency Yuanzheng transfected the constructs into hippocampal neurons. He found Kv3.1bHA1-554 and

67 Kv3.1bHA1-530 enhanced spiking frequency similar to Kv3.1bHA, whereas Kv3.1bHA1-

502 functioned like Kv3.1aHA (Fig. 3.3F).

Figure 3.3. Regulation of axonal targeting and action potential firing by Kv3.1b truncations. (A) diagram of Kv3.1a and Kv3.1b C-terminal regions. Residues 1–501 are identical for the two splice variants. This region includes cytosolic N-terminal T1 domain (red circle), six membrane spanning segments (6 black bars), and the most part of cytosolic C terminus of Kv3.1a (aC). Kv3.1b has a large 85-residue splice domain (sC) at its C terminus. Thus, the Kv3.1b C terminus (bC) consists of aC and sC. Five numbers indicate positions of 5 residues. Within the N-terminal 30 residues, “minus” and “plus” indicate negatively and positively charged residues, respectively. (B) cultured hippocampal neurons transfected with Kv3.1bHA deletion constructs at 5 DIV. The transfected neurons at 7 or 8 DIV were stained with an anti-HA antibody (green) under a nonpermeabilized condition and with an anti-microtubule-associated protein 2 (MAP2) antibody (red) under a permeabilized condition. White arrowheads indicate proximal axons. Scale bars, 100 μm. (C) summary of the average fluorescence intensities of Kv3.1 constructs expressed in neuronal soma under the permeabilized condition. (D) summary of axonal levels of the mutants stained under the permeabilized condition. One-way analysis of variance followed by Dunn test, **, p < 0.01. (E) summary of polarized targeting of the mutants stained under a nonpermeabilized condition. One-way analysis of variance followed by Dunnett’s test, **, p < 0.01. (F) summary of the relationship of the amount of sustained injected current and AP firing frequency. Between Kv3.1bHA and Kv3.1bHA1–502 t test: *, p < 0.05. The p values were adjusted by a Holm-Bonferroni method from repeat measures of Kv3.1bHA. (Immunostaining and quantification of B-E performed by Joshua Barry. Recordings in F performed by Yuanzheng Gu.)

Figure 3.3. Regulation of axonal targeting and action potential firing by Kv3.1b truncations.

70 When the C termini of Kv3.1a and Kv3.1b were compared to one another an intriguing difference was found. The net charge between residues 501 to 530 of Kv3.1b is -5, whereas the last 10 residues of Kv3.1a (502 to 511) is +3 (Fig. 3.3A). Since the net charge of the C-terminal ATM is +8 we hypothesized that an electrostatic interaction might play a role in the N/C-terminal interaction with the splice domain of Kv3.1b. I mutated two pairs of consecutive acidic residues in Kv3.1b to either two Lys (K) or Ala

(A). Mutating D529E/D530E (Kv3.1bHADE-KK or Kv3.1bHADE-AA) reduced Kv3.1bHA axonal levels more than mutating E517D/E518D (Kv3.1bHAED-KK or Kv3.1bHAED-AA)

(Fig. 3.4B).

Yuanzheng then examined the effect these point mutations had on spiking frequency. Kv3.1bHADE-KK did not enhance the maximal spiking frequency (Fig. 3.4C) whereas both Kv3.1bHAED-KK and Kv3.1bHAED-KK were similar to Kv3.1bHA in enhancing the spiking frequency (Fig. 3.4C). Kv3.1bHADE-AA, which has a similar targeting pattern to Kv3.1aHA, did not enhance the spiking frequency as strongly as

Kv3.1bHA (Fig. 3.4C). Yuanzheng analyzed the biophysical properties of these mutants expressed in HEK293 cells and found only Kv3.1bHADE-KK had a decrease in current and slower activation time constant, while the other three mutants were similar to Kv3.1bHA

(data not shown). This mutagenesis study suggests that it is the electrostatic attraction and repulsion between the splice domain and the T1 domain that regulates the uncovering of the ATM. Therefore axonal targeting of Kv3.1 channels is critical for maximal AP firing frequency.

Figure 3.4. Axonal targeting of Kv3.1b is critical for inducing the maximal AP firing frequency. (A) structure diagram of the role of charged residues of Kv3.1 splice domains in the N/C-terminal interaction and in exposing the ATM. (B) the axon-dendrite targeting of Kv3.1b point mutants. Example images of Kv3.1bHAand Kv3.1bHADE-AA- expressing neurons are on the left. The anti-HA staining (green) was performed under the nonpermeabilized condition. The anti-microtubule-associated protein 2 (MAP2) staining (red) labeled dendrites. Scale bars, 50_m. Summary of the HA staining results are on the right. One-way analysis of variance followed by Dunnett’s test, **, p < 0.01. White arrowheads, proximal axons. (C) relationship of injected current and AP firing frequency, between Kv3.1bHA and Kv3.1bHADE-AA t test: *, p < 0.05. The p values were adjusted by the Holm-Bonferroni method from repeat measures of Kv3.1bHA. (Immunostaining and quantification in B performed by Joshua Barry. Recordings performed by Yuanzheng Gu.)

Figure 3.4. Axonal targeting of Kv3.1b is critical for inducing the maximal AP firing frequency

73 Computer simulations reveal effect of Kv3 polarized targeting on spiking frequency

We then collaborated with Dr Robert McDougel and Dr David Terman and used computer simulations to examine whether axonal targeting of Kv3 channels is important for spiking frequency. They used the NEURON program to build three models with different amounts and localization of Kv3.1. When only Nav channels are expressed at the AIS a small number of APs was produced when a simulated depolarization occurred

(Fig. 3.5A, B). When Kv3.1 channels were enriched in the somatodendritic region more

APs were produced under the same depolarization event (Fig. 3.5A, B). When Kv3.1 channels were enriched in the axon this induced the highest number of APs (Fig. 3.5A,

B). Therefore these computer models showed that it is the axonal targeting of Kv3.1 that increased the maximal spiking frequency.

Figure 3.5. Modeling and simulations of the effect of Kv3.1 axonal targeting on spiking frequency. (A) simulated action potential traces for neurons expressing no Kv3.1 (top), dendritic Kv3.1 (middle), and axonal Kv3.1 (bottom). (B) the input-output relationship of the three simulated neurons. See ”Methods“ for details of the parameter values. (C) hypothetical models of a control neuron (top), a neuron expressing dendritic Kv3.1a (middle), and a neuron expressing axonal Kv3.1b (bottom). Red bars, NaV channels at the AIS; blue bars, Kv3.1 channels. (All computer modeling performed by Dr. Robert McDougel and Dr. David Terman.)

75 DISCUSSION

In this paper we show that axonal targeting and biophysical properties are critical for the maximal frequency firing of APs in neurons. I also showed a novel mechanistic insight into Kv3.1 axonal targeting, including the electrostatic interaction between the N and C termini of Kv3.1.

Maximal spiking frequency requires both the rapid biophysical properties and axonal targeting of Kv3.1. These results show that blocking Kv3 channels, in either mature neurons or young neurons expressing Kv3.1b, caused a decrease in spiking frequency which is consistent with the thought that Kv3 channels are essential for fast spiking (Rudy and McBain, 2001). Expression of Kv3.1 in young neurons induced fast spiking (Fig 3.2), whereas none of the young neurons expressing Kv1.2 or Kv4.2 induced fast spiking, even though their are expressed on the plasma membranes (Gu et al., 2003;

Rivera et al., 2003).

However, having the proper biophysical properties alone is not sufficient to induce fast spiking. Kv3.1a has identical properties as Kv3.1b but does not induce fast spiking as effectively as Kv3.1b. The maximal spiking frequency requires axonal targeting of Kv3.1, especially to the AIS. AIS targeting of Kv3.1a is much less than

Kv3.1b (Xu et al., 2007). Kv3.1a was able to increase spiking frequency, just not to the levels of Kv3.1b. This could be due to either: 1. electrical field passively spreading from the soma, or 2. low levels of Kv3.1a existing in the AIS. It cannot also be ruled out that there are unknown binding proteins the differentially regulate Kv3.1a and Kv3.1b in neurons.

76 My mutagenesis study showed that Kv3.1bHA mutants Kv3.1bHA1-502 and

Kv3.1bHADE-AA, which have similar targeting patterns to Kv3.1a, had unchanged channel activity but decreased AIS levels and failed to increase maximal spiking frequency (Fig.

3.3 and 3.4). This is consistent with my hypothesis. My major conclusion is that the axonal targeting of Kv3.1 channels regulates the maximal spiking frequency, which was confirmed by both experimental data and computer simulations.

Exposure of the ATM caused polarized targeting of Kv3.1, which is regulated by the electrostatic interaction between Kv3.1 N and C termini. Our earlier study suggested that the splice domain weakened the masking effect the N-terminal T1 domain had on the

ATM. The ATM is then uncovered, recognized by ankyrin G at the AIS, and allowed

Kv3.1b to be targeted into the axon (Xu et al., 2007). However, how the splice domain interfered with the T1-ATM interaction was unknown. In this new study I showed that the novel electrostatic interaction between the N and C termini was disrupted by the splice domain. Deletion analysis mapped the critical region of the splice domain down to residues 513 to 530 (Fig. 3.3). The net charge of this region (-5) is opposite of the last 10 residues of Kv3.1a (+3) (Fig. 3.3A). The ATM itself has a +8 net charge (Xu et al.,

2007), which could interact with the negative charges on the T1 domain, mediating the masking effect. The +3 charge on the Kv3.1a C-terminus may strengthen the T1-ATM interaction, while the -5 charge on the Kv3.1b splice domain may weaken it (Fig. 3.4A).

This conclusion was supported by the Kv3.1bHA point mutations (Fig. 3.4). This model of intramolecular interactions requires future study using such techniques as x-ray crystallography to examine this interaction more in depth. My results of altered axonal targeting of Kv3.1b by mutating D529E/D530E but not E517D/E518D suggests and

77 important role in conformational change, in addition to the electrostatic interaction of N-

/C-termini. This is consistent with a recent study showing complex binding surfaces of

N-/C-termini interaction in Kir2.1 channels (Ma et al., 2011).

Important physiological functions may be regulated by altering the spiking frequency, which in turn is regulated by Kv3 polarized targeting. Kv3 mediated fast spiking is critical for stabilizing images on the retina via the vestibule-ocular reflex circuit and sound location by the auditory circuit (Parameshwaran et al., 2001; von Hehn et al., 2004; Gittis et al., 2010). Regulating the Kv3 protein levels and phosphorylation of

Kv3 is a proposed mechanism in adjusting the spiking frequency of neurons in auditory circuits and the suprachiasmatic nucleus following different acoustic environments and circadian rhythm, respectively (Itri et al., 2005; Song et al., 2005).

In this paper we showed that the targeting of Kv3.1 channel is a novel strategy for regulating spiking frequency. The expression of the two splice variants of Kv3.1, Kv3.1a and Kv3.1b, is regulated developmentally (Perney et al., 1992). As neurons mature dendritic Kv3.1 channels shift to axonal ones (Xu et al., 2010) to enhance spiking frequency. In the future it would be interesting to identify what affect altering Kv3 polarized targeting has on activity dependent regulation of neuronal spiking.

METHODS cDNA Constructs

Kv3.1aHA, Kv3.1bHA, Kv3.1bHA1–502, YFP-Kv1.2, and Kvβ2 were previously described (Gu et al., 2006; Xu et al., 2007). YFPKv4.2 was made by inserting a cDNA fragment encoding YFP into the N terminus of Kv4.2 before its T1 domain between SalI

78 and NheI restriction enzyme sites, which were engineered between the codons for Lys36 and Arg37 using the QuikChange mutagenesis strategy. Kv3.1bHA1–513, Kv3.1bHA1–530, and Kv3.1bHA1–554, were made by engineering stop codons in Kv3.1bHA C terminus with QuikChange mutagenesis. Kv3.1bHADE-KK, Kv3.1bHAED-AA, Kv3.1bHAED-KK, and

Kv3.1bHAED-AA were made with QuikChange mutagenesis.

Hippocampal Neuron Cultures and Transfection

The E18 hippocampal neuron culture was prepared as previously described from rat hippocampi at the embryonic day 18 (see pg 44).

The P8 hippocampal neuron culture was prepared from rat hippocampi at postnatal day 8 (P8) to obtain interneuron-enriched cultures, using the same procedure as the E18 culture as described previously (Xu et al., 2010). Enrichment of GABAergic interneurons in P8 cultures may reflect differences in the birth and migration of

GABAergic interneurons versus pyramidal neurons. In addition, pyramidal neurons may die more readily during dissociation for culture, because P8 pyramidal neurons already have lengthy and complex dendritic and axonal arbors. Neurons were usually cultured for about 14 to 21 DIV before the experiments.

Current Clamp Recording of Action Potentials

The same internal solution and Hanks’ buffer were used for recording of primary cultured neurons. Both mature neurons (older than 16 DIV) and young neurons (7 to 10

DIV) were recorded from either the E18 or the P8 cultures. The membrane resistance, capacitance, and resting membrane potentials of the neurons were measured. These values are consistent within each age group. APs induced by either puffing (0.5–1 s in duration) 2μM glutamate (Glu) onto neuronal soma, or current injection from the

79 recording pipette, were recorded under the current clamp mode. For long pulse stimulations, 1000-ms duration currents of increasing amplitude (from 5 to 145 pA with increments of 10 pA) were injected. Due to the variation of expression levels of transfected channel constructs, only the neurons carrying clear after-hyperpolarization were used for quantification of spiking frequency. For short pulse stimulations, 2-ms duration currents of 800 pA with increasing frequency (from 50 to 300 Hz with increment of 50 Hz) were injected. Because the current injection is very short for short pulse stimulations, Yuanzheng found that only the large current (800 pA) can reliably induce the first AP in control neurons, consistent with previous studies (Atzori et al., 2000; Song et al., 2005; Yang et al., 2007).

Post Hoc Immunostaining of Hippocampal Neurons from P8 Culture

To examine the AP firing frequency of the endogenous Kv3.1 channels role,

Yuanzheng performed current clamp recording on the P8 neurons from 14 to 16 DIV. For each neuron, induced AP traces by long-pulse stimulation were recorded and its morphology was imaged with transmitted light. The neuron was fixed immediately after recording, permeabilized with 0.2% Triton X-100, and stained for endogenous Kv3.1b and NaV channels with a mouse monoclonal anti-Kv3.1b antibody (clone number

B16B/8; University of California Davis/NIH NeuroMab Facility, Davis, CA) and a rabbit polyclonal anti-pan NaV channel antibody (Millipore), respectively. The maximal AP frequency and immunostaining intensity of endogenous Kv3.1b in proximal axons are correlated.

Immunostaining, Imaging, and Quantification

80 The immunocytochemical procedures were previously described (Gu et al., 2006;

Xu et al., 2007). Briefly, neurons were stained under nonpermeabilized conditions

(without Triton X-100) to label the surface pool and under permeabilized conditions

(with 0.2% Triton X-100) to label total proteins. Fluorescence images were captured with a Spot CCD camera RT slider (Diagnostic Instrument Inc., Sterling Heights, MI) in a

Zeiss upright microscope, Axiophot, using Plan Apo objectives x 20/0.75 and x 100/1.4 oil, saved as 16-bit TIFF files, and analyzed with NIH ImageJ and Sigmaplot 10.0 for fluorescence intensity quantification. Exposure times were controlled so that the pixel intensities in dendrites and axons were below saturation, but the same exposure time was used within each group of an experiment. The quantification procedure was described previously (Xu et al., 2007; Xu et al., 2010). Only transfected neurons with clearly separated dendrites and axons, and isolated from other transfected cells, were chosen for analysis. To obtain the axonal polarity index (Faxon/Fdend) reflecting polarized targeting on the neuronal surface, I performed anti-HA staining under the nonpermeabilized condition. Using NIH ImageJ, I laid a line along the major axon to acquire its average fluorescence intensity (in arbitrary unit) (Faxon), and laid lines along proximal dendrites

10 μm away from the soma to obtain the average fluorescence intensity on dendritic membranes (Fdend). To obtain the relative axonal level of total proteins (Faxon/Fsd), I performed anti-HA staining under permeabilized conditions. Using NIH ImageJ, I laid a line along the major axon to acquire Faxon, and laid lines along proximal dendrites starting from the soma to obtain Fsd. The background fluorescence intensity was measured for each image and subtracted.

Simulation of Action Potential Firing Using NEURON Software

81 The computation model was implemented using NEURON 7.1 (Hines and

Carnevale, 1997). We assumed a simplified morphology, largely consistent with a cultured hippocampal neuron at 10 DIV. The cell was constructed using three cylinders, each representing a distinct region of the neuron: a dendrite (100μm in length and 4 μm in diameter), a soma (20 μm in length and 20 μm in diameter), and an axon (400 μm in length and 2 μm in diameter). The AIS is located in the first 10–45 μm of the axon.

Each simulation was compared three model neurons (control, dendritic Kv3.1, and axonal Kv3.1) with different Kv3 channel distributions but otherwise identical, that is, they had the same morphology, NaV channel distribution, and leak channel distribution.

In dendritic Kv3.1-expressing neurons, Kv3 current density is relatively higher on the somatodendritic surface, whereas in axonal Kv3.1-expressing neurons, Kv3 current density is relatively higher on the axonal surface including the AIS. This is largely consistent with the immunostaining results under the nonpermeabilized condition.

In Fig. 3.5 A and B, we provide one example of the simulation results. The parameters used in this figure are as follows: NaV channels carrying transient Na+ currents were present throughout the cell, with density in the AIS (37,300 pS/μm2) five times higher than along the rest of the axon (7,460 pS/μm2) and 20 times higher than the rest of the cell (1,865 pS/μm2). In the control neuron, Kv3.1 channels were absent. In the dendritic Kv3.1-expressing neuron, Kv3.1 channels were present on somatic and dendritic membranes (20,000 pS/μm2) and axonal membranes including the AIS (1,000 pS/μm2). In the axonal Kv3.1-expressing neuron, Kv3.1 channels were present on somatic and dendritic membranes (5,000 pS/μm2) and axonal membranes including the

82 AIS (15,000 pS/μm2). The ratios of Kv3.1 channels used here were adopted from the most highly polarized examples observed in my immunostaining results (Faxon/Fdend) under the nonpermeabilized condition. It was assumed here that the anti-HA immunofluorescence signal is in a linear relationship with the actual amount of Kv3.1 channels. Leaky channel conductance was present throughout the cell at 7.1 pS/μm2.

The biophysical properties of NaV channels, Kv3.1 channels, and leaky channels used in our model cells were adopted from Golomb et al. (Golomb et al., 2007) with modifications based on experimentally observed time constants. The current balance equation is,

CVt = (r/2Ra)Vxx-INa(V,h,x) - IKv3(V,n,x) - It(V) + Iapp (Eq. 1) where V is the membrane potential of the neuron, C = 1 μF/cm2 is the membrane capacitance, r is the section radius, Ra = 35.4 Ω-cm is the cytoplasmic resistivity, Iapp denotes external current injected into the neurons, and INa, IKv3, and Il represent the sodium (NaV), potassium (Kv3.1), and leak currents, respectively. These are modeled as:

3 2 INa(V,h,x) = gNa(x) m∞ (V)h(V - VNa); IKv3(V,n,x) = gk(x)n (V - VK); and Il(V) = gl(V -

Vl), where gNa(x), gK(x), and gl represent the maximum NaV, Kv3.1, and leak conductances, the first two vary with space depending on the region of the cell. The sodium, potassium, and leak reversal potentials are VNa = 50 mV, VK = -77 mV, and Vl =

-70 mV, respectively.

Sodium activation is assumed to be instantaneous and is given by: m∞(V) = 1/(1 + exp(-(V - θm)/σm)). The sodium inactivation variable h are governed by,

ht = (h∞(V) – h)/τh(V) (Eq. 2)

83 where h∞(V) = 1/(1 + exp(-(V - θh)/σh)) and τh(V) = h (0.5 + 10/(1 + exp(-(V - θth)/σth))).

We take θm = -24 mV, σm = 11.5 mV, θh = -58.3 mV, σh = -6.7 mV, σth = -16 mV, σth = -

12 mV, and h = 1.5.

The potassium activation variable n is governed by,

nt = (n∞(V) – n)/τn(V) (Eq. 3) where n∞(V) = 1/(1 + exp(-(V - θhn)/σn)) and τh(V) = ϕn (0.2 + 11.4/(1 + exp((V +

3)/6)))(0.07 + 11.4/(1 + exp(-(V - 1.3)/15))). We take θhn = -12.4 mV, σn = 6.8 mV, and

h = 0.5.

Long pulse experiments were performed as follows. First we administered a current at the midpoint of the soma for 5 s; then we counted the number of spikes; and finally divided the total count by 5 to get the firing rate. We established the robustness of the qualitative result that axonal Kv3.1 leads to a higher firing rate than dendritic Kv3.1 by keeping the expression ratios constant and randomly choosing base conductances in

2 the following ranges: gNa (peak expression) between 0 and 50,000 pS/μm , gK (lowest

2 2 expression) between 0 and 10,000 pS/μm , and gl between 0 and 20 pS/μm .

CHAPTER 4

KV3 CLUSTERS AND ACTIVATES KIF5

SUMMARY

As stated earlier KIF5 is the motor that transports Kv3.1, therefore KIF5 affects

Kv3.1. On the other hand, in this section I will discuss how Kv3.1 affects KIF5 via clustering and activating the KIF5. Endogenous KIF5 formed clusters, which may be due to the role of binding proteins. Biochemical assays showed that the high affinity binding between the KIF5B tail and the Kv3.1 T1 domain requires three basic residues in the

KIF5B tail. We*examined three other KIF5 cargo proteins, KLC1, SNAP25 and VAMP2 along with Kv3.1. Only Kv3.1 showed an increase in the number and activity of KIF5B-

YFP puncta clusters. These data show that KIF5 is clustered and activated by Kv3 which in turn regulates not only Kv3 channel transport but also KIF5 motor function regulation by the Kv3 cargo protein.

* Joshua Barry performed the mapping experiments, SPR experiment, immunostaining, co-transfection and western blotting. Dr. Mingxuan Xu performed immunostaining, mapping and competition assays. Andrew Dangel helped perform SPR experiment. Dr. Chandra Shrestha performed a competition assay. Dr. Yuanzheng Gu performed all electrophysiology recordings and cerebellar neuron culture. Peter Jukkola performed mouse perfusion, brain sectioning and immunostaining.

85 INTRODUCTION

For a neuron to function properly membrane proteins, like ion channels and receptors, proper localization to subcellular compartments is a necessity. Anterograde transport is thought to rely on ‘smart’ kinesin motors that can determine which microtubules lead down axons or dendrites (Burack et al., 2000; Hirokawa and

Takemura, 2005; Nakata et al., 2011). Other cytoskeletal and associated proteins also regulate KIF5 transport. Ankyrin-G and actin filaments act as a filter or ‘gate’ to maintain axon-dendrite polarity via selective transport regulation (Xu et al., 2007;

Sobotzik et al., 2009; Song et al., 2009; Xu et al., 2010; Maniar et al., 2012). However it is unknown whether the cargoes that kinesins carry can be ‘smart’ and direct the motor where to go.

Kinesin-1, also known as the conventional kinesin or KIF5, is made up of a heavy chain (KIF5) dimer and two kinesin light chains (KLCs) that bind to the C-terminal of the dimer. One motor is often sufficient to transport a vesicle along at microtubule, and sometimes a few motors work together to increase processivity and distance traveled, but not velocity (Miller and Lasek, 1985; Howard et al., 1989; Block et al., 1990; Hirokawa et al., 1991; Leopold et al., 1992; Schnitzer and Block, 1997; Vershinin et al., 2007;

Shubeita et al., 2008; Laib et al., 2009). The C-terminal tail is also the binding site for many other proteins (Xu et al., 2010). This leads to a problem. How does the ‘smart’

KIF5 distinguish between two different cargoes, especially if they have overlapping binding sites?

Axonal Kv channels regulate the initiation of APs, waveform, frequency and unidirectional propagation along axons, and neurotransmitter release at axonal terminals 86 (Gu and Barry, 2011). Each Kv channel is made of four voltage-sensing and pore forming α subunits. Each α subunit has six transmembrane domains and intracellular N- and C-terminal domains. The conserved T1 domain in the N terminal forms tetramers within Kv channel subfamilies, which is required for proper channel assembly (Li et al.,

1992; Xu et al., 1995; Jan and Jan, 1997; Bixby et al., 1999; Choe et al., 2002; Jahng et al., 2002; Long et al., 2005). Kv3.1 is involved in fast spiking (Rudy et al., 1999;

Kaczmarek et al., 2005; Bean, 2007) and is the first ion channel to be shown to directly bind to KIF5 (Xu et al., 2010; Barry and Gu, 2012).

Endogenous KIF5 often clusters along neurites. We wondered if Kv3 channels may regulate this clustering, and possibly the activity, of KIF5. We used protein biochemistry and live cell imaging assays to show that high-affinity multimeric binding between Kv3.1 T1 and the tail of KIF5 leads to KIF5 activation in clusters.

RESULTS

The distribution pattern of endogenous KIF5 motors in neurons

In young cultured hippocampal neurons (3 DIV) KIF5B was expressed in the soma and smoothly down the axon, with clusters in the axonal endings (Fig. 4.1A left).

In older (21 DIV) neurons KIF5B is in both axons and dendrites, with varying sized clusters of KIF5 along the neurites (Fig. 4.1A right). At 10DIV, in low density culture,

KIF5B clustered in a lot of axonal growth cones, where it colocalized with phalloidin (F- actin marker) (Fig. 4.1B). KIF5B was also more smoothly distributed along the distal axon (Fig. 4.1B) at a lower level than in the growth cone. Clusters of KIF5B were also

87 observed in the axon trunks, and they did not colocalize with F-actin or tubulin (Fig.

4.1C, D). If these clusters were actually moving vesicles they most likely contained tens or hundreds of KIF5 molecules, which argues against the current single vesicle/one or a few motors model.

Figure 4.1. Clustering of endogenous KIF5 motors in cultured hippocampal neurons. (A) Expression and distribution of endogenous KIF5 motors in cultured hippocampal neurons at 3 DIV (left, rabbit anti-KIF5B) and 21 DIV (right, mouse monoclonal anti-KIF5 H2). (B) Endogenous KIF5B motors cluster at the axonal growth cone. F-actin was labeled with phalloidin Alexa 546 (red in merged). KIF5B was stained with a rabbit anti-KIF5B antibody (green in merged) and β-tubulin was labeled with a mouse anti-β-tubulin antibody (blue in merged). Signals are inverted in single-channel images. (C) and (D) Clusters of KIF5B along axons with (indicated by white arrowheads) and without colocalizing F-actin (indicated by black arrows). Scale bars, 100 μm in (A) and 10 μm in (B)-(D). (Immunostaining performed by Mingxuan Xu).

With the observation of these KIF5 clusters we wondered what mechanism was underlying this clustering, and whether any KIF5 binding proteins were involved.

Mammalian KIF5, which contains three isoforms KIF5A, KIF5B, KIF5C, contains an N- terminal motor domain for movement along microtubules, a stalk domain for

88 dimerization through coiled-coil domains, and a C-terminal tail domain for cargo binding. Out of all the KIF5 tail domain binding proteins known only the Kv3 channel forms functional tetramers required for KIF5 binding (Xu et al., 2010). It is unknown how many KIF5 tails one T1 tertramer can bind, and what regions of KIF5 tail are critical for their interaction.

High-affinity and multimeric binding between the Kv3.1 T1 and KIF5B tail domains

To identify the specific Kv3.1 T1 binding site within the KIF5B tail Mingxuan

Xu, another postdoc in the Gu lab, and myself mapped the region using GST fusion proteins of KIF5B (Fig. 4.2A). Purified GST-T70 (aa 865-934) pulled down His-31T1 from bacterial lysate (Fig. 4.2B-E). When cut in two both N-terminal and C-terminal portions of T70 pulled down His-31T1, but not as strong as T70 (Fig. 4.2B-D). These two fragments share three basic residues (R892K893R894) with a net positive charge greater than +5. Further shortening of these two regions from either end eliminated the binding (Fig. 4.2B-E). GST-T70RKR, with the three basic residues mutated to three acidic ones (Ds) completely lost the binding to His-31T1 (Fig. 4.2E).

Figure 4.2. Direct binding between the Kv3.1 T1 and KIF5 tail domains. (A) Diagram of the Kv3.1 T1-binding site (T70) in KIF5 tail. KIF5 and Kv3.1 are shown as a dimer and a tetramer, respectively. For simplicity, only one Kv3.1 C-terminal domain is shown. In the sequence alignment of human KIF5A, KIF5B and KIF5C, conserved residues are highlighted in yellow. The numbers indicate residue positions in KIF5B. GST fusion proteins of KIF5B tail fragments that bind or fail to bind to His-31T1 in pull down assays are indicated in black or green, respectively. Residue numbers of tail fragments are indicated above the lines. The motor inhibiting site and three basic residues critical for binding to Kv3.1 T1 are indicated with blue and red, respectively. (B)-(E) In vitro binding assays to map the Kv3.1 T1-binding site in the KIF5B tail domain. (B) Mapping the minimal region of the Kv3.1 T1-binding site. (C) The N-terminal half of T70 binds to His-31T1. (D) Neither half of the fragment 892-934 binds to His-31T1. (E) Mutating R892K893R894 to three Ds to switch positive to negative charges completely eliminated the binding of T70 to His-31T1. Molecular weights are indicated on the left in kDa. Pulldown assays were repeated at least three times. (Mapping experiments performed by Mingxuan Xu and Joshua Barry).

Figure 4.2. Direct binding between the Kv3.1 T1 and KIF5 tail domains.

91 Mingxuan and I purified GST, GST-Tail, GST-Tail fragments and His-31T1 proteins and used them for in vitro protein-protein interaction experiments. Purified

GST-Tail and GST-T70, but not GST-T70RKR precipitated His-31T1 as seen on a protein gel (Fig. 4.3A). The N and C termini of GST-T70 did not pull down purified His-31T1

(Fig. 4.3A), which is probably due to the weaker binding affinity and the weaker sensitivity of colloidal blue staining compared to Western blotting.

I performed Surface Plasmon Resonance (SPR), in collaboration with Andrew

Dangel, to determine the binding affinity between GST-T70 and His-31T1 with purified

His-31T1, GST, GST-T70, GST-Tail892-934 and GST-T70RKR. The GST fusion proteins were immobilized on the sensor chip, while His-31T1 was flowed over the chip surface in binding buffer. His-31T1 bound with high affinity to GST-T70 (KD: 6.0  1.4  10-8

M; n = 4), fast association (Kon: 2.7  0.8  104 M; n = 4) and relatively slow disassociation (Koff: 1.6  0.1  10-3 M; n = 4) (Fig. 4.3B left). In contrast, GST-Tail892-

934 had nearly a thousand fold reduction in binding affinity (KD: 3.1  0.4  10-5 M; n =

3) (Fig. 4.3B middle). GST-T70RKR completely lost the binding to His-31T1 (Fig. 4.3B right). This SPR experiment confirmed direct binding between Kv3.1 T1 and KIF5B tail, the role of the three basic residues, and gave the first value of the binding affinity between an ion channel and kinesin motor.

We then wanted to estimate how many KIF5B tails bind to one Kv3.1 T1 tetramer. We explored this by using a pulldown assay with purified His-31T1 (~20 kD) and GST-T70 (~32 kD) at various molar ratios from 0:1 to 100:1. We kept the GST-T70 constant but increased the amount of His-31T1 where it saturated at an intensity ratio of

92 200:320 (AU) (His-31T1:GST-T70) (Fig. 4.3C). This suggests the stoichiometry is 1:1 and also suggests that the Kv3.1 T1 tetramer can therefore bind up to four KIF5B motors.

Figure 4.3. Binding affinity and stoichiometry of Kv3.1 T1 and KIF5B tail domains. (A) Purified GST-Tail and GST-T70, but not GST-T70RKR pulled down purified His- 31T1, indicated by the Colloidal Blue staining. Molecular weights (in kDa) are indicated on the left. (B) Binding response traces of His-31T1 to immobilized GST-T70 (left), GST-Tail892-934 (middle) and GST-T70RKR (bottom), in the SPR experiment. Solutions containing six different concentrations (0, 200 nM, 600 nM, 2 μM, 10 μM, 100 μM) of His-31T1 were flowed over the chip. The spikes in the traces at the highest concentration of His-31T1 are most likely due to buffer change. Triton X-100 (0.1%) was included in the stock buffer of high-concentration purified proteins to increase the solubility, but not in the binding buffer used in SPR. (C) Estimating the stoichiometry of the Kv3.1 T1 and KIF5B tail binding complex. In the left panel, purified GST-T70 (3.2 μg) was first coated on glutathione beads (30 μl total volume; 100% binding assumed), which were further incubated with different amounts of purified His-31T1. The molar ratios between His- 31T1 ( 20 kDa) and GST-T70 ( 32 kDa) were, 0:1, 0.4:1, 1:1, 4:1, 10:1, 30:1, 60:1, 100:1. The right panel shows the staining intensity graph of GST-T70 (black circles) and His-31T1 (red triangles). The protein gel was scanned as a TIFF image file and the intensity of protein bands were measured and subtracted with the background value. (Purified protein-protein interaction and SPR experiments in A and B performed by Joshua Barry. SPR experiment was a collaboration with Andrew Dangel. Stoichiometry experiment in C performed by Mingxuan Xu.)

94 Kv3.1 T1 competes with the KIF5B motor domain and microtubules but not with

KLC1 for binding to KIF5B tail

The C-terminal tail domain of KIF5 contains many overlapping binding sites for such things as cargo, autoinhibition and microtubules (Fig. 4.4A). To examine if these proteins and Kv3.1 T1 can affect one another’s binding to KIF5B tail we performed in vitro competition assays. GST-Tail was kept at a constant value while various concentrations of His-tagged proteins were used. While His-31T1 did not affect His-

KLC from binding to GST-Tail (Fig. 4.4B, C) it did compete with His-Motor, although the His-Motor/GST-tail interaction was weaker (Fig. 4.4D).

To examine the role Kv3.1 T1 plays in KIF5B tail and microtubule binding we used in vitro assembly and pulldown assays. GST-Tail and GST-T70, but not GST,

GST-T70RKR or GST-T63 (aa 758-820, the KLC1 binding site) bound to microtubules

(Fig. 4.4E and F). His-31T1 itself did not bind to microtubules (data not shown). His-

31T1 was able to compete GST-T70 away from microtubules in a concentration dependent manner (Fig. 4.4G). Therefore Kv3.1 channels may activate KIF5B motors by releasing the tail domain from its interactions with the motor domain and microtubules.

Figure 4.4. Kv3.1 T1 competes with microtubules but not KLC1 for binding to KIF5B tail. (A) Diagram of the KIF5B tail domain and five of its binding proteins. The KLC-binding site (T63) is located between residues 758 and 820, highlighted in blue. The Kv3 T1-binding site (T70) is between residues 865 and 934, highlighted in red, which overlaps with binding sites for KIF5 motor domain, microtubules, and SNAP25. (B) His-31T1 did not compete with His-KLC1 for binding to GST-Tail. GST-Tail (3.5 μg) was first coated on glutathione beads and further used to pull down purified His- KLC1 (5.6 μg, so that GST-Tail and His-KLC1 are in a 1:1 molar ratio) mixed with different amount of His-31T1. Molar ratios between His-31T1 and His-KLC1 used were 0:1, 0.3:1, 1:1, 3:1, and 10:1. (C) The Western blotting with an anti-6His antibody in an experiment similar to (B), except that lower GST-Tail (2.5 μg) amount, an additional condition (30:1), and 10% loading compared to protein gels, were used. (D) Increased amount of His-Motor reduced the amount of His-31T1 binding to GST-Tail. (E)-(F) Purified GST-Tail and GST-T70, but not GST, GST-T63, or GST-T70RKR, bound to microtubules. Microtubules were assembled in vitro with purified tubulins for 20 min, then incubated with purified GST fusion proteins (around 5 μg total) for another 30 min, and pelleted with high speed spin. Pellets were resolved with sample buffer equal to the supernatant in volume, and equal volume of supernatants and dissolved pellets were loaded on a SDS-PAGE gel. Protein bands were stained with Colloidal Blue (top). GST fusion proteins were further revealed with Western blotting using an anti-GST antibody (10% loading) (bottom). (G) Purified GST-T70 and His-31T1 were mixed in four different molar ratios, 1:0, 1:1, 1:4, and 1:8. In this experiment, microtubules were assembled in the presence of 100 μM paclitaxel to further stabilize assembled microtubules. The supernatants and pellets were resolved in SDS-PAGE and revealed by both Colloidal Blue staining (top) and Western blotting (10% loading) using an anti-GST antibody (bottom). In (E)-(G), S, supernatant; P, pellet. Black arrowheads, GST fusion proteins. Red arrowheads, His-31T1. Since there was no washing step after assembled microtubules were pelleted, very faint bands in the pellet lane is due to supernatant contamination, even when the protein does not bind to microtubules. All in vitro binding experiments were repeated at least three times. Molecular weights are indicated on the left in kDa. (Competition assays performed by Mingxuan Xu and Chandra Shrestha).

Figure 4.4. Kv3.1 T1 competes with microtubules but not KLC1 for binding to KIF5B tail.

97 Binding proteins of KIF5 differentially affect the localization of KIF5 tail fragments

I then wanted to evaluate the results from our biochemical assays by designing experiments to be used in neurons. I examined the role that four known KIF5 binding proteins play on KIF5 distribution and motility. Using cultured hippocampal neurons at

21 DIV I found some that expressed Kv3.1b and it colocalized with KIF5 in clusters (Fig.

4.5A top). KLC1 also colocalized with KIF5 in clusters (Fig. 4.5A row 2). SNAP25 partially colocalized with KIF5 in clusters, while VAMP2 colocalized significantly less with no clear cluster colocalization (Fig. 4.5A row 3 and 4). These immunostaining results showed that different KIF5 binding proteins colocalize with KIF5 in different patterns.

Since all four of these proteins bind to KIF5 tail domain I then wanted to examine their effect of KIF5B tail distribution by using a coexpression system. YFP-Tail is localized to the somatodendritic region when expressed alone in cultured hippocampal neurons, while YFP-KLC1 is in both axons and dendrites. CFP-Tail was brought into the distal axon by YFP-KLC1 by not by YFP alone (Fig. 4.5B). YFP-Kv3.1b, YFP-SNAP25 and YFP-VAMP2 all failed to bring CFP-Tail into the distal axon (Fig. 4.5C) even though they were all expressed in the axon and dendrites of transfected neurons.

Fluorescence resonance energy transfer (FRET) imaging performed by Yuanzheng was used to examine the physical interaction between KIF5B tail and KLC1 in living neurons.

CFP-Tail and YFP-KLC1 highly colocalized and gave strong FRET signal in distal axons

(Fig. 4.5D) and soma (Fig. 4.5E). These results from the neuron culture were consistent with those from our biochemical analysis. More importantly these results have uncovered two smaller regions of KIF5B tail that can be used as dominant-negative

98 constructs in both a KLC dependent and independent manner. Also, when overexpressed these two constructs cause less problems with neuronal survival.

Figure 4.5. KIF5-binding proteins differentially regulate KIF5B tail localization. (A) Co-staining of cultured hippocampal neurons at 20 DIV for endogenous KIF5 (mouse H2 antibody in green) and its binding proteins (Kv3.1b, KLC1, SNAP25, and VAMP2; in red). (B) CFP-Tail was restricted in somatodendritic regions in 8-DIV neurons expressing CFP-Tail and YFP (top). Co-expression of YFP-KLC1 (green in merged) brought CFP- Tail (blue in merged) into distal axons (bottom). Arrows, axons; Arrowheads, dendrites. (C) CFP-Tail intensity profiles along axons in the presence of various YFP fusion proteins. Only the expression of YFP-KLC1, but not YFP-Kv3.1b, YFP-SNAP25 and YFP-VAMP2, targeted CFP-Tail into distal axons. (D) Strong FRET signals (inverted in single channel and red in merged) were detected along axons between CFP-Tail (blue) and YFP-KLC (green) (17 out of 19 neurons had strong FRET signals). (E) Strong FRET signals were also detected in the soma of transfected neurons. (Immunostaining and cotransfection experiments in A and B performed by Joshua Barry, FRET experiments in C-E performed by Yuanzheng Gu.)

100 Opposite effects of the binding of Kv3.1 and KLC1 on KIF5B distribution

It remains unknown how various binding proteins to KIF5 tail domain affect KIF5 activity and potentially cluster the motor. KIF5B-YFP has a similar distribution pattern to endogenous KIF5. KIF5B-YFP is more smoothly concentrated along the distal axon

(Fig. 4.6A top), although it does cluster at the axonal endings and along axonal trunks.

Mutating the three basic residues (R892K893R894) in KIF5B-YFP caused KIF5BRKR-

YFP to cluster in various sizes along axonal endings and axon trunks (Fig. 4.6A bottom).

It was difficult to find the soma and dendrites expressing this construct, and its clustering pattern is more pronounced than tailless KIF5B (Xu et al, 2010).

Mingxuan and I used cotransfection to examine the effects of four different KIF5 binding proteins on the distribution patterns of KIF5B-YFP and KIF5BRKR-YFP. CFP-

KLC1 did not change the localization of KIF5B-YFP. While CFP-Kv3.1b caused

KIF5B-YFP to form variously sized clusters along axons (Fig. 4.6B, D, E). CFP-KLC1 converted KIF5BRKR-YFP from being highly clustered to a uniform pattern similar to

KIF5B-YFP, which may suggest KLC1 has an inhibitory role in KIF5 activation. CFP-

Kv3.1b did not change the clustered pattern of KIF5BRKR-YFP (Fig. 6 4.C-E). Only few of the clusters contained CFP-Kv3.1b (Fig. 4.6C), which may be due to endogenous KIF5 dimerizing with the mutant to allow binding to Kv3.1. Therefore Kv3.1 clusters KIF5B motors while KLC1 disperses them (Fig. 4.6 D-E). CFP-SNAP25 and CFP-VAMP2 partially colocalized with both KIF5B-YFP and KIF5BRKR-YFP, but did not change their distribution pattern (Fig. 4.6E).

101

Figure 4.6. Mutating the three charged residues or the presence of Kv3.1 clusters KIF5B-YFP. (A) Relatively smooth distribution of KIF5B-YFP (green) along axons of hippocampal neurons (top), which were transfected at 5 DIV, fixed and stained for axonal marker, Tau1 (red), two days later. Highly clustered pattern of KIF5BRKR-YFP (green) along axons (labeled for Tau1 in red) (bottom). Arrowheads, clusters of KIF5BRKR-YFP along the axon segment. (B) Distribution patterns of KIF5B-YFP (green in merged) when co-expressed with either CFP-KLC1 (top) or CFP-Kv3.1b (bottom) in hippocampal neurons. White arrows, colocalizing clusters. (C) Distribution patterns of KIF5BRKR- YFP (green in merged) when co-expressed with either CFP-KLC1 (top) or CFP-Kv3.1b (bottom) in hippocampal neurons. YFP is in green and CFP is in blue in merged images. Signals are inverted in single-channel images. White arrows, colocalizing clusters. Black arrows, KIF5BRKR-YFP clusters without CFP-Kv3.1b. (D) Profiles of fluorescence intensity along axons in (B) (top) and (C) (bottom). (E) Summary of the clustering effect of CFP-tagged KIF5-binding proteins on KIF5B-YFP (top) and KIF5BRKR-YFP (bottom). Scale bars, 10 μm in (A); 100 μm in (B) and (C). (Transfection experiments carried out by Joshua Barry and Mingxuan Xu.)

102

Figure 4.6. Mutating the three charged residues or the presence of Kv3.1 clusters KIF5B-YFP.

103 Kv3.1b but not other KIF5-binding proteins increased the moving frequency of

KIF5B-YFP anterograde puncta.

To examine the effects these proteins have of KIF5 motility Yuanzheng performed live-cell imaging of KIF5B-YFP with the four cargos tagged with CFP. Only

CFP-Kv3.1b caused a marked increase of the frequency of moving puncta containing

KIF5B-YFP (data not shown). KIF5BRKR-YFP, however, had more than half of their puncta already moving, primarily in the anterograde direction. Co-expression with CFP-

CFP-Kv3.1b, CFP-SNAP25 or CFP-VAMP2 had no effect on the frequency of moving puncta, but CFP-KLC1 significantly reduce the frequency. Most importantly these imaging results showed that there was an increase in the moving puncta of KIF5B-YFP when co-expressed with Kv3.1b.

KIF5B-YFP number increased on carrier vesicles when co-expressed with Kv3.1

Yuanzheng’s experiment examining the increased movement of KIF5B-YFP puncta when co-expressed with Kv3.1b led to an interesting discovery. The fluorescent intensity of these moving puncta was fairly strong, suggesting they might contain many

YFP molecules. Our lab previously reported measuring YFP-Kv1.2 axonal transporting puncta using quantitative microscopy which was calibrated with various YFP-fusion proteins expressed in yeast strains (Wu and Pollard, 2005; Gu and Gu, 2010).

Fluorescent intensities of KIF5B dimers were calculated using the previously calibrated standards (Gu and Gu, 2010). When KIF5B-YFP was expressed alone each anterogradely moving puncta contained 29 KIF5B dimers. However, when coexpressed with CFP-Kv3.1b the number jumped to roughly 187. A significant increase was also

104 seen for retrograde and stationary puncta (data not shown). KIF5BRKR-YFP anterograde puncta were increased compared to KIF5B-YFP (161 vs 29), co-expression with Kv3.1b did not increase this value (data not shown). Co-expression of CFP-SNAP25 or CFP-

VAMP2 had no clear effect on the KIF5B-YFP moving puncta. These results suggested

Kv3 channels can cluster KIF5 motors during intracellular transport.

KIF5 clusters in cerebellar neurons from Kv3.1 knockout mice reduced

Peter Jukkola and I used Kv3.1 knockout (KO) mice to examine KIF5B distribution. Western blotting and PCR-based genotypic confirmed KO mice (Fig. 4.7A,

B). Western blotting confirmed complete loss of Kv3.1b in KO mice, while KIF5,

KLC1, GRIP1 and SNAP25 were unchanged (Fig. 4.7A). Kv3 channels are highly expressed in the cerebellum, and Kv3.1 channels are expressed in granule cells (Weiser et al., 1994; Grigg et al., 2000; Rudy and McBain, 2001). Kv3.1b is present in parallel fibers in the molecular layer but not in Purkinje neurons (Puente et al., 2010). Peter

Jukkola found that in coronal sections of the cerebellum Kv3.1b and KIF5B were present and colocalized in the molecular and granule cell layers (Fig. 4.7C). In Kv3.1 KO mice

Kv3.1b staining is reduced close to the background (Fig. 4.7D), while KIF5B remains unchanged (Fig. 4.7E). KIF5B clusters were significantly reduced (Fig. 4.7F), but clusters still remained, possibly due to other proteins/mechanisms that may cause KIF5 clustering to occur.

105

Figure 4.7. KIF5 clusters reduced in cerebellar neurons of Kv3.1 knockout mice. (A) Expression of Kv3.1b, KIF5, and KIF5-binding proteins in brains of Kv3.1 knockout (-/-) and wildtype (+/+) mice. Soluble fractions (left) and crude membranes (right) are compared. Molecular weights (in kDa) are indicated on the left. (B) Genotyping of Kv3.1 heterozygotes (+/-), homozygotes (-/-), and wildtype mice. DNA ladders are on the left in bp. (C) Kv3.1b and KIF5B staining patterns in coronal sections of cerebellum from WT (C) and Kv3.1 KO (D) mice. P, Purkinje cell layer; GL, granule cell layer; ML, molecular layer. (E) To quantify the KIF5B clusters from the WT (left) and Kv3.1 -/- (right) sections, images were thresholded and binarized to show pixel clusters. (F) Summery of the clusters per image (40 objective lens). The "n" numbers are indicated within the bars. Unpaired t-test: *, p < 0.05. Scale bars, 300 μm in (C) and (D), and 100 μm in (E). (Western blotting in A performed by Joshua Barry. All other experiments performed by Peter Jukkola.)

106 Yuanzheng Gu generated a cerebellar neuron culture using wild-type and Kv3.1 knockout mice. This culture is performed similarly to the hippocampal neuron culture and favors the survival of granule cells. At 20 DIV KIF5B colocalized in puncate with

Kv3.1b in wild-type cultures, but in Kv3.1 knockout mice KIF5B was more smoothly expressed along the axon with some punctae still visible (data not shown). The results from the neuron culture are consistant with the cerebellar brain sections results, however it must be taken into account that KIF5 clusters are still present and may be due to other proteins and mechanisms that can cluster KIF5.

DISCUSSION

This study has revealed a novel mechanism that a cargo (Kv3) can regulate the activity of KIF5 by relieving two inhibitory mechanisms mediated by the tail-motor and tail-microtubule binding (Fig. 4.8A). KIF5 motors are clustered by Kv3 tetramers through direct and high-affinity multimeric binding (Fig. 4.8B). This study also showed that different KIF5 binding proteins can ride the motor in different ways. This is probably critical for the specificity of KIF5 mediated transports.

107

Figure 4.8. Summary of the mechanism and consequence of the Kv3-KIF5 binding. (A) Interaction diagram of KIF5B, KLC1, Kv3.1 and microtubules. (B) Hypothetical diagrams. Direct binding to an oligomer, but not a monomeric adaptor, clusters and activates KIF5 motors, and regulates the motor number on a vesicle carrier.

Kv3.1 is the first identified ion channel that directly binds to KIF5, and it also requires proper tetramerization for this interaction (Xu et al., 2010) which raised an interesting possibility that Kv3 channels may be somewhat responsible for KIF5 clustering which was observed in endogenous KIF5 staining (Fig. 4.1). My mutagenesis experiment identified three basic residues within the 70 residue KIF5 tail that are crucial for binding the Kv3.1 T1 domain (Fig. 4.2). The SPR experiment determined the binding affinity between Kv3.1 T1 and its binding region within KIF5B (Kd = 6.0 ± 1.4 x 10-8 M)

(Fig. 4.3B). This Kd is comparable to the binding affinity between KIF5 and GRIP1 (Kd

= 1.9  10-8 M), and higher than the binding affinity between Sunday Driver/JIP3 and

KIF5 (Kd = 1.8  10-6 M). Also the pulldown assay suggests that one Kv3.1 channel tetramer can bind up to four KIF5 heavy chains (Fig. 4.3C). The competition experiments show that purified His-31T1 competed with His-Motor and microtubules but

108 not with His-KLC1, suggesting Kv3.1 may activate the KIF5B motor by relieving the inhibition of the motor domain and the microtubule binding to KIF5 tail (Fig. 4.4). These biochemical data have revealed critical information regarding how Kv3 channels may activate and cluster KIF5 motors. It is important to note that the interactions revealed with protein biochemical assays are subject to variety of regulations in vivo. Therefore, we have carried out various experiments in living neurons.

Kv3.1 is unique among the KIF5-binding proteins examined in this study in regulating KIF5B function. Although KLC1 was also shown to release the tail-motor domain binding and tail-microtubule binding (Wong and Rice, 2010), Kv3.1 and KLC1 differ in multiple ways in regulating KIF5 motor activity. First, they bind to different regions in KIF5B tail (Fig. 4.4). Second, CFP-Kv3.1b but not CFP-KLC1 clustered

KIF5B-YFP along axons (Fig. 4.6). Third, interestingly, CFP-KLC1 but not CFP-Kv3.1b dispersed the clustered pattern of KIF5BRKR-YFP (Fig. 4.6). In Fig. 4.6, the changes of distribution patterns were very dramatic and clear. KLC1 apparently did not compete with Kv3.1 channels for binding to KIF5 (Fig. 4.4E), nor clusters KIF5 (Fig. 4. 6). In contrast to the sophisticated roles of KLC1 (Cai et al., 2007; Wong and Rice, 2010), the action of Kv3.1 appears to be straightforward, which is to activate and cluster KIF5B motors. I also examined two other KIF5-binding proteins, SNAP25 and VAMP2.

Although they have overlapping binding sites with Kv3 in KIF5 tail, both failed to cluster or activate KIF5 motors (Fig. 4.6E). Therefore, although the KIF5/kinesin-1 motor can be activated by other binding proteins, this study has clearly demonstrated that the Kv3 channels tetramers uniquely cluster KIF5 motor, contributing to the specificity of both cargo loading and cargo-regulated activation.

109 We used Kv3.1 KO mice to examine how Kv3.1 deletion affects KIF5 clustering.

KIF5 clusters were reduced in Kv3.1 KO, but still abundant. This may be due to the following reasons: 1. Other Kv3 channels, with highly conserved T1 domains may also cluster KIF5 due to the conserved T1 domain. 2. There may be other unidentified proteins that can cluster KIF5 motors. 3. Other mechanisms underlying KIF5 clustering, for example the clustering of KIF5BRKR-YFP may be due to elimination of autoinhibition. However, it is still unclear what causes KIF5BRKR-YFP to cluster.

The decrese in the clusters of KIF5 in the cerebellar brain sections may be hard to see in the brain sections. This may be due to the fact that when imaging brain sections you are not capturing a single layer of neurons like in a culture, there are many layers of neurons captured within that one image. The clusters that appear large in the Kv3.1 knockout mice may be due to multiple neurons have clustered KIF5 ending up near each other. When the analysis was performed thresholding was used to quantify only punctae of a certain size or larger. If this is the case with many neurons having their puncta close together it could be considered one large puncta instead of being counted as smaller puncta. The cerebellar neuron culture which compared wild-type versus Kv3.1 KO neurons showed that KIF5 puncta are significantly decreased and KIF5 is more smoothly expressed along the axon with some puncta still remaining. This may be due to other proteins or other unknown mechanisms that are able to cluster KIF5.

As stated earlier in this chapter, it was suggested that one vesicle could contain tens or hundreds kinesin motors. This is much higher than the less than 10 motors per vesicle supported by experiments and mathematical modeling (Shubeita et al., 2008;

Gazzola et al., 2009; Erickson et al., 2011). From Yuanzheng’s co-expression

110 experiment he showed that co-expression of KIF5B-YFP with CFP-Kv3.1b could increase the number of anterograde moving puncate from 29 KIF5 dimers per carrier vesicle to 167. It is important to mention this is with an overexpresion system so this value may be close to the upper limit for a carrier vesicle.

When compared to my model in Figure 4.8 how can one vesicle cluster tens to hundreds of kinesin motors? A carrier vesicle would not just carry one channel per vesicle, that is a severe waste of time and energy to transport a single channel down the axon. Each vesicle could contain many channels, possibly of various types. These various channels (including Kv3.1b) could cluster a multitude of kinesin motors onto each vesicle even if the motors are not all active at the same time. Many of the motors may function as a reserve pool to increase processivity. The motors that are actively walking along microtubules can be further regulated by microtubule-binding proteins

(Dixit et al., 2008).

Various ion channels must be precisely targeted to the correct location for neuronal excitability and synaptic transmission. How these proteins are properly transported to their neuronal compartments is still a big question in neurobiology. Some ligand-gated ion channels like NMDA, AMPA and GABAA receptors use adaptor proteins (Barry and Gu, 2012). These adapters are monomeric, and are unlikely to form multimeric complexes or cluster kinesin motors. This study has shown that there is a direct binding between Kv3.1 and KIF5. This is different from all other known channels transported by KIF5, and Kv3.1 activates and clusters KIF5, which may be a novel mechanism regulating cargo transport specificity. Kv3 channels may regulate axonal

111 transport of other cargos by clustering KIF5, which is an interesting area for further research.

METHODS cDNA constructs

GST-Tail, GST-T70 (GST-Tail865-934), GST-T63 (GST-Tail758-820), His-

31T1, Kv3.1aHA, Kv3.1bHA, CFP-Kv3.1aHA, CFP-Kv3.1bHA, YFP-T70, YFP-T63,

KIF5B-YFP were previously described . GST-Tail865-986, GST-Tail892-934, GST-Tail870-896,

GST-Tail865-891, GST-Tail892-912, GST-Tail875-896, GST-Tail913-934, GST-Tail856-886, GST-

Tail892-929, GST-Tail892-924, GST-Tail897-934, GST-Tail902-934, and GST-Tail875-919 were made by inserting the PCRed cDNA fragments corresponding to the indicated regions of

KIF5B tail into the pGEX4T-2 vector. GST-T70RKR, YFP-T70RKR, and KIF5BRKR-YFP were made by mutating R892K893R894 to DDD with Quickchange based on GST-T70,

YFP-T70 and KIF5B-YFP, respectively. His-KLC1 and His-Motor were made by inserting the KLC1 full-length cDNA (OpenBiosystem, Huntsville, AL) and the cDNA containing KIF5B residues 1 to 399 into pRSET B vector. C(Y)FP-KLC1 was made by inserting the KLC1 full length cDNA into pEC(Y)FP-C1 (Clontech Laboratories, Inc,

Mountain View, CA) between BglII and HindIII. C(Y)FP-SNAP25 and C(Y)FP-VAMP2 were made by inserting the full length cDNAs into pEC(Y)FP-C1 between BglII and

EcoR1. All constructs were confirmed by sequencing.

Antibodies and immunostaining

112 Antibodies used include mouse anti-6×His antibodies (Invitrogen, Carlsbad, CA;

UC Davis/NIH NeuroMab facility (clone # N144/14), Davis, CA), mouse anti-GST and anti-Kv3.1b antibodies (clone # N100/13 and N16B/8, respectively; UC Davis/NIH

NeuroMab Facility), mouse anti-β-tubulin and anti-KIF5 H2 antibodies (Millipore,

Billerica, MA), mouse anti-GRIP1 antibody (Abcam, Cambridge, MA), rabbit polyclonal anti-microtubule-associated protein 2 (MAP2) and anti-Tau1 antibodies (Chemicon,

Temecula, CA), rabbit polyclonal anti-Kv3.1b antibody (Alomone Labs, Jerusalem,

Israel), rabbit polyclonal anti-KLC1 antibody (Santa Cruz Biotechnology Inc., Santa

Cruz, CA), rabbit anti-SNAP25, anti-VAMP2 and anti-KIF5B antibodies (Abcam), rat monoclonal anti-HA antibody (Roche, Indianapolis, IN), Cy2, Cy3, and Cy5 conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). The procedures of immunocytochemistry were described previously (see pg 45). To distinguish axons and dendrites of neurons, an anti-Tau1 (Tau1 is an axonal marker) antibody or an anti-MAP2 (MAP2 is a dendritic marker) antibody was used in costaining.

F-actin was labeled with phalloidin Alexa 546 and nuclei were stained with Hoechst

33342 (Invitrogen).

Hippocampal neuron culture and transfection

Hippocampal neuron culture was prepared as previously described (see pg 44).

For transient transfection, neurons in culture at 5-7 DIV were incubated in Opti-MEM containing 0.8 μg of cDNA plasmid and 1.5 μl of Lipofectamine2000 (Invitrogen) for 20 min at 37°C.

113 Protein purification and in vitro binding assays

Expression of GST- or 6×His-tagged fusion proteins was induced in BL21 E. coli cells with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 hr at 37°C.

Bacterial pellets were solubilized with sonication in the pulldown buffer (50 mM Tris-

HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, and a complete protease inhibitor tablet) at 4°C, and centrifuged at 50,000 × g for 30 min at 4°C. The supernatants were incubated either with glutathione beads (GE Healthcare Bio-Sciences AB, Sweden) or with Co2+ beads (TALON metal affinity resin, Clontech Laboratories Inc.) at 4°C for 3 hrs. After extensive washing, the beads were coated with purified fusion proteins and were eluted with the elution buffer containing either 20 mM glutathione or 150 mM imidazole. The elution was further dialyzed with the pulldown buffer at 4°C overnight.

In binding assays to map the Kv3.1 T1-binding site in Fig. 4.2, glutathione beads coated with purified GST fusion proteins were further incubated with bacterial lysate supernatant containing His-31T1 at 4°C for 2 hrs. After extensive washing, the precipitants were eluted with 2 sample buffer, resolved in SDS-PAGE, and subjected to

Western blotting with an anti-His antibody.

In pulldown assays using purified proteins, purified GST fusion proteins (around

2.5-5 μg) were first coated onto glutathione beads (beads total volume: 30 μl). Coated beads were further incubated with purified His-tagged proteins in different molar ratios in the pulldown buffer without protease inhibitors (total volume 500 μl) at room temperature for 1 hr. After extensive washing, the precipitants were eluted with 2 sample buffer, resolved in SDS-PAGE, and subjected to Western blotting or Colloidal

Blue staining. In Fig. 4.3C, after binding, the beads were quickly washed once ( 15 sec) 114 with the IP buffer and immediately incubated with the sample buffer. Each in vitro binding assay was performed at least three times.

Surface Plasmon Resonance (SPR) experiments to measure binding affinities of protein-protein interactions

SPR experiments were performed at 25°C on a Biacore T100 instrument (GE

Healthcare, Piscataway, NJ) with CM5 sensor chips (GE Healthcare). One microgram of monoclonal anti-GST antibody was immobilized on flow cells covalently coupled as recommended by the manufacturer using the amine-coupling kit (GE Healthcare).

Purified GST (0.6 μg) flowed over in the running buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) and was captured by the antibody in cell #1 as a control, and other GST fusion proteins (0.6 μg) were immobilized in cells #2-4. Purified His-31T1 at different concentrations was injected at a flow rate of 20 μl/min for 180 sec. After 300 sec of dissociation, the chip was fully regenerated with 1 M NaCl for 600 sec. All sensorgrams show data in which the background signal (GST in cell #1) was subtracted from the total signal (GST fusion proteins). The KD for His-31T1 binding to GST fusion proteins were calculated by either fitting the curves to obtain Kon and Koff (for GST-T70) or plotting saturation binding curves using the equilibrium response value at the plateau of all curves

(for GST-Tail892-934). Since we estimated the binding stoichiometry of GST-T70 and

His-31T1 is about 1:1 (therefore 4:4), in analyzing SPR experimental data I adopted the ratio 1:1, assuming no allosteric effect during binding.

Microtubule assembly and binding assays

To reconstitute microtubule in vitro, purified tubulin (Sigma) (20 μl in 2 mg/ml) was incubated in the polymerization buffer containing 80 mM PIPES (N,N0-piperazine

115 diethane sulfonic acid), pH 6.9, 0.5 mM MgCl2, 1mM GTP (guanosine-50-triphosphate), and 5% glycerol, at 37°C for 30 min. Then purified proteins (around 5 μg) were added and incubated for another 30 min. Microtubules and binding proteins were pelleted at

50,000 × g for 30 min at room temperature and resuspended in sample buffer. To determine the amounts of tubulin and other protein in the microtubule or supernatant fractions, 15 μl of supernatants and resuspended pellets were analyzed by SDS-PAGE. In the competition experiment of GST-T70 and His-31T1, 100 μM paclitaxel was added to promote and stabilize microtubule formation.

Fluorescence microscopy and quantification

Fluorescence images were captured with a Spot CCD camera RT slider

(Diagnostic Instrument Inc., Sterling Heights, MI) in a Zeiss upright microscope,

Axiophot, using Plan Apo objectives 20/0.75 and 100/1.4 oil, saved as 16-bit TIFF files, and analyzed with NIH Image J and Sigmaplot 10.0 for fluorescence intensity quantification. Exposure times were controlled so that the pixel intensities in dendrites and axons were below saturation, but the same exposure time was used within each group of an experiment. The quantification procedure was described previously (Xu et al.,

2010). Only transfected neurons with clearly separated dendrites and axons, and isolated from other transfected cells, were chosen for analysis. Using NIH Image J, I laid a line along the major axon to acquire its average fluorescence intensity (in arbitrary unit)

(Faxon), and laid lines along proximal dendrites which connect with soma to obtain the average fluorescence intensity of the somatodendritic region (Fsd) to represent the total level. Thus, the ratio (Faxon/Fsd) reflects the relative axonal level. The background fluorescence intensity was measured for each image and subtracted.

116 Live cell and FRET imaging

Neurons growing on 25 mm coverslips were loaded into the imaging chamber

(Molecular Devices, Downingtown, PA) and incubated with imaging buffer (HE-LF medium (Brainbits, Springfield, IL) plus 2% B-27, 0.5 mM Glutamine, and 25 μM

Glutamate) at room temperature. The timelapse imaging setup was built upon a Nikon

(Nikon Inc., Melville, NY) TE2000 inverted microscope. Images were captured with a

CCD camera Coolsnap HQ (Photometrics, Tucson, AZ) through CFP or YFP filter sets with 1 second exposure time. The filters were changed through filter wheels controlled through Lamda 10-3 (Sutter Instrument, Novato, CA) by MetaMorph software

(Molecular Devices). The time-lapse imaging was performed with 2-second interval for

100 frames.

The strategy and protocol of FRET imaging were described previously in detail .

In this system, 64.7%  0.5% (n = 18) of CFP, and 1.47%  0.08% (n = 12) of YFP fluorescence bleed through the FRET channel . Therefore, FRETcorrected = FRETraw –

(0.65  CFP) – (0.015  YFP). The calculation of FRETcorrected was performed with the

MetaMorph software.

Kv3.1 knockout mouse genotyping

Kv3.1 knockout (KO) mouse line was kindly provided by Dr. R. Joho at UT

Southwestern Medical Center and have been maintained using a PCR-based genotyping procedure as previously described (Ho et al., 1997; Sanchez et al., 2000; Hurlock et al.,

2009). The Kv3.1 KO mice were backcrossed with BL6 for ten generations. Seizure can be observed occasionally from some Kv3.1 homozygotes (-/-) but not heterozygotes (+/-) mice. The following primes are used: forward primer 31F775 (for both WT and 117 knockout, 5'- GCG CTT CAA CCC CAT CGT GAA CAA GA -3'), reverse primer

31R991 (for WT, 5'- GGC CAC AAA GTC AAT GAT ATT GAG GG -3'), and reverse primer PNR278 (for knockout, 5'- CTA CTT CCA TTT GTC ACG TCC TGC AC -3').

Three Kv3.1 KO (-/-) and three control Bl6 mice of either sex at the age of 2-4 months were used in this study.

Immunofluorescence staining on mouse brain sections and cluster quantification

After cardiac perfusion and tissue fixation, mouse cerebellum was removed, post- fixed, and then embedded in optimal cutting temperature media (Sakura Finetek USA,

Inc., Torrance, CA) and stored at -80°C until sectioning. Coronal sections (40-μm thickness) of mouse cerebellum were cut with a Microm HM550 cryostat (Thermo

Scientific, Waltham, MA) and collected on Superfrost Plus microscope slides

(FisherScientific, Pittsburgh, PA). Immunostaining and imaging were performed as previously described . The whole images captured with a 40 objective lens were processed for quantification using Metamorph by flattening the background, thresholding the images to include only the 5% highest intensity pixels, and binarizing the image.

Kif5B puncta were quantified in ImageJ using the “Analyze Particles” tool to count all pixel clusters ranging in size from 9-300 pixels.

118 CHAPTER 5

ZN2+ REGULATES KV3 CHANNEL ASSEMBLY, TRAFFICKING AND

ACTIVITY.

SUMMARY

As stated previously Kv3 channels are critical for fast spiking neurons. In this chapter I discuss the discovery that zinc reversibly inhibited fast spiking in cultured neurons by inhibiting Kv3 channels. We identified a new zinc-binding site in the C- terminus of Kv3.1 which is involved in regulating Kv3.1 activity and trafficking but not the zinc inhibition. Another zinc-sensing site was discovered that is between the first transmembrane segment and the first extracellular loop which is regulated by zinc, and is the site of the zinc inhibition. This study shows novel mechanisms that the divalent heavy metal zinc regulates the Kv3 channel activity and localization.

* Joshua Barry performed the mapping assay, cloning, pulldown experiments, neuronal transfection, imaging and quantification. Dr. Yuanzhen Gu performed all electrophysiology recordings and quantification.

119 INTRODUCTION

Zinc is both an essential element for our survival, but too much can be lethal. Zinc is found throughout the brain where it is usually stored in glutamatergic neuron presynaptic vesicles, where it is co-released with glutamate (Huang, 1997; Lin et al.,

2001; Smart et al., 2004; Frederickson et al., 2005). Zinc can regulate various ligand and voltage-gated ion channels, which in turn regulates neuronal excitability and synaptic transmission (Frederickson et al., 2005; Mathie et al., 2006; Kay and Toth, 2008; Sensi et al., 2009)..

Zinc can stimulate the activity of ATP-sensitive K+ channels, transient receptor potential cation channel A1, and high-conductance voltage- and Ca2+-activated K+ channels (Prost et al., 2004; Hu et al., 2009; Hou et al., 2010), while inhibiting the activity of acid-sensing ion channel 1b (Jiang et al., 2011). These results raised an interesting question, does zinc binding to extracellular or intracellular domains correlate with stimulation or inhibition? Various isoforms of Kv channels are suppressed by zinc to various degrees (Lovinger et al., 1992; Poling et al., 1996; Zhang et al., 2001; Mathie et al., 2006; Aras et al., 2009). However, the exact zinc binding site mechanism underlying

Kv3 regulation is still unknown.

The zinc-binding site (HX5CX20CC) within the T1 domain of Kv3 families Kv2 to

Kv4 is the most well known. Proper tetramerization of the Kv channel itself requires the conserved N-terminal T1 domain to tetramerize (Li et al., 1992; Xu et al., 1995; Bixby et al., 1999; Jahng et al., 2002). T1 tetramerization requires zinc. In Kv4 channels the zinc binding site, within the T1 domain, is involved in regulated channel activity (Jahng et al.,

2002; Kunjilwar et al., 2004; Wang et al., 2007). My labs previous research showed that

120 polarized axon-dendrite targeting of Kv3 channels required regulation by ankyrin G and

KIF5 (Xu et al., 2007; Xu et al., 2010; Gu and Barry, 2011). The N- and C- termini interaction of Kv3.1 is zinc dependent (Xu et al., 2007), while direct binding of KIF5B tail with the T1 domain requires zinc-mediated T1 tetramerization (Xu et al., 2010). This

KIF5-T1 interaction is critical for the axonal transport of Kv3. These results suggest that the T1 domain, with its conserved zinc binding site, could be essential for the regulation of both channel activity and trafficking.

Fast spiking in some neurons require the unique channel properties of Kv3 channels, high activation threshold and rapid activation and deactivation kinetics (Rudy and McBain, 2001; Bean, 2007; Gu and Barry, 2011). In this study, we identified multiple zinc-binding sites in Kv3.1 channels, and further systematically analyzed the role of these sites in channel activity and localization using protein biochemistry and cell biology techniques.

RESULTS

Zinc reversibly reduces the firing frequency of cultured neurons.

Cerebellar granule cells express Kv3.1 and Kv3.3 channels (Weiser et al., 1994;

Grigg et al., 2000; Rudy and McBain, 2001). Yuanzheng Gu performed all electrophysiology experiments. Injecting square-pulse currents into soma induced many action potentials that were significantly and reversibly suppressed by 300 μM zinc (Fig.

5.1A). The inhibitory effect was first visible at 100 μM of zinc (Fig. 5.1D). Some neurons of cultured hippocampal neuron cultures generated high firing frequency due to

121 Kv3 channel presence (Gu et al., 2012) which were also reversibly inhibited by extracellular zinc (Fig. 5.1B). Since 100 μM zinc is unlike to have an inhibitory effect on voltage-gated sodium (Nav) channels (Nigro et al., 2011) we were curious if zinc’s reversible and inhibitory effect was mediated by inhibition of Kv3 channels. To test this

Yuanzheng applied zinc to Kv3.1b transfected young hippocampal neurons, and observed the same reversible and inhibitory effects as seen with the cerebellar and hippocampal neurons (Fig. 5.1C).

122

Figure 5.1. Extracellular application of zinc reversibly reduces spiking frequency of cultured neurons. (A) Action potential firing of a cultured cerebellar neuron at 14 DIV pipette. (B) Spiking frequency of a cultured hippocampal neuron at 21 DIV was reduced by 1 mM zinc. The scale bars are the same in (A-C). (C) Spiking frequency of an 8-DIV hippocampal neuron transfected with Kv3.1bHA and YFP was markedly reduced by 1 mM zinc. (D) Inhibitory effects on spiking frequency by different concentration of zinc. The "n" numbers are indicated with parentheses. (All recordings performed by Yuanzheng Gu.)

123 A novel metal-binding site in the conserved C-terminal region of Kv3.1

When purifying various tagged proteins we discovered that GST-tagged Kv3.1a

C-terminus (GST-31aC) could be purified with Co2+ beads. These beads are usually used to purify 6xHis-tagged proteins. This result suggested that there is an additional divalent heavy metal binding site(s) in the Kv3.1a C-terminus (Fig. 5.2A). We made a series of

GST-fusion proteins with different fragments of Kv3.1a C-terminal domain to examine their binding to Co2+ beads (Fig. 5.2A).

I discovered both GST-31a and GST-31b C-termini were pulled down by Co2+ beads, while GST and GST-31sC (the C-terminal splice domain of Kv3.1b which is 75 residues) were not (Fig. 5.2B, C). Surprisingly GST-31T1, the N-terminal T1 domain which contains a known zinc binding site, was not pulled down by Co2+ beads (Fig. 5.2B,

C). This may be due to the zinc binding sites are buried within the T1 tetramer, which requires proper assembly of four T1 domains.

Looking more closely at the 31aC sequence we found three H residues that are possible divalent heavy metal binding sites (Fig. 5.2D-G). The first H (H459) was found within the axon targeting motif (ATM) (Xu et al., 2007). A small N-terminal fragment of

Kv3.1 C-terminus, GST-442-469, but not GST-442-452, was pulled down by Co2+ beads

(Fig. 5.2D, E). Mutating the H459 to A eliminated GST-449-469 pulldown by Co2+ beads (Fig. 5.2F, G). This was surprising because this small region has a net charge of

+8, with only one H residue and no acidic residues. This region may form an oligomer upon binding of Co2+ or Zn2+, or negatively charged lipids might mediate this binding.

This study demonstrated both N and C termini of Kv3.1 channel contains zinc binding sites.

124 The second and third H residues (H480H481) are located in a region immediately downstream of the ATM. GST-468-489 and GST-468-511, but not GST-468-489HH or

2+ GST-468-511HH with the two Hs mutated to As, were pulled down by Co beads (Fig.

5.2D-G). Therefore it appears there are two zinc binding sites within the Kv3.1a C- terminal.

125

Figure 5.2. A novel binding site for divalent heavy metal ions in the Kv3.1 C- terminus. (A) Structural diagram of Kv3.1a and Kv3.1b. The residues of Kv3.1a cytoplasmic domain are shown (31aC (blue), aa 442-511 of Kv3.1a; 31bC (blue + green), aa 442-585 of Kv3.1b; 31sC (green), aa 500-585 of Kv3.1b; 31T1 (red), the N-terminal T1 domain aa 1-186). The ATM is highlighted in yellow. The numbers underneath the sequence indicate the residue numbers. Asterisks indicate the three His residues. “Δ” shows the end of the sequence. Black thick line indicates the region that does not bind to Co2+ beads. Blue thick lines indicate the regions that bind to Co2+ beads.; (B) The whole C-termini of Kv3.1a and Kv3.1b, but not the splice domain or the N-terminal T1 domain, bind to Co2+ beads. Pulldown proteins (upper) and inputs (lower) are revealed by Colloidal blue staining. (C) The Western blots of (B). 10% of loading in (B) was used in Western blotting. (D) The fragments in Kv3.1a C-terminus, containing at least one H residue, bind to Co2+ beads. (E) Western blots of (D). (F) Mutating the H residues in three different fragments eliminated their binding to Co2+ beads. (G) Western blots of (F). Each pulldown experiment was performed at least three times with consistent results. (All experiments performed by Joshua Barry).

126 My mapping experiments uncovered three critical H residues, H459, H480 and

H481, in binding to Co2+ beads (Fig. 5.2). Mutating any of them reduced, but not eliminated, the binding of GST-31aC to Co2+ beads (Fig. 5.3A). To examine that it is the

H residues that bind to zinc, and not GST itself, I fused 31aC to the yellow fluorescent protein (YFP) C-terminus (YFP-31aC). I used Quikchange to generate the following three 31aC mutants (YFP-31aCH, YFP-31aCHH, YFP-31aCHHH) (Fig. 5.3B). I then used zinc beads to pulldown supernatants of HEK293 transfected with YFP-31aC, YFP-

31aCH, YFP-31aCHH or YFP-31aCHHH. Zinc beads pulled down YFP-31aC, while YFP-

31aCH and YFP-31aCHH was less efficient, and YFP and YFP-31aCHHH was not pulled down at all (Fig. 5.3B). These results confirmed the discovery of two zinc binding sites in the C-terminal region of Kv3.1 channel, one within the ATM and the other downstream of the ATM.

127

Figure 5.3. Three H residues in Kv3.1a C-terminus are required for binding to Co2+ or Zn2+. (A) Mutating H residues reduced the binding of GST-31aC to Co2+ beads. Structural diagram of GST-31aC and positions of three Hs are on the upper left. Co2+ beads pulled down GST-31aC and mutants carrying single or double H mutations, but not GST and GST-31aCHHH (all three H residues mutated to A). Mutating one or two H residues reduced the pulldown. Pulldown results of Colloidal blue staining (upper) and Western blotting with an anti-GST antibody (lower, only 10% in loading) are shown on the top, with inputs at the bottom. (B) Mutating H residues also reduces the binding of YFP-31aC to Zn2+ beads. 31aC (wildtype or mutants) was fused to YFP. The constructs were transfected into HEK293 cells. The supernatants of cell lysates were pulled down with Zn2+ beads. Molecular weights are indicated on the left in KDa. Each pulldown experiment was performed at least three times with consistent results. (All experiments performed by Joshua Barry.)

128 H459 is required for axonal targeting and rapid activation of Kv3.1b channels

Kv3.1aHA and Kv3.1bHA are localized to dendritic and axonal membranes respectively, where the C-terminal ATM plays a critical role (Xu et al., 2007). Since

H459 is located within the ATM domain I wanted to examine whether the three H residues affect the channel axonal targeting. Single mutant Kv3.1bHAH459A and triple mutant Kv3.1bHAHHH, but not double mutant Kv3.1bHAHHAA, reduced axonal level of

Kv3.1bHA (Fig. 5.4). Earlier results also showed that mutating H459 may contribute to eliminating axonal targeting of CD4, but not binding to T1 or ankyrin G. Therefore the new zinc binding site in Kv3.1 C-terminus is likely involved in axonal targeting by regulating the N-/C-terminal interaction and/or Kv3.1/ankyrin G interaction.

Yuanzheng performed whole-cell voltage-clamp recording on HEK293 cells transfected with Kv3.1bHA or the three mutants to examine their biophysical properties and zinc sensitivity. Mutating H459 increased the activation time constant and caused a shift of the G-V curve to the right (data not shown). However, all three mutants were still sensitive to zinc inhibition (data not shown).

129

Figure 5.4. H459, but not H480 and H481, in the C-terminal domain, is required in Kv3.1b axonal targeting. (A) Distribution patterns of Kv3.1bHA and three point mutants in neurons under the permeabilized condition. Anti-HA staining (left) is in dark green and anti-MAP2 staining (right) in red. Signals are inverted. (B) Distribution patterns of Kv3.1bHA and three point mutants in neurons under the non-permeabilized condition. Arrows, axons; Arrowheads, dendrites. Scale bars, 100 μm. (C) Summary of the staining results. One-way analysis of variance followed by Dunn test, **, p < 0.01. (All experiments performed by Joshua Barry).

130

The junction of the 1st TM segment and the 1st extracellular loop in Zn2+ sensing

Since zinc’s inhibitory effect was quite fast, generally within seconds, we hypothesized the binding site may be on the external side or in the TM portion of the channel. Yuanzheng Gu performed a mutagenesis screen of six H and C residues in the extracellular loops or TM segments of Kv3.1. I created the mutant constructs and

Yuanzheng expressed them in HEK293 cells for voltage-clamp recordings (Fig. 5.5), where all were able to conduct current. The only mutant that had significant resistance to

1mM zinc was C208A.

Figure 5.5. Mutagenesis studies in searching for potential zinc-binding sites in the membrane portion of Kv3.1b. Diagram of the membrane portion Kv3.1. All six C and H residues in the transmembrane segments and extracellular loops are indicated with red asterisks with residue numbers labeled.

Construction of a Kv3-expressing and fast spiking neuron with relative resistance to the Zn2+ inhibition

Both biophysical properties and axonal localization are important for Kv3 mediated fast spiking in neurons (Gu et al., 2012). I further examined the axon-dendrite

131 targeting of Kv3.1bHAC208A on the neuronal surface. Kv3.1bHAC208A was restricted to somatodendritic membranes (Fig. 5.6A) with some puncta on the cell surface (Fig. 5.6A,

B). When coexpressed with KIF5-YFP, Kv3.1bHAC208A localized to axonal membranes

(Fig. 5.6B-D).

Expression of Kv3.1bHA can convert a slow to a fast spiking young hippocampal neuron (Fig. 5.6E) (Gu et al., 2012). When Kv3.1bHAC208A is coexpressed with KIF5B-

YFP in cultured hippocampal neurons it increased the firing rate of transfected neurons

(Fig. 5.6F). Yuanzheng attempted to use zinc concentration as close to its in vivo physiological or pathological concentration as possible. At 50 μM zinc WT Kv3.1bHA had a higher inhibition than Kv3.1bHAC208A (Fig. 5.6E-G). Therefore in this study we successfully recreated a fast-spiking neuron with significant resistance to zinc inhibition.

132

Figure 5.6. Constructing a fast-spiking neuron with relative resistance to the zinc inhibition (A) Kv3.1bHAC208A was restricted on somatodendritic membranes in cultured hippocampal neurons revealed by double staining of anti-HA (green) under the non-permeabilized condition and anti-MAP2 (red). (B) Kv3.1bHAC208A enriched on somatodendritic membranes. Neurons at 5 DIV were co-transfected with Kv3.1bHAC208A (red) and YFP (green), fixed two days later, and stained with an anti- HA antibody under non-permeabilized condition. (C) Kv3.1bAHAC208A localized to axonal membranes in the presence of KIF5B-YFP. Scale bars, 100 μm. (D) Summary of axonal targeting of Kv3.1bHAC208A in the absence or the presence of KIF5B-YFP. 50 μM zinc significantly reduced the action potential firing rate of the neuron expressing Kv3.1bHA (E), but not the neuron expressing Kv3.1bHAC208A and KIF5B-YFP (F). (G) Summary of the effect of zinc on spiking frequency. The "n" numbers are indicated within the bars. (Immunostaining and quantification in A to D performed by Joshua Barry. Recordings and quantification in E through G performed by Yuanzheng Gu.)

133

Figure 5.6. Constructing a fast-spiking neuron with relative resistance to the zinc inhibition

134 DISCUSSION

Using protein biochemistry, mutagenesis, cell biology and electrophysiology we analyzed the underlying mechanism of zinc-mediated regulation of Kv3.1 channels. We identified multiple zinc binding sites involved in channel assembly, biophysical properties and localization (Fig. 5.7). The T1 domain zinc binding site is required for channel tetramerization and also biophysical properties and localization. The new site identified within the C-terminus is important for channel activity and axonal targeting.

Zinc inhibition is mainly mediated through a site between the 1st transmembrane domain and 1st extracellular loop. This study has revealed novel mechanistic insights into the multifaceted regulation of Kv3 channel activity and localization by zinc (Fig. 5.7).

We accidentally discovered a new divalent metal binding site in the C-terminus of

Kv3.1. We first found Co2+ beads pulled down GST-31aC expressed in bacteria. We then found GST-31bC but not GST-31sC (the splice domain of Kv3.1b) was pulled down by Co2+ beads, this in interesting because 31sC contains 1 histidine and 4 cysteine residues (Fig 5.2). Therefore, there must be a binding site within the conserved region in the C-termini of Kv3.1a and Kv3.1b. Finally, we identified three histidine residues, one in the ATM (H459) and the other two downstream of the ATM (H480H481). All three were required for pulldown by Zn2+ beads.

The H459 residue is conserved within the Kv3 subfamily (Kv3.1 to Kv3.4), while

H480H481 are not. The three residues appear to play different roles in regulating activity and localization of Kv3.1. Mutating H459 altered the channels’ activation kinetics and voltage-conductance relationship, along with decreasing the axonal level of the channel

135 proteins. Mutating the other two histidines did not affect either channel activity or axonal targeting (Fig 5.4 and data not shown).

Figure 5.7. Distinct binding sites for divalent heavy metal ions in the tetrameric Kv3.1 channel complex. (A) Structural diagram of Kv3.1b subunit. All the potential zinc-binding sites are indicated with red asterisks and the residue numbers are labeled. (B) Diagram of a tetrameric Kv3 channel complex regulated by extracellular and intracellular divalent heavy metal ions.

136 Since Kv3.1 C-terminus binds to the N-terminal T1 domain in a zinc-dependent manner (Xu et al., 2007), the C-terminal zinc-binding sites are most likely involved in this interaction. The C-terminus of Kv3.1 is most likely quite flexible, so it could undergo a conformational change before or after zinc binding, or during its interaction with the T1 domain. This is an interesting question that remains to be determined in future experiments. Despite these novel findings, exactly how Kv3 function is regulated by the extracellular and intracellular zinc signaling is still unknown. Neuronal activity or pathogenic processes might affect zinc homeostasis and therefore might affect intracellular trafficking and activity of Kv3 channels, which are another set of topics for future study.

The Kv3.1 C-terminus but not its T1 domain was pulled down by Co2+ beads (Fig.

5.2). This may be due to the fact that the metal binding sites of the T1 domain are buried within the T1 tetramers. The Zn2+-binding site in the T1 tetramer is composed of residues from two T1 monomers. A single T1 monomer alone cannot bind zinc. In the pulldown assay GST-31T1 already formed tetramers (Xu et al., 2010). Unlike the T1 domain, the C-terminal site may bind to other divalent heavy metal ions besides Zn2+ and

Co2+. Mutating either the N- or C-termini failed to reduce zinc inhibition, which suggested other sites may be involved.

The rapid inhibitory effect of zinc suggested the site might be on the extracellular surface of the channel complex. When performing the mutation screen we were surprised to find the zinc-binding site between the 1st transmembrane segment and 1st extracellular loop was the site that ameliorated the zinc inhibition, specifically the C208A mutant. An episodic ataxia type-1 mutation was recently identified in the C-terminus of the 1st

137 transmembrane segment (F184C) of human Kv1.1 channel, which is associated with a higher sensitivity to zinc (Cusimano et al 2004, Imbrici et al 2007). This residue is in a similar position to C208 in Kv3.1b, which suggests this junction between the 1st transmembrane segment and 1st extracellular loop could transfer a conformational change to the channel core to alter its biophysical properties.

Mutating C208 to A decreased its axonal targeting (Fig 5.6). Exactly how this mutant affects the channel polarized targeting remains an interesting question for further study due to its location within the 1st transmembrane segment. The axonal targeting of the mutant can be rescued by co-expressing KIF5B-YFP, which is just like Kv3.1aHA

(Xu et al., 2010), which suggests the T1 tetramerization is not abolished in the mutant.

In hippocampal neurons, zinc released from presynaptic vesicles can cause synaptic concentrations to reach 10-30 μM (Frederickson et al., 2005; Mathie et al.,

2006). It can reach even higher concentrations under pathological conditions like epilepsy, ischemic injury and Alzheimer’s disease (Weiss et al., 2000; Galasso and Dyck,

2007; Hershfinkel et al., 2009; Medvedeva et al., 2009; Pithadia and Lim, 2012). Kv3 regulation may still be important under certain pathological or physiological conditions.

Zinc released at excitatory synapses may regulate Kv3 channels located on presynaptic and postsynaptic membranes, to feedback-regulated neurotransmitter release of Kv3- expressing fast-spiking neurons.

Our research contributes to understand how the input-output relationship of neurons modifies neurophysiological functions under normal and abnormal conditions.

We developed sensitive protein biochemistry, cell biology and electrophysiology assays to analyze different aspects of Kv3 channel trafficking and activity, which can provide

138 mechanistic insights into the activity dependent regulation of Kv3 channels and their function in various central nervous system disorders.

METHODS cDNA constructs

Kv3.1aHA, Kv3.1bHA, Kv3.1bHAH77A, KIF5B-YFP, GST-31aC, GST-31bC,

GST-31sC, and GST-31T1 were previously described . GST-442-452, GST-442-469,

GST-442-489, GST-468-489, and GST-468-511 were made by inserting cDNA fragments encoding different regions of Kv3.1a C-terminal domain into the pGEX4T-2 vector between SalI and NheI restriction enzyme sites. GST-442-469H, GST-468-489HH, and GST-468-511HH were made with Quickchange mutagenesis by mutating His residues to Ala. GST-31aCH, GST-31aCHH and GST-31aCHHH were made with Quickchange based on GST-31aC. YFP-31aC, YFP-31aCH, YFP-31aCHH and YFP-31aCHHH were generated by inserting Kv3.1a C-terminus and its mutants into pEYFP-C1 vector. Kv3.1bHAH459A,

Kv3.1bHAHHAA and Kv3.1bHAHHH were made with Quickchange based on Kv3.1bHA.

Kv3.1bHAC208A, Kv3.1bHAH212A, Kv3.1bHAC252A, Kv3.1bHAH327A, Kv3.1bHAH381A,

Kv3.1bHAH383A, and Kv3.1bHAH381AH383A were made with Quickchange based on

Kv3.1bHA. All constructs were confirmed with sequencing.

Primary hippocampal and cerebellar neuron cultures

The E18 hippocampal neuron culture was prepared as previously described (see pg 44). For transient transfection, neurons in culture at 5-7 DIV were incubated in Opti-

MEM containing 0.8 μg of cDNA plasmid and 1.5 μl of Lipofectamine2000 (Invitrogen,

Carlsbad, CA, USA) for 20 min at 37°C. At least three independent transfections were

139 performed for each condition. The cerebellar neuron culture was made from cerebellum of rat pups at postnatal day 1 or 2, with the same procedure of the hippocampal neuron culture.

Current clamp recording from cultured neurons and local perfusion

Glass pipettes with tip diameter around 1 μm for patch clamp recording were pulled with Model P-1000 Flaming/Brown micropipette puller (Sutter Instrument,

Novato, CA, USA). Glass pipettes with diameter around 50 μm, made by adjusting the pulling parameters and filled with Hank’s buffer containing different concentrations of

ZnCl2, were used for perfusion of whole neurons. The procedure was described previously (Gu et al., 2012).

The internal solution and Hank’s buffer were previously described for recording of primary cultured neurons . Hank’s buffer: 150 mM NaCl, 4 mM KCl, 1.2 mM MgCl2,

10 mg/ml glucose, 1 mM CaCl2, 20 mM HEPES (pH 7.4). The internal solution of electrical pipettes: (in mM) 122 KMeSO4, 20 NaCl, 5 Mg-ATP, 0.3 GTP and 10 HEPES

(pH 7.2). The current clamp procedure was previous described .

Expression of fusion proteins and pull down by Co2+ and Zn2+ beads

Expression of GST fusion proteins was induced in BL21 E. coli cells with 1 mM

IPTG (Isopropyl β-D-1-thiogalactopyranoside) for 4 hr at 37°C. Bacterial pellets were solubilized with sonication in the IP buffer at 4°C, and centrifuged at 50,000 × g for 30 min at 4°C. The supernatants were incubated with Co2+ beads (Clontech Laboratories

Inc., Mountain view, CA) at 4°C for 3 hrs. After extensive washing, the beads coated with purified fusion proteins were eluted with the sample buffer containing 1 mM EDTA.

YFP fusion proteins were expressed in HEK293 cells. The supernatant of cell lysates was

140 further incubated with Zn2+ beads. The precipitated proteins were eluted with the sample buffer.

Western blotting and colloidal blue staining

The precipitants were resolved by SDS-PAGE, transferred to a PVDF membrane, and subjected to Western blotting with a mouse anti-GST antibody (1:1000 dilution).

Protein bands in the gels were stained with a colloidal blue staining kit (Invitrogen), using the procedure described by the manufacturer.

Immunostaining, imaging and quantification

The immunocytochemical procedures were previously described (Gu et al., 2006;

Xu et al., 2007). Briefly, neurons were stained under the non-permeabilized condition

(without Triton X100) to label the surface pool and under the permeabilized condition

(with 0.2% Triton X100) to label total proteins. Fluorescence images were captured with a Spot CCD camera RT slider (Diagnostic Instrument Inc., Sterling Heights, MI) in a

Zeiss upright microscope, Axiophot, using Plan Apo objectives 20/0.75 and 100/1.4 oil, saved as 16-bit TIFF files, and analyzed with NIH Image J and Sigmaplot 10.0 for fluorescence intensity quantification. The quantification procedure was also described previously (Xu et al., 2007; Xu et al., 2010).

141

CHAPTER 6

CONCLUSIONS

The body of work in my thesis has advanced the understanding of the function and underlying mechanisms of polarized targeting of membrane proteins, and how they affect neuronal function. A neuron’s ability to function as a signal propagator requires proper localization of many different membrane proteins. Membrane proteins perform many roles within a neuron’s life from axonal guidance to proper AP firing, and much research has been done examining their localization. But how and why they get to where they are going is still not completely understood.

NgCAM polarized targeting and bundling of axons or dendrites

A neuron’s ability to propagate electrical signals requires it to form connections with other neurons. When thinking of neuron-neuron connections, most people immediately think of the axon-dendrite interaction of the synapse. But axon-axon and dendrite-dendrite interactions also occur (Bastiani et al., 1984; Tosney and Landmesser,

1985; Escobar et al., 1986; Lin et al., 1994; Yu et al., 2000; Curtetti et al., 2002; Krieger et al., 2007; Hanson et al., 2008). This bundling is due to the function of CAMs, especially L1-CAM in mammals. The Ig domains are known to be required for the

142 heterophilic and homophilic interaction of the CAMs between neurons (Maness and

Schachner, 2007).

My work showed that a single CAM can regulate not only axonal but also dendritic bundling due to its polarized targeting to the axon or dendrite respectively. I showed that this targeting can be regulated by either deletion of the FN repeats or blocking activity of protein kinases. Overall my research showed that the polarized targeting of even a single CAM can affect the axonal and dendritic bundling of neurons.

However, this research must be taken with a grain of salt due to the fact that overexpression of a protein is not the same as studying said protein endogenously. In the case of L1-CAM overexpression of L1-CAM is five times higher than endogenous levels

(Dequidt et al., 2007), which was similar to what we observed with our mature hippocampal neuron culture.

Maximal spiking frequency regulated by Kv3 polarized targeting.

The polarized targeting of NgCAM can induce axonal or dendritic bundling which could, in turn, affect a neuron’s activity. But how are membrane proteins targeted to their proper subcellular compartment? My lab recently showed that the axon-dendrite targeting of Kv3.1 is due to an axonal targeting motif located within the C-terminal of both splice variants of Kv3.1 (Kv3.1a and Kv3.1b) (Xu et al., 2007). But what effect does this targeting have on neuronal activity?

To examine this Yuanzheng Gu, our postdoc, examined the biophysical properties of Kv3.1a and Kv3.1b, and found them identical, except Kv3.1a was less effective at enhancing the maximal spiking frequency of hippocampal neurons when transfected into

143 young hippocampal neurons. My deletion studies mapped the important region that is required for the polarized targeting of Kv3.1b between amino acids 513 and 530. We hypothesized that there might be a possible electrostatic repulsion between the C-terminal splice domain of negatively charge Kv3.1b and its negatively charged N-terminal to unmask the axonal targeting motif of Kv3.1b. This is due to the net charge of Kv3.1b being -5 whereas Kv3.1a has a net charge of +3 and the shared axonal targeting motif has a charge of +8. Therefore, Kv3.1b axonal targeting increased maximal spiking frequency. Computer simulations also confirmed that axonal targeting of Kv3 channels increased maximal spiking frequency (Gu et al., 2012).

Clustering and activation of KIF5 by Kv3

While proper targeting, and in some cases like Kv3.1b the polarized targeting, of the membrane proteins is a requirement for neuronal activity, the only way to get the proteins to their destination is through trafficking via motor proteins. My lab recently showed that Kv3 was transported as a tetramer by the classical kinesin-1/KIF5 motor (Xu et al., 2010), showing the motor affected the cargo. But KIF5 binds to many cargoes, could the cargoes themselves affect the function of their motor(s)? That was the question

I wanted to examine.

KIF5 was shown to form clusters along both axons and dendrites in developing neurons. Our biochemical assays showed that there was a high-affinity multimeric binding between KIF5B and the T1 domain of Kv3.1, which required three basic residues in the KIF5B tail (R892K893R894). Also, Kv3.1 T1 competed with microtubules and the motor domain of KIF5, but not with kinesin light chain, to bind to the KIF5B tail. Kv3.1

144 channels likely activate KIF5B motor activity via releasing the tail binding to the microtubules and the motor itself. A recent study showed that artificially clustered kinesin-1 can cause a cilia-like beating of active microtubule bundles (Sanchez et al.,

2011). Therefore, clustered KIF5 motors can alter the behavior of microtubules and possibly indirectly affect trafficking of other proteins. This raises an interesting possibility, that Kv3 channels may regulate axonal transport of other cargos by clustering

KIF5, which in an interesting area for further study.

Zinc’s role in Kv3 activity and trafficking

Zinc is required for the proper tetramerization of Kv 2-4 family T1 domains, which is necessary to form the tetramerized Kv channel itself (Li et al., 1992; Xu et al.,

1995; Bixby et al., 1999; Jahng et al., 2002). We first discovered that local perfusion of zinc reversibly reduced spiking frequency, most likely due to suppressing Kv3 channels.

We eliminated any affect the N-terminal zinc binding sites had on this inhibition due to the fact that the zinc binding site is buried within the T1 tetramer. However, our research showed that there was a novel heavy metal binding site in the C-terminal of Kv3.1.

When purifying GST fusion proteins we discovered that the C-terminal of Kv3.1 bound to Co2+ beads. This led us to explore the exact function of this metal binding site, and to

2+ determine if there were others besides the primary one know to be in the T1 Zn binding site. My work with the C-terminal showed that there were two metal binding sites within the C-terminal of Kv3.1a. Mutagenesis of H residues within these regions and within the full channel showed that one of these new binding sites, H459 which is located within the axonal targeting motif, was critical for both channel activity and axonal targeting.

145 We then looked closer at the N-terminal and discovered C208 was important not only for the axon-dendrite targeting of Kv3.1b(when C208 was mutated to A the axonal targeting of Kv3.1b was lost) it was also important to alleviate the zinc inhibition seen when extracellular zinc was added to Kv3.1bHA expressing neurons. When

Kv3.1bHAC208A was expressed in neurons it was primarily targeted to the somatodendritic region just like Kv3.1aHA. Upon co-expression with KIF5B-YFP

Kv3.1bHAC208A was able to be trafficked to the axon where they were relatively resistant to the inhibition caused by zinc, compared with wild type Kv3.1bHA.

Future Directions

My thesis shows that polarized targeting of membrane proteins can be caused by different mechanisms, whether it is electrostatic repulsion or zinc binding. This polarized targeting is required for proper neuronal activity and function. But there is still much to be discovered about how this polarized targeting occurs and what regulates it. For example, how membrane proteins are trafficked anterogradely by kinesin is being studied heavily, whereas, very little is known about how retrograde transport of membrane proteins by dynein occurs, or if membrane proteins are transported by myosin. Also, what types of regulation are occurring between the motor and its cargo? Is there activity dependent regulation possibly through phosphorylation or dephosphorylation events?

The results from my experiments above may act as a starting point to determine if similar or different mechanisms are required for proper localization of other membrane proteins.

146

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