The Metabotropic Glutamate Receptor Mglur1 Regulates the Voltage-Gated Potassium Channel Kv1.2 Through Agonist-Dependent and Agonist-Independent Mechanisms

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Graduate College Dissertations and Theses Dissertations and Theses

2019 The etM abotropic glutamate receptor mGluR1 regulates the voltage-gated potassium channel Kv1.2 through agonist-dependent and agonist- independent mechanisms Sharath Chandra Madasu University of Vermont

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Recommended Citation Madasu, Sharath Chandra, "The eM tabotropic glutamate receptor mGluR1 regulates the voltage-gated potassium channel Kv1.2 through agonist-dependent and agonist-independent mechanisms" (2019). Graduate College Dissertations and Theses. 982. https://scholarworks.uvm.edu/graddis/982

This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. THE METABOTROPIC GLUTAMATE RECEPTOR MGLUR1 REGULATES THE VOLTAGE-GATED POTASSIUM CHANNEL KV1.2 THROUGH AGONIST-DEPENDENT AND AGONIST-INDEPENDENT MECHANISMS.

A Dissertation Presented

Sharath Chandra Madasu

The Faculty of the Graduate College

The University of Vermont

In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Cellular Molecular and Biomedical Science

January, 2019

Defense Date: September 27, 2018 Dissertation Examination Committee:

Anthony D. Morielli, PhD., Advisor John Green, PhD., Chairperson Karen Lounsbury, Ph.D. Benedek Erdos, PhD. Cynthia J. Forehand, Ph.D., Dean of the Graduate College

ABSTRACT

The voltage gated potassium channel Kv1.2 plays a key role in the central nervous system and mutations in Kv1.2 cause neurological disorders such as epilepsies and ataxias. In the cerebellum, regulation of Kv1.2 is coupled to learning and memory. We have previously shown that blocking Kv1.2 by infusing its specific inhibitor tityustoxin-kα (TsTX) into the lobulus simplex of the cerebellum facilitates eyeblink conditioning (EBC) and that EBC itself modulates Kv1.2 surface expression in cerebellar interneurons. The metabotropic glutamate receptor mGluR1 is required for EBC although the molecular mechanisms are not fully understood. Here we show that infusion of the mGluR1 agonist (S)-3,5-dihydroxyphenylglycine (DHPG) into the lobulus simplex of the cerebellum mimics the facilitating effect of TsTX on EBC. We therefore hypothesize that mGluR1 could act, in part, through suppression of Kv1.2. Earlier studies have shown that Kv1.2 suppression involves channel tyrosine phosphorylation and endocytocytic removal from the cell surface. In this study we report that an excitatory chemical stimulus (50mM K+- 100µM glutamate) applied to cerebellar slices enhanced Kv1.2 tyrosine phosphorylation and that this increase was lessened in the presence of the mGluR1 inhibitor YM298198. More direct evidence for mGluR1 modulation of Kv1.2 comes from our finding that selective activation of mGluR1 with DHPG reduced the amount of surface Kv1.2 detected by cell surface biotinylation in cerebellar slices. To determine the molecular pathways involved we used an unbiased mass spectrometry-based proteomics approach to identify Kv1.2-protein interactions that are modulated by mGluR1. Among the interactions enhanced by DHPG were those with PKC-γ, CaMKII, and Gq/G11, each of which had been shown in other studies to co-immunoprecipitate with mGluR1 and contribute to its signaling. Of particular note was the interaction between Kv1.2 and PKC-γ since in HEK cells and hippocampal neurons Kv1.2 endocytosis is elicited by PKC activation. We found that activation of PKCs with PMA reduced surface Kv1.2, while the PKC inhibitor Go6983 attenuated the reduction in surface Kv1.2 levels elicited by DHPG and PMA, suggesting that the mechanism by which mGluR1 modulates cerebellar Kv1.2 likely involves PKC. mGluR1 has been shown to signal independently of the agonist through a constitutively active, protein kinase A-dependent pathway in the cerebellum. Using HEK293 cells we show that co-expression of mGluR1 increases the surface expression levels of Kv1.2. This effect occurs in absence of mGluR1 agonists and in the presence of a noncompetitive mGluR1 inhibitor YM298198. Co-expression of known downstream effectors of the agonist driven mGluR1 pathway such as PKC-γ, CaMKIIα, Grid2 had no effect on Kv1.2 surface expression or on the ability of mGluR1 agonist to modulate that expression. In contrast, the inverse agonist BAY 36-7620 significantly reduced the mGluR1 effect on Kv1.2 surface expression, as did pharmacological inhibition of PKA with KT5720. Therefore, mGluR1 is involved in regulation of surface Kv1.2 via dual mechanisms, the agonist dependent mechanism reduces surface Kv1.2 via PKC, while agonist independent constitutive mechanism increases surface Kv1.2 via PKA.

ACKNOWLEDGEMENTS

I would like to dedicate this dissertation to my parents, Dr. Ram Murthy and

Sandhya Rani Madasu. They have always been unconditionally supportive of me and my endeavors and their love and kindness kept me going all throughout my life. They have always reminded me that I was never alone and always had my back providing me encouragement, comfort especially when experiments turnout to be not what I expected. I would also like to thank my uncle and aunt Ravinder and

Jyothi Eeraveni who like my parents they have taken care of me here in my adopted home of United States. My wife Priyanka deserves the credit for putting up with me especially when experiments did not pan out, when I missed all the movie nights and anniversaries because I was doing one last experiment or analyzing last set of data all the time. I would like to thank all my family members (it’s a long list) for all their love and support.

My friends at the Cellular Molecular and Biomedical Sciences have contributed a lot to my life and success here at UVM. All the fun BBQs, night outs and trips we had really made me feel at home. This work would have not been fun without my lab mates in crime Dr. Eugene Cilento who taught me the basics of mass spectrometry, flow cytometry and imaging. We have worked on the early experiments for co-immunoprecipitation that lead to discovery of the Kv1.2 interactome this thesis is mostly based on. Most importantly for keeping the work environment fun. Dr. Kutibh Chihabi for his fun and positive attitude, he taught me the flow cytometry which is all I have used in chapter 3.

I would like to thank Department of Pharmacology which has been my home since

I joined UVM. I would like to thank Dr. Mark Nelson, the Chair of the

Pharmacology department for supporting my graduate education and for being an inspiration to many budding scientists as myself.

My dissertation research would be incomplete without the support of COBRE facilities and the faculty. In particular I would like to thank Dr. Ying- Wai Lam, from the mass spectrometry core. He has taken time to teach me the intricacies of quantitative mass spectrometry and has worked with me on the proteomics part of the projects and added plethora of valuable data which this dissertation builds on.

All of this would have not been possible if it was not for Dr. Anthony Morielli, my advisor and mentor. I still remember his talk at the Faculty presentations during the student orientation. His energy filled story about how he during his post-doctoral studies stumbled into Kv1.2 is what interested me to this line of research. Tony dedicates a lot of time to wards scientific progress of his students and makes sure they have adequate support to succeed, he has been a pillar of support for me during my PhD life at UVM. Most of all he has been a terrific mentor and friend. Thank you, Tony.

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TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS………………………………………………………...... ii

LIST OF TABLES…………………………………………………………………….. x

LIST OF FIGURES…………………………………………………………………… xi

CHAPTER 1 COMPREHENSIVE LITRATURE REVIEW…………………………. 1

1.1 Overview.………………………………………………………………………….. 1

1.2 Potassium channel structure and function…….…………………………………... 4

1.3 The Potassium channel Kv1.2……….……………………………………………. 8

1.4 Kv Channel composition in the brain…….……………………………………….. 8

1.5 Electrophysiological properties of Kv1family channels.………………………….. 10

1.6 Physiological role of Kv1.2……………………………………………………….. 11

1.7 Protein sequence and composition affects the surface expression of heteromeric Kv channels…………………………………………………………………………… 14

1.8 Glycosylation regulates stability and surface expression of Kv1.2 channels……... 15

1.9 Posttranslational Modifications (PTMs) affect endocytic trafficking of Kv1.2…... 16

1.10 Secretin receptors regulates surface Kv1.2 via PKA in the cerebellum…………. 19

1.11 Metabotropic glutamate receptors mGluR1……………………………………… 19

1.12 mGluR1 signaling………………………………………………………………... 21

1.13 mGluR1 is constitutively active via intracellular loops 2, 3 and its interactions with Homer……………………………………………………………………………. 21

1.14 Identification of Metabotropic Glutamate receptor – 1 (mGluR1) its role in LTD and Cerebellar learning a brief history………………………………………………... 22

1.15 mGluR1 in cerebellar learning………………………………………………...... 25

1.16 Regulation of potassium channels via mGluR1 in the brain…………………….. 26

1.17 mGluR1 regulates potassium channels in the cerebellum and affects intrinsic excitability…………………………………………………………………………….. 28

References for Chapter 1.……………………………………………………………... 30

Chapter 2: mGluR1 regulates EBC and Kv1.2 potassium channels in the cerebellum 48

2.1 Abstract……………………………………………………………………………. 49

2.1 Introduction………………………………………………………………………... 50

2.3 Materials & Methods……………………………………………………………… 52

Antibodies……………………………………………………………………………... 52

Chemicals...…………………………………………………………………………… 52

Slice preparation………………………………………………………………………. 52

Drug treatment………………………………………………………………………… 53

Biotinylation…………………………………………………………………………... 54

Inclusion criteria………………………………………………………………………. 54

Western Blots………………………………………………………………………….. 54

Membrane fractionation and Co immunoprecipitation………………………………... 55

Mass Spectrometry……………………………………………………………………. 56

Trypsin digestion and peptide labeling by tandem mass tag (TMT)………………... 56

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) ………………… 57

Data analysis………………………………………………………………………… 58

Immunofluorescence…………………………………………………………………... 59

Eyeblink conditioning…………………………………………………………………. 60

Subjects..…………………………………………………………………………….. 60

Surgery………………………………………………………………………………. 60

Apparatus……………………………………………………………………………. 61

Procedure……………………………………………………………………………. 62

Behavior Analysis…………………………………………………………………… 62

2.4 Results……………………………………………………………………………... 63

2.4.1 DHPG infusion into the cerebellar cortex facilitates EBC……………………… 63

2.4.2 mGluR1 activation and increases Tyrosine phosphorylation on Kv1.2………… 64

2.4.3 mGluR1 activation reduces surface Kv1.2……………………………………… 65

2.4.4Kv1.2 interacts with components of mGluR1 signaling pathway……………….. 66

2.4.5 Kv1.2, PKC-γ and mGluR1 are expressed in the molecular layer of the cerebellum……………………………………………………………………………... 67

2.4.6 Broad spectrum PKC inhibitor attenuates mGluR1 mediated loss of surface Kv1.2………………………………………………………………………………….. 67

2.5 Discussion…………………………………………………………………………. 68

Acknowledgments…………………………………………………………………….. 72

Author contributions ………………………………………………………………….. 72

Conflicts of Interest…………………………………………………………………… 72

Figures for Chapter 2.………………………………………………………………… 73

References for Chapter 2.…………………………………………………………… 80

CHAPTER 3: CONSTITUTIVELY ACTIVE MGLUR1 INCREASES SURFACE EXPRESSION OF KV1.2 VIA PKA SIGNALING PATHWAY……………………. 86

3.1 Abstract……………………………………………………………………………. 86

3.2 Introduction………………………………………………………………………... 87

3.3 Methods…………………………………………………………………………… 89

Materials…………………………………………………………………………….. 89

Cell culture and transfection………………………………………………………… 89

Flow cytometry……………………………………………………………………… 90

Western Blots…...…………………………………………………………………… 90

Immunofluorescence Microscopy…………………………………………………... 91

3.3 Results……………………………………………………………………………... 91

3.4.1 Expression of functional mGluR1 receptors in HEK293 cells………………….. 91

3.4.2 mGluR1 agonist DHPG activates calcium signaling and increases ERK phosphorylation……………………………………………………………………….. 91

3.4.3 Detection of Surface Kv1.2 by Flow Cytometry………………………………... 92

3.4.4 mGluR1activation by agonist does not affect Kv1.2 surface expression….……. 93

3.4.5 Over expression of PKC-γ, Grid2, CaMKIIα does not affect surface expression of Kv1.2……………………………………………………………………………….. 93

3.4.6 Modulation of Kv1.2 via constitutively active mGluR1………………………... 94

3.4.7 mGluR1 increases surface Kv1.2 via PKA mediated mechanism and not via PKC……………………………………………………………………………………. 94

3.5 Discussion…………………………………………………………………………. 95

Figures for Chapter 3………………………………………………………………….. 99

References for Chapter3………………………………………………………………. 105

CHAPTER 4: Discussion……………………………………………………………... 109

4.1. Overview …………………………………………………………………………. 109

vii

4.2. mGluR1 activation improves EBC and reduces surface Kv1.2 Channels in cerebellum……………………………………………………………………………... 110

4.3 Potential mGluR1 regulation of Kv1.2 in other parts of the brain and its effects on learning………………………………………………………………………………... 112

4.4 Kv1.2 protein interactions………………………………………………………… 113

4.5 Grid2, PKC-γ, CaMKII and the regulation of Kv1.2 ….…………………………. 115

4.6 mGluR1 mediates PKA dependent increase in surface Kv1.2……………………. 118

4.7 Conclusion: Kv1.2 interactors potential targets for Ataxia and role for Kv1.2 in Alzheimer’s, Epilepsy and Chronic pain……………………………………………… 119

References for Chapter 4……………………………………………………………… 121

Comprehensive Bibliography ………………………………………………………… 127

Appendix :TMT MS Table for Chapter 2……………………………………………... 140

viii

LIST OF TABLES

Table 1...………………………………………………………………………………… 11

TMT MS Table for Chapter 2…………………………………………………………. 140

LIST OF FIGURES

Figure 1.1 Classification of potassium channels based on mode of activation and of transmembrane segments……………………………………………………………… 4

Figure1.2 Phylogenetic tree of Potassium channels…………………………………... 7

Figure 1.4 General Schematic representation of cerebellar circuit showing major cells involved in EBC and LTD…………………………………………………………….. 25

Figure 2.1 Eye blink conditioning Experiments………………………………………. 73

Figure 2.2: Identification of Kv1.2 Tyrosine Phosphorylation………………………... 74

Figure 2.3 Kv1.2 Endocytosis in the cerebellum…………………………………….... 75

Figure 2.4: Quantitative Mass spectrometry…………………………………………... 76

Figure 2.5 TMT Experiment - western blot validation………………………………... 77

Figure 2.6: Immunofluorescence images of Rat Cerebellum…………………………. 78

Figure 2.7. PKC dependent Kv1.2 Endocytosis in the cerebellum…………………… 79

Figure 3.1: Expression of mGluR1 in HEK cells……………………………………... 99

Figure 3.2: Functional expression of mGluR1 in HEK cells………………………...... 100

Figure 3.3. Gating strategy for estimating surface Kv1.2 via flow cytometry………... 101

Figure 3.4. mGluR1 increases surface expression of Kv1.2…………………………... 102

Figure 3.5. mGluR1 increases surface expression of Kv1.2 independent of Agonist… 103

Figure 3.6. Constitutively active mGluR1 increases surface expression of Kv1.2 via PKA...…………………………………………………………………………………. 104

Chapter 1: Comprehensive literature Review

1.1 Overview

Among the earliest adaptations in the evolution of nervous systems is the capacity to adapt

to the external environment by encoding and storing over the long-term, changes in neuron

function. Understanding the cellular and molecular mechanisms of learning and memory

has been a major focus of modern neuroscience research. Several lines of study indicate

that changes in the electrical properties of neurons is one way by which learning and

memory can be encoded in the nervous system. Early studies that provided evidence that

changes in neuronal excitability are involved in a learned response date from the 1970’s,

for example Charles Woody et al demonstrated that classically conditioned cats showed long-term and persistent changes in the coronal – pericruciate cortex neurons. In

conditioned cats these cortical neurons had lower thresholds for generating an EMG response indicating that an increase in neuronal excitability is involved in the learning process [1-3]. During the same time Crow and Alkon showed similar long term changes in the mollusk Hermissenda crassicornis photo receptor cells and proposed that a persistent

depolarization in the type B photoreceptors cells was responsible for these cellular and

behavioral changes [4]. Later, their studies demonstrated a classical conditioning-induced

decrease in voltage dependent K+ and Ca2+ dependent K+ currents in the photoreceptors of

the mollusk [5, 6]. Similar associative learning and memory tasks and learned responses

were found to occur in mammalian cerebellum, in particular an associative learning process

called delay eyeblink conditioning in rabbits, rats, and mouse [7-12].

Cerebellar EBC is the only form of mammalian learning for which the complete brain circuit has been delineated, including stimulus input pathways, sites of plasticity, and

behavioral output pathways. The fear conditioning pathways involving the amygdala are

also very well-understood at the circuit level, but many details are still missing on the

aversive unconditioned stimulus pathway that drives synaptic plasticity. Part of the reason

for this is that cerebral circuits, for example in the hippocampus or amygdala, are more

complex than the relatively simple circuitry of the cerebellum. Correspondingly, cerebellar

learning itself involves fewer complex stimuli, typically a pure tone paired with localized

stimulation of the eye, and a relatively simple learned response, an eye blink to the tone in

anticipation of the eye stimulation. Thus, EBC readily lends itself to research on the

mechanisms of neural plasticity in learning because the neural links that connect

environmental stimuli to behavior are known and are relatively simple.

EBC, as well as other forms of cerebellar learning, is critically dependent on Group

1 metabotropic glutamate receptors (mGluR1/5) [10, 13]. A key hypothesized mechanism

by which these receptors influence cerebellar learning is through the induction of long- term depression (LTD) at parallel fiber-Purkinje cell (PF-PC) synapses [14-16]. However, mice with genetic modifications that eliminate LTD exhibit normal EBC [17, 18], arguing that LTD contributes to EBC but is not required for it. In contrast, mice lacking mGluR1α fail to undergo either LTD or EBC [10], indicating that although EBC does not require

LTD, it does require mGluR1 activity. Therefore, mGluR1 receptors may influence EBC, at least to a significant degree, through one or more LTD-independent mechanisms. There are currently no compelling reports in the literature proposing an alternative mechanism linking cerebellar synaptic plasticity to cerebellar learning. In this dissertation I address this gap. Previous studies show that blocking a voltage gated potassium channel Kv1.2 by direct infusion of its specific inhibitor Tityus toxin (TsTX) or via activation of secretin

receptors which cause endocytosis of Kv1.2 facilitates EBC [19], and that EBC itself can alter the expression of Kv1.2 at the cell surface [20]. In this dissertation I test the general hypothesis that mGluR1 may contribute to EBC not only by affecting AMPA receptor trafficking in LTD, but also by modulating Kv1.2. In Chapter 2 we show that direct stimulation of mGluR1 enhances EBC and activation of mGluR1 with an agonist

Dihydroxy phenyl glycine (DHPG) reduces the surface expression of Kv1.2. Using pharmacological inhibitor of proteins kinase C (PKC) Go6983 we show that mGluR1 acts via PKC to reduce surface Kv1.2. We used quantitative proteomics to identify the protein interactors of Kv1.2 that are involved in mGluR1 signaling pathway. We show that known protein interactors of mGluR1 such as PKC-γ, CaMKIIα, increase binding to Kv1.2 when mGluR1 is activated by DHPG in the cerebellum. In Chapter 3 we sought to study mGluR1 regulation of Kv1.2 in a model cell system (HEK293 cells) as a way of analyzing components in the signaling pathway in detail. We measured the surface expression of

Kv1.2 when mGluR1, PKC-γ, CaMKIIα, and Grid2 are over expressed in a HEK293 cell line stably expressing Kv1.2 and Kvβ2. We found that in HEK cells, mGluR1 increases surface expression of Kv1.2 in an agonist independent manner. Neither the mGluR1 specific agonist DHPG nor the natural agonist glutamate affects the increase and co- expression of the mGluR1 signaling proteins PKC-γ, CaMKIIα, or Grid2 had minimal impact on mGluR1 mediated increase. Only an inverse agonist of mGluR1 Bay 36-7620 and PKA inhibitor KT5720 reduced the mGluR1 mediated increase in surface Kv1.2.

These results suggest that mGluR1 is constitutively active and signals via PKA to increase surface Kv1.2. Overall, we have identified two molecular signaling mechanisms for Kv1.2

regulation via mGluR1. Our work provides alternative mechanisms that link Purkinje cell

plasticity with cerebellar learning via inhibition of Kv1.2.

1.2 Potassium channel structure and function

Potassium ion channels are membrane proteins which form a selectively permeable pore,

through which only K+ flows across the cell membrane along its electrochemical gradient.

Figure 1.1. Classification of potassium channels based on mode of activation and number of transmembrane segments. Adapted from Edward S. A. Humphries et al [21].

Potassium channels have central roles in maintaining and stabilizing the resting membrane

potential, action potential repolarization and in modulating neurotransmitter release at axon

terminals. The family of potassium ion channels are encoded by around 80 genes [21, 22].

The first potassium channel was cloned in Drosophila by Papazian [23] followed by

cloning from mouse brain in 1988 [24]. Since then several other potassium channels and

their subunits have been identified. Based on the number of transmembrane segments and 4

mode of activation they are classified into 4 classes (Figure 1.1). The following is a brief

summary:

Inward rectifying 2-transmembrane K-channels: These are the structurally simplest

form of potassium channels with two transmembrane domains with the pore forming loop

between transmembrane segments 1 and 2. Four such subunits form a functional ion

channel. The name “inward rectifying” refers to the fact that is these channels allow K+

ions to flow into the cells much more easily, than out of the cells. Thus these channels help

set the resting membrane potential close to equilibrium potential for K+, rather than for

example contributing to the repolarization phase of an action potential [21]. These include

Kir channels Kir 1.1 – Kir7.1 and sulphonyl urea receptors SUR1 and SUR2.

+ 2-pore 4-transmembrane K channels K2p: K2p channels have 4 transmembrane domains

with 2 pore forming loops and are referred to as tandem pore K+ channels. Two such

subunits form a dimer to form a functional potassium channel. Channels such as TWIK-1,

TREK-1-2, TASK-1-5, TRAAK, THIK- 1-2 encoded by KCNK genes 1-18 fall under the

K2p channel family. These are the leak or background potassium channels and are major

determinants of resting membrane potential. K2p channels such as TASK and TREK

respond and open to various physiological stimuli such as pH, temperature, hypoxia [25,

26].

Calcium activated K+ channels: Most of these channels have six transmembrane

segments, although some of these channels possess a seventh transmembrane domains

called S0 which is necessary for channel regulation via its interactions with β subunits.

Tetramers of these subunits form a functional channel. Family members include Slo1, Slo3,

BK channels, SKCA channels, IKCa1, Slack and Slick channels. Some of these channels,

for example BKCa, respond to both changes in membrane voltage and to changes in intracellular calcium levels. In contrast, SK/IKCa channels are voltage insensitive and are regulated by interactions with calmodulin a protein which functions as an intracellular calcium sensor [21].

Voltage gated potassium channels: These ion channel subunits have 6 transmembrane domains with pore forming s5-s6 linkers. These channels respond to changes in voltage of the cell membrane via the voltage sensor on S4 transmembrane domain. There are twelve known family members, Kv1 through Kv12 (Fig 1.2) [27]. Four alpha subunits from the same subfaimily combine to produce a complete channel. Kv channels are involved in regulating repolarization, action potiential propogation and frequency of the action potential as described below. For rest of the dissertation we will be focussing on Kv1 family of potassium channels and in particular we will focus Kv1.2 potassium channels.

Figure 1.2. Phylogenetic tree of Potassium channels. Adapted from Guide to pharmacology. Adapted from Gonzales et al (2012) [27]

1.3 The potassium channel Kv1.2

Kv1.2 is the alpha (α) subunit of voltage gated potassium channel of shaker class which

also includes Kv1.1, K1.3, 1.4, 1.5 etc. as shown the in phylogenetic tree (Fig1.2). The

alpha subunits have six transmembrane spanning regions and four such α subunits form

homo and/or heterotetramers with other Kv1 family α subunits. Different subunit

compositions have different pharmacological properties, function and expression as

discussed latter. The major Kv1α subunits in the brain are Kv1.1, Kv1.2, Kv1.4. These six transmembrane one pore K+ potassium channels are activated by depolarization of the

cell/neuron they are expressed in. Four pore-loop containing α subunits combine together and get arranged in a tetramer to form a functional ion channel. The s5-s6 contains the P- loop which lines the pore. The sequence motif G(Y/F) G forms the selectivity filter which allows only K+ ions through the channels. The S4 segment of these channels is usually lined by positively charged residues arranged in a regular spaced array of 5 to 7 positive charges. This allows the S4 segment to function as a voltage sensor and its re-arrangement

in response to the changes in membrane voltage allows the pore to open or close. It is

estimated that K+ channels allow 106 to 108 K+ ions to pass through the electrochemical

gradient per second when they are open [28].

1.4 Kv channel composition in the brain:

Analysis of crude extracts from the fore brain and synapoto-neurosomal preparations reveal

that the assembly of Kv1 channels is tightly regulated and may play key role in the diseases

associated with mutations of these subunits themselves. Kv1.2, Kv1.4, Kv1.6 and Kv1.1

are widely expressed throughout the body. Kv1.1 and Kv1.2 form the majority the ion

channels found in the brain. In the mammalian brain certain combinations are preferred

over others. For example, over 85% of the material bound to α-Dendrotoxin (α-DTX) a

specific toxin that binds to the extracellular pores of Kv channels containing Kv1.1, Kv1.2,

Kv1.6 α subunits was also precipitated by Kv1.2 antibody, followed by Kv1.1 (47%),

Kv1.6 (16%), Kv1.4 (8%) anti-bodies. Moreover, it was observed that in the brain most

Kv1.1 co-immunoprecipitated with Kv1.2 suggesting that Kv1.1 primarily associates with

Kv1.2 in the brain [29].

Kv1.1 has highest expression in the brain stem nuclei and white matter and lowest in the cerebellum and hippocampus. In the hippocampus Kv1.4 expression is highest followed by Kv1.2 (Kv1.4>Kv1.2 in hippocampus). Kv1.6 is expressed in hippocampus and cerebellum. In the hippocampus axon terminals of the performant porection and hilar interneurons, the terminals of mossy fibers and Schaefer collateral with in the CA3 and

CA1 regions express high levels of Kv1.1, Kv1.2, KV1.4 [29]. Within CA3, Kv1.1 is known to co localize with Kv1.4 excluding Kv1.2 [29]. Kv1.1 is known to form channel complexes with either Kv1.2 or Kv1.4 in the striatal efferent of pallidal neurons and in neurons of pars reticulate of the substantia nigra. In the cerebellum Kv1.1 has high expression in the Basket cell axon terminal and can form complexes with Kv1.2. Kv1.5 has intense staining in the somato-dendritic area of the PC cells. The cerebellar nuclei showed strong expression of K1.5, Kv1.6 and Kv1.6 was also found in nucleus medialis, interposes and lateralis cells along with weak Kv1.2 staining [30-32].

1.5 Electrophysiological properties of Kv1family channels:

Kv channels are delayed rectifier channels with outward currents, the homo tetramers of

Kv1.1, Kv1.2, Kv1.5 and Kv1.6 are non-inactivating currents, Kv1.3, Kv1.4 and Kv1.7 conduct inactivating currents in absence of the beta subunits [29]. Apart from the type of

inactivation (N-, C- type inactivation and non-inactivating) there are subtle variations in

the activation threshold and kinetics. Therefore, depending on the cell type and the subunits

the cells express the net activation kinetics depends on the combination and stoichiometry

of the channel complex. For example, the Kv1.1 subunits have low activation threshold

(V1/2 in mV) and fast activation kinetics compared to other subunits and has been identified

to be in the following order: K1.1

activation kinetics (τ in ms) could vary as Kv1.1

Since Kv1.1 has the low activation threshold and fastest activation kinetics, Kv1.1 would have major impact on the channel kinetics compared to channels which do not have Kv1.1 subunits. Therefore, channels containing Kv1.1 would be the first to activate upon depolarization and prevent depolarization and excessive firing of neurons. Kv1.1 and

Kv1.2 concatemer studies by Bagechi et al [33] and Sokolov et al [34] show that Kv1.1 subunits altered the activation kinetics more towards the faster gating and shifted the V1/2

towards negative potential in a dose dependent manner [29]. Here they also observed that

Kv1.2 homomeric channels had lower conductance than Kv1.1 and that a single Kv1.2

subunit could lower the conductance of Kv1.1/Kv1.2 channel complex [33].

Kv1.1 Kv1.2 Kv1.3 Kv1.4 Kv1.5 Kv1.6 Kv1.7 Kv1.8 Activation V1/2 (mV) -35 5 and 27 -32 22 and 34 -14 -20 -20 and -8 3.6 κ (mV) 8.5 13 6 5 6 to 12 8 8 τ (ms) 5 (-34 mV) 6 (60 mV) 16.5 16.5 7.1 6 and 8 6 18 (60mV) Inactivation V1/2 (mV) -51 -33 and -15 -63 -62 -25 and -10 -43 κ (mV) 3 -15 7.7 -12.8 3.5 >3 τ (ms) 11 (- 40 mV) - 0.2 (-40mV) 8.5 0.46 (40mV) very slow 10 Single channel conductance, pS 10 14 and 18 13 5 8 9 21 10 and 12 Table 1: Biophysical characteristics of Kv1 α subunits adapted from Saak V Ovespain et

al [29].

1.6 Physiological role of Kv1.2

The Kv1 channels regulate the resting membrane potential and excitability of the cells, the timing and frequency neuronal action potential firing during repetitive spike trains. Kv channels are also known to regulate section of neurotransmitters such as dopamine and GABA [29]. These ion channels predominantly form heteromers and are responsible for low threshold, sustained or delayed rectifier currents, they are mostly expressed in the preterminal regions, axon initial segments and juxta paranodal regions of unmyelinated axons [35-42]. Kv1.2 is highly expressed in neurons particularly in the juxtaparanodal regions of the myelinated neurons where Kv1.2 is known to modulate excitability by preventing recurrent nodal excitation. In microglia Kv1.2 is known to

regulate IL-1β and TNF alpha levels in response to hypoxia [43]. In the CA3 pyramidal

cells Kv1.2 expressed in the distal apical dendrites and is known to contribute to the

intrinsic excitability, on set of action potentials and contributes to the direct synaptic input

to CA3 pyramidal neurons [44, 45]. In the cerebellum Kv1.2 is expressed in the Purkinje

cell dendrites and shows diffuse expression pattern in the molecular layer [46, 47].

Khavandgar et al showed that Kv1.2 containing potassium channels prevent random spontaneous Ca2+ spikes and dendritic hyper excitability without affecting action potentials

and contribute to integration of parallel fiber inputs on the Purkinje Cells [48]. Blocking

Kv1 channels using α-DTX and Tityustoxin (TsTX) results in brief transient increase in activity and caused spontaneous bursts that were random and infrequent. But it did not affect the baseline firing rate of the PCs. Blocking Kv1.2/Kv1.1 channels potentiated and prolonged the responses to PF stimulation on PCs, suggesting that PCs express α-DTX

sensitive Kv1.2/Kv1.1 channels post synaptically and regulate the intrinsic excitability of

the PCs [48]. Kv1.1 and Kv1.2 are highly expressed in the pinceaux of the cerebellum

these are the axons of basket cells which surround the AIS of the Purkinje cell [19, 30, 49,

50].

Recent clinical evidence suggests that Kv1.2 plays a key role in maintenance of

normal human health. Several denovo mutations in Kv1.2 in humans were identified such

as Thr374Ala where the patient suffered from intractable seizures, microcephaly,

developmental delay, progressive brain atrophy [51], a deletion mutation which resulted in dominant loss of function where repositioning of critical arginine (p.Arg297Gln) was predicted results in episodic ataxia and pharmaco-responsive ataxia in the patient [52].

Similarly, a denovo missense mutation Val408Ala was observed in a child with infantile onset seizure variant of RTT [53]. Kv1.2 is known to play a physiological role in sleep, where knocking down KCNA2 the gene for Kv1.2 resulted in sleep depravity in zebra fish larvae [54]. Kv1.2 is also known to be down regulated in dorsal root ganglion resulting in enhanced pain upon nerve ligation. The reduction in Kv1.2 levels in the nerve ligation models is at a genetic level by suppressing the gene by DNA methyl transferases DNMT3a

in the primary afferent neurons [55] and by histone methyl transferases G9a in the dorsal

root ganglion [56]. Overexpression of Kv1.2 to rescue loss of Kv1.2 in the dorsal root

ganglion after sciatic nerve ligation injury resulted in attenuation of pain hypersensitivity

without the loss of acute pain or motor functions, it was also reported that nerve injury also

results in elevation of long non coding RNA that targets the Kv1.2 mRNA and thus results

in degradation and low Kv1.2 protein levels [57-59]. Apart from pain Kv1.2 has been implicated in epilepsies and spastic paraplegia [60-62], several denovo mutations in Kv1.2 gene were identified which results in epileptic encephalopathy [52] a threonine to Alanine

(Thr374Ala) mutation was identified in a patient with early onset epileptic encephalopathy

[51], more over a recurrent mutation in KCNA2 gene was identified as cause for ataxia and spastic paraplegia. Arg294His mutation which leads to loss of function was identified from analysis of over 2000 patients [63]. In a girl with infantile onset seizures variant of RTT, a mutation of KCNA2 gene Val408Ala was identified [53]. Investigators from the University

of Leipzig and University of Tübingen have identified 4 de novo mutations in six unrelated

patients [64]. A denovo Arg297Gln mutation was identified as a potential cause of of

myoclonic epilepsies in a 7-year-old boy [65]. In general, both gain of function and loss of

function mutations cause epileptic encephalopathy [66-68]. It has been shown that VEGF when in injected into rat brains inhibited outward potassium channels and increased tyrosine phosphorylation of Kv1.2 by activating PI 3-kinase pathway [69] and protects neurons from hypoxic death. Dysfunctional Bone morphogenetic protein – 2 signaling associated with pulmonary hypertension was found to downregulate KCNA2 gene in human pulmonary artery smooth muscle cells (PASMC) via attenuation of c-Myc

expression and suggested that increased Kv channel activity might be involved in

proapoptotic and anti- proliferative effects of BMP-2 on PASMC.

As the above-mentioned mutations and deletions of the Kv1.2 gene are rare in humans, and Kv1.2 is tightly regulated at the genetic level. Once the gene is transcribed and proteins synthesized, ion channels are regulated by the effective expression to the cell surface and back into the cell via processes such as forward trafficking and endocytosis.

We will briefly review the known methods of Kv1.2 channel regulation that occurs in the

cells.

1.7 Protein sequence and composition affects the surface expression of heteromeric

Kv channels:

One of the mechanism of Kv channel regulation and subsequently their contribution in

neuronal excitability is protein synthesis and export to the cell surface from Endoplasmic

Reticulum (ER) – Golgi complex biosynthetic pathway. One of the major rate limiting

steps in the biosynthetic trafficking of Kv channels is ER to Golgi transport. This process

is influenced by specific amino acid motifs. A consensus sequence for this motif in most

proteins is “KKXX” motif where “X” can be any amino acid was found to interact with

COP1 coatomer proteins and aid in retrieval of proteins from Golgi to ER [70, 71]. These

unique protein sequences prevent misfolded proteins and ER lumen proteins from being

exported to cell surface. Similarly, in Kv1.1 the ER retention signal is coded by amino

acids A352, E353, S369 and Y379 [72, 73] which line the external region of the pore (turret

region) play an important role in the surface expression. Kv1.1, Kv1.2 and Kv1.6 which

expresses similar amino acid sequence were found to have lower surface expression

compared to Kv1.3, Kv1.4and Kv1.5 [74] which don’t express such ER retention motifs.

Moreover, it was shown that co-expression with Kv1.4 α subunits increased the surface expression of Kv1.2 and Kv1.1 containing channels in a dose dependent manner. It was also found that Kv1.1 mostly had a dominant negative effect on surface expression of homotetramer and on the heteromeric channels [74] due to its ER retention signal.

Manganas and Trimmer observed that most of Kv1.1 was localized to the ER, while Kv1.4 which lacks the ER-retention signal was mostly in the cell surface, while Kv1.2 was more evenly distributed in the ER and the cell surface [74]. The other factor affecting the surface expression of Kv channels is the forward trafficking signal (FTS) on the cytoplasmic C- tail with a consensus sequence of VXXSL. For example, the GVKESL sequence in Kv1.4 and GVNNSN sequence in Kv1.2, DLRRSL in Kv1.5 code for forward trafficking.

Moreover, it has been shown that variations in the FTS influence the extent of surface expression. For example, the VXXSL sequence of Kv1.4 was more efficient in surface trafficking than that of Kv1.5; when the Kv1.5 FTS was replaced with that of Kv1.4 surface expression of Kv1.5 ion channel was increased relative to wild-type. Mutating the Serine and/or Leucine in the FTS reduced the expression of channels by more than 2-fold, suggesting these are important for expression, while replacing the valine had moderate effect. When the Leucine of Kv1.4’s GVKESL was replaced with VXXSN the surface expression of Kv1.4 was reduced and was found to be similar to that of Kv1.2. Based on the evidence thus far, the individual surface expression of the α subunits alone may be in the following order K1.4>Kv1.5>Kv1.2>Kv1.1 [74, 75].

1.8 Glycosylation regulates stability and surface expression of Kv1.2 channels

Glycosylation of Kv1.2 plays a vital role in regulating the surface trafficking. Indeed Kv1.2 undergoes N-Linked glycosylation to form mature channels to exit from the ER. Kv1.2

channels have highly conserved N-linked glycosylation sites in the first extracellular loop

and play an important role in stability of endocytosed channel. Thayer et al identified that

in Cos-7 cells and hippocampal cells the non-glycosylated channels when endocytosed

undergo degradation faster than glycosylated channels [76]. They also propose that the

sialic acid residue at the terminal sugar branches on N207 glycosylation site has a critical

role in protection. They observed that mutation of N207Q had lower surface expression in

cultured hippocampal neurons. In their Cos-7 expression system Kv1.2 undergoes N-linked

oligomannosidic type glycosylation which is sensitive to Endo-H, PNGase F but is

insensitive to sialidase T, while surface Kv1.2 is sensitive to sialidase V, PNGase F but

resistant to Endo H [76]. Kv1.2 has three extracellular loops: S1-S2, S3-S4, S5-P linker

regions, while glycosylation was identified in S1-S2 linker region amino acid N207, the

amount of glycosylation is affected by the sequences in other linker regions. The pore

region (S5-S6) amino acids also influence the glycosylation of Kv1.2; in CHO cells

expressing Kv1.2, Ser 371 is important for glycosylation and the Ser 371T mutation increased the amount of glycosylation which is sensitive to Endo H, which cleaves high mannose glycans and N-glycosidase F which cleaves all N-linked glycans [77, 78].

Kv1.2 Arg 354 influences intermediate glycosylation and when this Arg is mutated to a

Kv1.4 equivalent proline (R354P) Kv1.2 glycosylation increased. Similarly, a Val 381Y or Val 381K mutation within Kv1.2 (Kv1.1 and Kv1.2 equivalent respectively), resulted in decreased glycosylation suggesting that the Val 381 is important for glycosylation. From these data they concluded that Ser 381 inhibits glycosylation, while V381 strongly promotes glycosylation and Arg 354has an intermediate effect on glycosylation [77, 78].

1.9 Posttranslational Modifications (PTMs) affect endocytic trafficking of Kv1.2

Once the channel is expressed on the cell surface, another form of regulation occurs which

is dynamic and is dependent on interactions between Kv1.2 and several other proteins

which modify the amino acids Ser/Thr, Tyr, and Lys. Posttranslational modifications

(PTMs) of these sites include phosphorylation, dephosphorylation and ubiquitination.

These PTMs in turn affect intra- and inter-molecular Kv1.2 interactions thereby

influencing the channel’s biophysical properties as well as its trafficking to and from the

cell surface by endocytic trafficking and recycling. M1 muscarinic receptor activation was

found to induce phosphorylation of Kv1.2 and suppress the channel [79], in neuroblastoma

cells Kv1.2 channels are suppressed in a similar tyrosine phosphorylation dependent

manner by G-protein coupled receptors. Yeast two hybrid and co-immunoprecipitation

experiments showed that Kv1.2 binds to RhoA GTPase and over expression of RhoA

suppresses Kv1.2 currents. In cells expressing M1 muscarinic receptors, blocking RhoA

with C3 exoenzyme attenuated the M1 mediated suppression of Kv1.2 suggesting a role

for RhoA in coordinating M1 receptor mediated Tyr phosphorylation and suppression of

Kv1.2 [80]. The inhibitory effect of phosphorylation could be to inactivate the channel by closing the channel or by removing the channel from the cell surface. The mechanisms of

phosphorylation mediated suppression of Kv1.2 includes changes in the protein

interactions. For example, it was shown that Kv1.2 binds to actin binding protein cortactin

in phosphorylation dependent manner. Phosphorylation of Tyr 415 and Tyr 417 decreases

the interaction between Kv1.2 and cortactin. Kv1.2 deficient in cortactin binding had lower

ionic currents compared to the wild type suggesting that cortactin interactions and tyrosine

phosphorylation play a key role in channel function [81, 82]. The Y132F mutation which

reduces channel suppression also reduces endocytosis of Kv1.2 [83]. The mechanisms of

Kv1.2 endocytosis is varied, having dynamin-dependent and independent components. For example, RhoA mediates endocytosis of Kv1.2 through cholesterol dependent and clathrin- dependent mechanisms. Stimulation of LPA receptors which activates Rho Kinase suppresses Kv1.2 in clathrin dependent manner. On the other hand, in unstimulated cells the RhoA activated kinase ROCK affects steady state Kv1.2 trafficking through a cholesterol dependent mechanism [84].

Protein Kinase A (PKA) which was known to increase potassium currents in the cardiac cells of the atrium is also known to be involved in the trafficking of Kv1.2 and in maintaining the homeostatic balance of surface channel via cAMP dependent pathway.

Forskolin which activates cAMP production also increases Kv1.2 currents and its surface expression via both PKA dependent and independent pathways [43]. Moreover, Connors et al showed that forskolin treatment caused an electrophoretic shift in Kv1.2 immunoblots consistent with an increase in PTM modification such as phosphorylation. Indeed, Kv1.2 was phosphorylated at Serine 440 and 449 in HEK cells treated with forskolin. However,

PKA is not the kinase involved in the phosphorylation of those sites, as PKA inhibitors

failed attenuate the electrophoretic shift of Kv1.2 with forskolin treatment, possibly another

kinase which is dependent on cAMP could have been responsible [43]. At about the same

time, another group showed that Kv1.2 is phosphorylated at ser 440, Ser441 and Ser 449.

They also found that Ser440/441 are phosphorylated only in surface channel and not in ER

localized channel. They showed that Ser 449 is phosphorylated in newly synthesized Kv1.2 which is located in the ER. They also found that mutation of Ser 440/441 reduces surface expression of Kv1.2, while mutation of Ser449 reduces surface expression and seems to reduce phosphorylation of Ser440/441 sites [35]. Hence, Ser449 may be involved in

coordinating the phosphorylation of ser440/441 sites with kinases during the maturation

phase of Kv1.2 and favor forward trafficking of Kv1.2. Johnson et al showed that Ser449

can be phosphorylated by PKA therefore PKA might promote surface expression via

coordinated phosphorylation of Ser449 then Ser 440/441 [43, 85].

1.10 Regulation of Kv1.2 in the cerebellum

Williams et al (2012) showed that cerebellar Kv1.2 is regulated by G-protein

coupled receptor secretin, secretin receptor activation activates adenylate cyclase via Gs G

protein, increases cAMP and activation of PKA. In the same study the authors showed that blocking secretin receptors was sufficient to increase surface expression of Kv1.2 in the cerebellar cortex. Activation of PKA resulted in reduces surface expression of Kv1.2 in the same region [19]. Williams et al also showed that forskolin reduces surface expression of

Kv1.2, while the same stimulus increases surface expression of Kv1.2 when dynamin and

PKA are inhibited, suggesting that PKA independent mechanisms of regulation of Kv1.2 by cAMP in the cerebellum do exist. The most likely source of endogenous secretin are the

PCs themselves [86]. Such a regulation of Kv1.2 channels appears to affect cerebellar function as well. Williams et al showed that infusion secretin into the cerebellum or infusion of the and Kv1.2 specific toxin TsTX improved Eye blink conditioning (EBC) a cerebellum dependent learning task [19]. More recent studies show that that cerebellar learning itself affects surface expression of Kv1.2 in the cerebellum [20, 87].

1.11 Metabotropic glutamate receptors mGluR1

During cerebellar EBC, the PC is conjunctively stimulated by both the parallel fibers and climbing fibers, both release glutamate an excitatory neurotransmitter. At the same time, parallel fibers also stimulate the basket cell body via glutamate. The conjunctive

stimulation results in glutamate spill over which is shown to activate the metabotropic

glutamate receptors mGluR1. The mGluR1 receptors then induce a synaptic plasticity

phenomenon called Long term depression (LTD) at PC-PF synapses by endocytosing

AMPAR2 ion channels. The phenomenon of LTD was postulated to be the mechanism that is responsible for cerebellar learning. However, it is not known if activation of secretin results in LTD or if inhibition of Kv1.2 via its specific inhibitor tityus toxin affects mGluR1 mediated LTD. It is also not known if mGluR1 activation that results in LTD affects Kv1.2.

Since activation of mGluR1 and inhibition of Kv1.2 both result in enhanced EBC we hypothesized that direct mGluR1 activation affects surface Kv1.2.

Metabotropic glutamate receptors are G-protein coupled receptors that are broadly classified into Group I, Group II, Group III based on the downstream signaling mechanisms and sequence similarity. Group I receptors consists of mGluR1 receptors and its isoforms a-f and mGluR5a and b. These receptors are known to couple to Gαq/11, Gs G protein and lead to activation of phospholipase C and adenylyl cyclase. Group II receptors include mGluR2, mGluR3 and couple to Gαi to inhibit adenylyl cyclase. Group III receptors include mGluR4, mGluR6 isoforms a-c, and mGluR7 isoforms a-e and mGluR8 isoforms a-c. These receptors also signal via Gαi and inhibit adenylyl cyclase. All mGluR receptors share similar structural organization, the N -terminus (amino terminal domain, ATD) is extracellular and has a Venus fly trap domain [88] which serves as a ligand binding domain and binds to glutamate. Following the ATD is the cysteine rich domain which is essential for dimerization and activation. This is followed by the classical 7 transmembrane α-helical domains and c -terminal cytoplasmic tail [89]. The C-terminal domain of mGluR1a is the longest and the other mGluR1 isoforms are alternatively spliced and therefore have shorter

c – terminal domain. The longer c-terminal tail of mGluR1a has proline rich PPXXF

domain which is important for interaction with Homer proteins. Homer proteins are known

to regulate constitutive activation of mGluR1 and to connect with IP3 receptors [90].

1.12 mGluR1 signaling

Upon agonist binding to mGluR1, the active receptor initiates multiple signaling mechanisms via Gαq, Gαs, Gα11. In neurons the most widely described signaling of mGluR1 is via Gαq signaling. Activation of Gαq/11 results in activation of phospholipase

C and hydrolysis of phosphotidyl inositol 4.5 – biphosphate (PIP2) into Inositol triphosphate (IP3) and Diacylglycerol (DAG). IP3 is soluble second messenger that binds

2+ to IP3 receptor and releases calcium from endoplasmic reticulum. Ca thus released binds

PKC and initiates its translocation to cell membrane, Ca2+ also activates CaMKII. DAG is a membrane anchored lipid that binds to and activates the translocated PKC. Thus activated,

PKC translocate to the membrane and phosphorylates several ion channels and mGluR1

itself affecting their activity [89]. Homer proteins are also known to couple active mGluR1 to PI3K, which leads to accumulation of PIP3 and recruitment and activation of Akt1. mGluR1 is also shown to activate Gαs in HEK cells via its intra cellular loop2 which interacts with Gαs [91] and increases cAMP.

1.13 mGluR1 is constitutively active via intracellular loops 2, 3 and its interactions

with Homer

In heterologous expression system it has been shown that mGlur1a and mGluR5 have basal agonist independent activity. mGluR1a is shown to increase both inositol

phosphate (IP) and cAMP levels in HEK cells without activation. This constitutive activity

was attenuated in mutant where Arg 775 was replace with a lysine (R775K) and

substitution of Phe 781 with either polar amino acid (F781S) or hydrophobic residue

(F781P) completely removes both agonist and constitutive activity of increasing IP levels.

While N782I and E783Q mutants lack the constitutive activity only [91]. It has also been shown that cAMP – PKA prevents desensitization of mGluR1a and 1b by preventing internalization by GRK2 (GPCR kinase 2) and arrestin2 [92], therefore potentiating the agonist independent signaling of the receptor [93]. Homer proteins are intracellular scaffolding proteins that interact with C-terminal PPXXF domains of receptors and act as a scaffold for other signaling proteins. Homer proteins are classified into long form which include homer 1b, 1c,2 and 3 proteins while Homer 1a is short form. Homer 3 protein is known to interact with mGluR1/5 and prevents constitutive activation [90] while homer 1a binds competitively with mGluR1 replacing Homer3 and activates the receptors.

1.14 Identification of Metabotropic Glutamate receptor – 1 (mGluR1) its role in

Long term Depression (LTD) and Cerebellar learning a brief history

The Purkinje cell (PC) is an inhibitory neuron of the cerebellar cortex. It that receives excitatory inputs form parallel fibers (PF) originating from granule cells in the cerebellar cortex [94]. PCs in turn form inhibitory synapses on neurons in deep cerebellar nuclei and vestibular nuclei. The identification of the cellular mechanisms in the cerebellar cortex that results in EBC was identified from the work of Marr, Albus and Ito. Earlier

Marr (1969) proposed that conjunctive activation of parallel and climbing fibers that synapse on to the same climbing fiber results in Long term potentiation (LTP) of parallel fiber synapses. Latter Albus (1971) suggested that a depression event rather than a potentiation event occurs at the PF-PC synapse following conjunctive stimulation. Maso

Ito et al showed that the mossy fibers that carry the signals form vestibular organs and

climbing fibers that carry signals from retina converge on to PC dendrites. In vestibulo ocular reflex conditioning experiments Ito observed that the PC firing decreased because of LTD as predicted by Albus. Contemporary work form Gilbert and thatch showed that in monkeys which adapt to change in arm load, the change in PC firing was as proposed by

Albus. Ito in 1982 showed the first direct evidence that conjunctive stimulation of vestibular nerve and the inferior olivary nerve resulted in PC-PF synapse depression [16].

In the same body of work Ito showed that application of glutamate in conjunction with olivary stimulation also lead to depression of Purkinje cells and desensitized them to glutamate indicating a role for glutaminergic receptors in LTD. Ito latter showed that repeated stimulation of parallel fibers and in conjunction with climbing fibers also results in LTD in decerebrate rabbits. Sakurai showed similar results in rat cerebellar slice preparation [15, 95]. Based on these findings, Ito proposed the Mar-Albus- Ito theory that long-term depression (LTD) of PC-PF synapses is a mechanism for EBC [14, 16, 96].

Kano and Kato (1987) compared the sensitivity of LTD to various glutamate receptor agonists in order to identify the receptor involved in the LTD, observing that the quisqualate subtype of glutamate receptors were highly involved in LTD [97]. The identity of the specific receptor was not clear because the order of potency to induce LTD that Kano and Kato observed quisilate>glutamate>>>kainite did not fit the profile of any glutamate receptor known at the time. Sugiyama et al inject rat mRNA into Xenopus oocytes and identified a novel receptor which induced inositol phosphate hydrolysis and increase in

Ca2+ influx that was observed in LTD [98]. Masu etal cloned the mGluR1 receptors and showed that PCs and cells in dentate gyrus had high levels of the mRNA for mGluR1 and suggested that it could play an important role in the neuronal functions of these cells [99].

Crepel et al showed that application of an agonist for mGluR1 trans-ACPD conjunctive

with parallel fiber stimulation resulted in depression of PF mediated excitatory currents in

cerebellar slice cultures providing the first evidence for the role of mGluR1 in Purkinje

cells LTD [13]. More concrete evidence for the role of mGluR1 and LTD for EBC came

from Aiba et al (1994) who showed that mouse with knock out of mGluR1 protein showed lack of LTD and EBC [10].

1.15 mGluR1 in cerebellar learning

PC cells show strong expression of mGluR1 [100-103], many studies have shown that mGluR1 is essential for normal functioning of the cerebellum [104-109]. Mutations or knock out of the mGluR1 results impaired LTD, severe motor incoordination, ataxia and impaired EBC which could be alleviated by PC specific mGluR1 rescue [10, 110-113].

In the PC dendrites mGluR1 activation leads activation of its canonical pathway leads to activation of Gq → PLCβ4 → DAG → PKC & CAMKII [102, 114-122]. The activated PKC phosphorylates AMPAR2 at Ser 880. This phosphorylation leads to

AMPAR2 dissociation from GRIP and causes endocytosis of AMPAR2 channels in the cerebellum that leads to LTD [123-128]. It has also been shown that mGluR1 activation also leads to pre-synaptic inhibition of excitatory inputs via retrograde activation of CB1 receptors by 2-Acylglycerol which is the metabolic end product of DAG, which limits glutamate release on the PC cells thus contributing to the LTD [129]. But recent evidence shows that LTD is not necessary for EBC [17, 130] and also that mGluR1, PKCα and

CaMKII are essential. Observations that showed conditioned responses could be achieved by direct parallel fiber stimulation and GABA inhibition [130], mutant mouse models demonstrate that animals that have deficient AMPAR2 endocytosis exhibited normal EBC

responses [17]. However, modified protocols were able to induce LTD in these AMPAR2

endocytosis deficient mouse [18]. In these modified protocols increased CF (climbing

fiber) stimulations (from 1 stimulation to 2 or 5) or somatic depolarization of Purkinje cells

or inclusion of cesium in the patch pipette to block potassium channels resulted in rescuing

the LTD. Intracellular infusion of PKC inhibitor Go6979 blocked LTD in both mutants and

WT mouse. This evidence supports the idea that endocytosis of AMPAR2 is not essential

for LTD and for EBC responses. Therefore, other mechanisms which involve mGluR1,

PKC and CAMKII could be involved in cerebellar learning. Since we have previously

shown that direct inhibition of Kv1.2 by TsTX or inhibition of Kv1.2 via secretin receptor

- PKA pathway results in enhanced EBC responses. Because secretin is released when PC

are depolarized due to glutaminergic activation, we speculate that mGluR1 mediated

learning mechanisms might include Kv1.2 regulation. In this thesis I test the hypothesis

that mGluR1 modulates the expression of Kv1.2 at the cell surface in cerebellar neurons since modulation of Kv1.2 trafficking is a mechanism for modulating channel function.

Figure 1.4. A) General Schematic representation of cerebellar circuit showing major cells

involved in EBC and LTD (+): activation via Glutamate, (-): inhibition via GABA. B)

Signaling pathways involved in cerebellar learning process. Adapted from: Kano M and

Watanabe T. Type-1 metabotropic glutamate receptor signaling in cerebellar Purkinje

cells in health and disease [131].

1.17 Regulation of potassium channels via mGluR1 in the brain

It is well established that ion channels are regulated by the GPCRs. In rat

subthalamic nucleus mGluR1 activation causes increase K-ATP channel currents via Nitric oxide, PKG and intra cellular Ca2+ release mechanism [132]. In the hippocampus mGluR1

activation leads to calcium waves and activation of SK channels and TRPC channels [133].

In lamprey spinal cord neurons mGluR1 activation is shown to regulate the Na+ activated

+ K potassium channels (KNa channels) via PKC and such regulation is differential and is

dependent on the intracellular Ca2+ levels. When intracellular calcium is chelated mGluR1 activation potentiates the Na+ dependent K+ channels currents while under unchelated

conditions mGluR1 activation suppresses them [134]. mGluR1 activation inhibited I(A)

potassium current via PKCα pathway in the cholinergic interneurons in the striatum [135].

In oocytes expression of Kv1.1 and Kv β1.1 when subjected to treatment with PMA or

when co-transfected with mGluR1 and activation via glutamate causes dephosphorylation

and inactivation of Kv1.1/β1.1 channels [136]. mGluR1 receptors are also known to

regulate HCN channels which mediate the IH currents. The retinal ganglion cells and the

cingular cortex pyramidal neurons express both mGluR1 an HCN channels. In these cells

mGluR1 activation with the application of DHPG caused suppression of the HCN channels

and resulted in decreased IH currents in PKC dependent pathway [137, 138]. Such loss of

IH currents results in increased excitability of these neurons, comparable results were

observed in the hippocampal CA1 neurons where mGluR1 activation resulted in loss of IH

currents [139, 140]. In the hippocampus it has been suggested that downregulation of HCN channels by mGluR1 results in increased intrinsic excitability and thus mGluR1 activation affects the homeostatic regulation of intrinsic excitation. In retinal ganglion cells, the mGluR1 activation via DHPG also reduced KIR channel currents. For example, in retinal

Muller cells which co express mGlur5 and HCN, Kir4.1 channels, DHPG application also reduced the Kir 4.1 currents [141, 142].

1.18 mGluR1 regulates potassium channels in the cerebellum and affects intrinsic

excitability

In PCs, activation of mGluR1 results in a biphasic current response which consists of a

transient and sustained phase of inward current. The transient phase is due to TRPC3

channels [143] while the sustained phase is at least partially via Erg potassium channels

and activation of Ca2+/Na+ exchangers and SK channels [140, 144-146]. In neonatal mouse

PCs cells mGluR1 activation with DHPG (50mM DHPG + 40mM K+) decreased the

maximal Erg current to 70% of the peak current and shifted he activation curve to positive

by around 10 mV [145]. When Erg channels were blocked by inhibitor E-4031 followed

by application of DHPG the transient current was still present, but the sustained phase was

almost attenuated suggesting that Erg channels play a role in the sustained component of

the response and it also suggests that mGluR1 activation increased the excitability of PC

[145]. mGluR1 also suppresses Kv4.2 in the cerebellar PC [147]. mGluR1/5 was shown to

regulate the acid sensitive K2p3.1 potassium channels via endocytosis in the rat cerebellar

granule cells through PKC – 14-3-3β pathway [148]. D-myo-inositol-1,4,5-triphosphate

and phosphatidyl-4,5-inositol-biphosphate depletion are involved in TASK channel

inhibition, whereas diacylglycerols and phosphatidic acids directly inhibit TREK channels

[149]. In cultured cerebellar granule cells mGluR1 activates BK channels [150]. mGluR1

receptors via its constitutive activity via PKA results in increased HCN currents in the PC

of the cerebellum [45]. In cerebellar slices that were deprived of activity by incubation with

tetrodotoxin for 2 days, the amount of mGluR1 protein expression was found to be

upregulated. Such activity depravation also resulted in decreased intrinsic excitability. The

reduced intrinsic excitability of PC was alleviated by mGluR1 inverse agonist BAY 36-

7620, PKA antagonist KT5720 and HCN inhibitor ZD-7288, suggesting that mGluR1 via

PKA increased the HCN activity [45].

Although several lines of evidence presented above suggests that mGluR1 activation affects the regulation of several ion channels in the cerebellum, the primarily accepted mechanism for cerebellar learning is LTD of the PF-PC synapses by AMPAR2

endocytosis event though, mechanism has been challenged by recent findings [17, 18].

Previous work from our lab has shown that direct blocking of Kv1.2 is sufficient to

facilitate EBC and EBC itself regulates surface Kv1.2. The effect of Kv1.2 inhibition is

independent of PF-PC LTD and provides an alternative mechanism for cerebellar EBC.

However, it is not known if mGluR1 activation enhances EBC and if it regulates Kv1.2 directly. Here we test the hypothesis that mGluR1 activation enhances EBC and regulates cerebellar Kv1.2, we also identify potential mechanisms of such regulations in the cerebellum. In chapter 2 we identify that direct mGluR1 activation enhances EBC, reduces surface Kv1.2 via PKC. We also employ quantitative mass spectrometry and identify signaling proteins involved in mGluR1signaling pathway that bind to Kv1.2. In chapter 3

we co-expressed in HEK293 cells some of the proteins found to interact with Kv1.2 in the brain, and found that constitutive activity of mGluR1 regulates Kv1.2 via cAMP and PKA.

References for Chapter 1

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microstimulation at coronal-pericruciate cortex of cat with classical conditioning

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Chapter 2: mGluR1 regulates EBC and Kv1.2 potassium channels in the

cerebellum.

Sharath C. Madasu, Department of Pharmacology, Cellular Molecular and Biomedical

Science, The University of Vermont, Burlington Vermont, VT, USA.

Megan L. Shipman, Department of Psychological Science, The University of Vermont,

Burlington Vermont, VT, USA.

Dr. Ying- Wai Lam, Department of Biology, Vermont Genetics Network Proteomics

Facility, The University of Vermont, Burlington Vermont, VT, USA.

Dr. John T. Green, Department of Psychological Science, The University of Vermont,

Burlington Vermont, VT, USA.

Dr. Anthony D. Morielli*, Department of Pharmacology, Cellular Molecular and

Biomedical Science, The University of Vermont, Burlington Vermont, VT, USA.

Correspondence:

Dr. Anthony Morielli [email protected]

Key words: mGluR1, Kv1.2 potassium channels, mass spectrometry, proteomics,

Cerebellum, PKC, Calmodulin kinase, Grid2, eyeblink conditioning, synaptic plasticity,

Long term Depression, Tandem Mass Tags,

2.1 Abstract:

The voltage gated potassium channel Kv1.2 plays a key role in the central nervous system

and mutations in Kv1.2 leads to neurological disorders such as epilepsies and ataxias. In

the cerebellum regulation of Kv1.2 is coupled to learning and memory. We have previously

shown that blocking Kv1.2 by infusing its specific inhibitor Tityustoxin-kα (TsTX) into

the lobulus simplex of the cerebellum facilitates eyeblink conditioning (EBC) and that EBC

modulates Kv1.2 surface expression in cerebellar interneurons. The metabotropic

glutamate receptor mGluR1 is required for EBC although the molecular mechanisms are

not fully understood. Here we show that infusion of the mGluR1 agonist (S)-3,5-

dihydroxyphenylglycine (DHPG) into the lobulus simplex of the cerebellum mimics the

faciliatory effect of TsTX on EBC. We therefore hypothesize that mGluR1 could act, in

part, through suppression of Kv1.2. Earlier studies have shown that Kv1.2 suppression

involves channel tyrosine phosphorylation and its endocytocytic removal from the cell

surface. In this study we report that an excitatory chemical stimulus (50mM K+-100µM

glutamate) applied to cerebellar slices enhanced Kv1.2 tyrosine phosphorylation and that

this increase was lessened in the presence of the mGluR1 inhibitor YM298198. More direct

evidence for mGluR1 modulation of Kv1.2 comes from our finding that selective activation

of mGluR1 with DHPG reduced the amount of Kv1.2 detected by cell surface biotinylation

in cerebellar slices. To determine the molecular pathways involved we used an unbiased

mass spectrometry-based proteomics approach to identify Kv1.2-protein interactions that are modulated by mGluR1. Among the interactions enhanced by DHPG were those with

PKC-γ, CaMKII and Gq/G11, each of which had been shown in other studies to co- immunoprecipitate with mGluR1 and contribute to its signaling. Of particular note was the

interaction between Kv1.2 and PKC-γ since in HEK cells and hippocampal neurons Kv1.2

endocytosis is elicited by PKC activation. Here we show that activation of PKCs with PMA reduced surface Kv1.2, while the PKC inhibitor Go6983 attenuated the reduction in surface

Kv1.2 levels elicited by DHPG, suggesting that the mechanism by which mGluR1 modulates cerebellar Kv1.2 likely involves PKC.

2.1 Introduction:

Purkinje cells (PCs) are the sole output of the cerebellar cortex and a major site of signal integration for motor coordination and motor learning. Khavandagar et al showed that dendrotoxin-sensitive voltage gated potassium channel (Kv1) play an important role in regulating the excitability of PCs by preventing the occurrence of random calcium spikes

and hyperexcitability of PCs. Kv1.2 is highly expressed in PCs [1, 2] and regulates their

excitability. Basket cells (BC) are inhibitory interneurons in cerebellar cortex that synapse

with PCs and Kv1.2 is also expressed in the basket cell axon terminal where it regulates

the release of the inhibitory neurotransmitter GABA. Therefore, regulation of Kv1.2 ion

channels in the PC and BC influences the excitability of PC and therefore the output of the

cerebellum.

Genetic mutation studies have shown that I402T missense mutation of Kv1.2 in the

cerebellum decreases Kv1.2 surface expression in the basket cell axon terminal and

increases firing of basket cells, reducing the firing frequency of PCs. These mouse models showed movement disorders including abnormal gait, measured in coordinated footsteps, that indicate cerebellar deficiencies [3]. Kv1.2 knockout animal models show seizures and reduced life span [4] and mutations in Kv1.2 have been found to cause severe deficits in growth, development, ataxia, epilepsies in mouse models and seizures in human patients

[5-8]. These findings suggest that regulation of Kv1.2 could play an important

physiological role in shaping neuronal excitability and overall health.

An important mechanism of Kv1.2 regulation involves endocytic trafficking to and

from cell surface, we have previously shown that the surface expression of Kv1.2 is regulated by tyrosine and serine/threonine kinases [2, 9-12]. In the cerebellum, Kv1.2

expression at the cell surface is regulated by the secretin receptor through a process

involving protein kinase A (PKA)[13]. Such regulation likely contributes to a range of

cerebellar functions, including learning. Infusion of secretin into the lobulus simplex region

of the cerebellar cortex enhanced eyeblink conditioning, and this effect was mimicked by

infusion of the specific Kv1.2 inhibitory peptide Tityustoxin-kα (TsTX)[13]. A later study

showed that EBC alters the surface expression of Kv1.2 in regions of the cerebellar cortex

[14]. Together, these studies indicate that regulation of Kv1.2 expression at the cell surface

is a component of the complex mechanisms underlaying cerebellum-dependent learning.

mGluR1, a G-protein-coupled receptor which is highly expressed in PCs is required

for EBC. Knock out of mGluR1 impairs EBC [15-19], mGluR1 rescue restored EBC in

these knockout mice [16, 18]. Moreover, mGluR1 knockout mice also exhibited impaired

parallel fiber – PC cell long term depression, indicating that mGluR1 is important for PC

synaptic plasticity [16].

Given that both mGluR1 and Kv1.2 affect PC excitability and Kv1.2 regulation is

associated with EBC, we hypothesized that mGluR1 may regulate Kv1.2 in the cerebellum.

Here we show that mGluR1 activation with the specific mGluR1/5 agonist (S)-3,5-

Dihydroxyphenylglycine (DHPG) enhances EBC in adult rats. An LTD inducing stimulus

which known to activate mGluR1, increases tyrosine phosphorylation on Kv1.2 and some

of its co-immunoprecipitated proteins. Using a cell surface biotinylation assay we show that mGluR1 activation via DHPG and PKC activation via PMA reduces surface Kv1.2 in cerebellar slices. To identify molecular pathways involved in this regulation we took an

unbiased mass spectrometry approach and found that known interactors of mGluR1 such as PKC-γ, calmodulin kinase (CaMKII) and Gq/11 [20] co-immunoprecipitate with Kv1.2 and that those interactions are altered by mGluR1 activity. Using pharmacological inhibitor of PKC Go6983 (PKCi in figures) we observed that PKC inhibition attenuates decrease in surface Kv1.2 mediated by mGluR1 and PMA, suggesting that mGluR1 affects Kv1.2 at least in part through a PKC-dependent mechanism.

2.3 Materials & Methods

Antibodies: Kv1.2 antibodies clone K14/16 from Neuromab UC Davis. While Pan-

CaMKII, AMPAR2, p-Tyr 100, PKA p-Ser/Thr substrate antibody and Calbindin were purchased from Cell signaling. PKC - γ and p-PKC - γ thr514 antibodies were purchased from Genetex, Grid2 extracellular Antibody was from Alomone labs. Anti – mouse Dx700 was purchased from Invitrogen and anti – rabbit 800 infrared dye conjugated 2o antibodies

were purchased from Rockland INC.

Chemicals: DHPG, Go6983, EGLU, NCQX, DAP –V, tetrodotoxin were purchased from

Tocris and all other chemicals were purchased from sigma unless specified. HEPES was purchased from Affymetrix, Sulfo NHHS-S-S- Biotin, HALT protease inhibitor,

Neutravidin beads were purchased from Thermofisher. BPV-phen, mg-132 were purchased from Millipore.

Slice preparation: 400 μm thick cerebellar slices were generated from cerebellum of 4-6- week-old male Sprague dawley rats using a Lecia 1200 vibratome using an adaptation of

the protective recovery method [21]. Briefly, slices were generated in ice-cold Holding

and Slicing ACSF, allowed to recover in Recovery ACSF at 34OC for 7 min and then

transferred to Holding and Slicing ACSF at 25oC for at least 1 hour. Recovery ACSF:

(quantity in milli moles mM), 93 NMDG (N-Methyl D Glucosamine), 2.5 KCl, 1.2

NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 5 sodium ascorbate, 2 thiourea, 3 sodium

pyruvate, 12 N-acetyl cysteine, pH adjusted to 7.4 then 10 MgSO4*7H2O, 0.5 CaCl2.

Holding and Slicing ACSF: 92 NaCl, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25

glucose, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 12 N-acetyl cysteine, pH adjusted to 7.4 then 10 MgSo4.7H2O, 0.5 CaCl2. Recording ACSF: 124 NaCl, 2.5 KCl,

1.2 NaH2PO4, 24 NaHCO3, 5 HEPES, 12.5 glucose, 2 MgSo4.7H2O, 2 CaCl2. pH 7.4. The

osmolarity of all ACSF solutions was adjusted to 290 milliosmoles.

Drug treatment: The slices were then treated with 50 μM DHPG for 10 min or 1 μM PMA

30 min in presence of 100nM TTX, (in µM) 100 EGLU, 50 DAP-V, 100 NCQX made recording ACSF and were incubated for 1 hour in ACSF minus DHPG after drug treatment.

For PKC inhibition, the slices were pre-incubated with 1μM PKC inhibitor Go6983 (PKCi in figures) for 1 hour then the respective DHPG (10 min) or PMA (30 min) was added in presence of Go6983. After 10 min, the DHPG was replaced with recording ACSF containing the Go6983. After the drug treatment, the slices were put in Ice cold HBSS to stop endocytosis and were biotinylated as below.

Biotinylation:

2mg/ml Sulfo-NHS-SS-Biotin was made up in ice-cold HBSS (thermos) and once slices were treated with drugs for indicated times, biotin was applied to slices for 15 min (all in

Ice). Then the unreacted biotin was quenched using ice cold 50mM tris buffered HBSS (pH

7.5). The slices were then transferred to RIPA (containing protease inhibitor HALT 1X,

1mM DTT, 10µM proteasome inhibitor Mg132, Sodium Fluoride 100µM, 2mM EDTA,

Sodium Orthovanadate 100µM, 5mM NEM, 0.1µM BpV-Phen) and were lysed by sonication using brief 8 sec pulses twice. The 800 µL of lysate was incubated with 40μl of nutravidin bead slurry overnight to separate surface proteins, the remaining 200µL lysate was saved as total protein. The nutravidin bound proteins were washed 3 times with RIPA for 10 min and were eluted by boiling in lamelli buffer containing 100 mM DTT and were subjected to electrophoresis 10% Bis-poly acrylamide gel. Then total and surface levels of

Kv1.2, AMPAR2 (1:1000) and calbindin (1:2000) were probed by western blots. To estimate surface protein a ratio of the integrated intensities (Li-Cor Odyssey) of biotinylated Kv1.2 to total Kv1.2 was measured. The total Kv1.2 was obtained by taking the ratio of integrated intensities of total Kv1.2 (of individual slices) to the average of total

Kv1.2 across the blot to account for blot to blot variability. The surface protein ratio from individual experiments were normalized to control. So, the controls are averaged to 1 indicating 100% surface channel. All such individual experiments were pooled and averaged together, so N represents individual experiments and n represents number of slices in each condition.

Inclusion criteria: Calbindin was used as intracellular marker. Only the slices which had no calbindin signal in the eluted or surface portion were considered healthy non permeabilized slices and were used for quantification.

Western Blots:

Proteins were transferred on to nitro-cellulose membrane and were blocked by 5% BSA and strips separating molecular weights of 55KD- 90KD to be probed using mouse anti -

Kv1.2(2µg), rabbit anti - AMPAR2 (1:1000) and 10KD – 55KD to be probed using rabbit

anti – Calbindin (1:2000) antibodies overnight. The membranes were then washed with

TBST and incubated with anti – mouse Dx700 and anti – rabbit 800 infrared dye conjugated

2o antibodies. The images were developed using Li-cor odyssey infrared scanner in its linear range of detection. The blots were quantified using the accompanying Odyssey software and integrated intensities of individual bands were measured for quantification.

Calbindin was used as a cytosolic protein marker indicating live and healthy slices.

Membrane fractionation and Co immunoprecipitation:

Slices were generated and recovered as described above. The slices were then treated with

100 µM DHPG or PBS for 10 min or 60 min and were snap frozen in liquid nitrogen and stored in -80oC until further use slices form 6 - 8 different animals were pooled in an

experiment according to their treatment conditions. The frozen slices were then

homogenized using a Dounce homogenizer in ice cold HEPES buffer at 1ml/100mg of

brain tissue containing HALT 1X, 1mM DTT, 10µM proteasome inhibitor Mg132, 100µM

Sodium Fluoride, 2mM EDTA, 100µM Sodium Orthovanadate, 5mM NEM, 0.1µM BpV-

Phen, 5 mM BATA. The homogenates were subjected to centrifugation at 1000g for 15 min to remove the nuclear pellet. The supernatant was further subjected to centrifugation at 200,000g for 30 min at 4oC and resulting pellet was resuspended in half of the initial

volume of HEPES buffer and was re-centrifuged. At the end of second centrifugation the

pellet was resuspended in 1 ml lysis buffer and was centrifuged at 1000g’s for 10 min to

remove insoluble contents. The supernatant contains the membrane fraction and is used for

immunoprecipitation. 80µg of Kv1.2 antibodies were cross linked to 8mg of epoxy coated

Dyna magnetic beads according to manufacturer’s instructions. Equal amount of protein

from the membrane fraction was incubated with the crosslinked antibody bead complex

overnight (10 – 16 hours). The immunoprecipitated proteins were separated washed thrice

for 10 min at 4oC with lysis buffer. The proteins were eluted using high pH buffer as per manufacturer’s instructions and the eluate boiled in Lammeli buffer with 100mM DTT and were separated on 10% SDS poly acrylamide gel and stained with Coomassie Blue.

Mass Spectrometry:

Trypsin digestion and peptide labeling by tandem mass tag (TMT). The gel regions containing the separated proteins but not the antibody heavy and light chains for each condition were minced, combined and subjected to disulfide reduction using 10mM

DTT in 100mM triethylammonium bicarbonate (TEAB, Thermo) for 1 hour at 560C. The

reduced cysteines were then alkylated with 55mM iodoacetamide for 45 min at room

temperature in the dark. The gel pieces were washed with TEAB for 10 min at room

temperature, dehydrated with 100% acetonitrile and washed with TEAB for 10 min and

were dehydrated again in 100% acetonitrile followed by in-gel trypsin digestion (6 ng/µl

of sequencing grade trypsin enough to cover entire gel piece) overnight at 37oC. The

digested peptides were extracted by incubation in 5% formic acid for 1 hour, followed by

incubation in 5% formic acid in 50% acetonitrile. The extracted peptides were subjected to

evaporation in a SpeedVac. The peptides from each condition were labeled with one of the

isobaric tags from the TMT- sixplex (Thermo Fisher) according to the manufacturer’s

instructions. Briefly, the peptides resuspended in 102.5 uL TEAB were incubated with 41

uL TMT reagents for 1 h at room temperature (see the table insert in Figure 3a for the

information on specific tags that were used) after which 8 μl of 50% hydroxylamine was

added to quench the reaction. Equal amount of the labelled peptides were pooled and dried

down. The combined TMT-labeled peptides were resuspended in 10 μl of 2.5% acetonitrile

and 2.5% formic acid.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS). The

purified labeled and combined peptides were resuspended in 2.5% acetonitrile (CH3CN)

and 2.5% formic acid (FA) in water for subsequent LC-MS/MS based peptide identification

and quantification. Analyses were performed on the Q-Exactive mass spectrometer coupled to an EASY-nLC (Thermo Fisher Scientific, Waltham, MA, USA). Samples were loaded onto a 100 μm x 120 mm capillary column packed with Halo C18 (2.7 μm particle size, 90 nm pore size, Michrom Bioresources, CA, USA) at a flow rate of 300 nl min-1.

The column end was laser pulled to a ~3 μm orifice and packed with minimal amounts of

5um Magic C18AQ before packing the column with the 3-μm particle size chromatographic materials. Peptides were separated using a gradient of 2.5-35%

CH3CN/0.1% FA over 150 min, 35-100% CH3CN/0.1% FA in 1 min and then 100%

CH3CN/0.1% FA for 8 min, followed by an immediate return to 2.5% CH3CN/0.1% FA and a hold at 2.5% CH3CN/0.1% FA. Peptides were introduced into the mass spectrometer via a nanospray ionization source and a laser pulled ~3 μm orifice with a spray voltage of

2.0 kV. Mass spectrometry data was acquired in a data-dependent “Top 10” acquisition mode with lock mass function activated (m/z 371.1012; use lock masses: best; lock mass injection: full MS), in which a survey scan from m/z 350-1600 at 70, 000 resolution (AGC target 1e6; max IT 100 ms; profile mode) was followed by 10 higher-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS) scans on the most abundant ions at 35,000 resolution (AGC target 1e5; max IT 100 ms; profile mode). MS/MS scans were acquired with an isolation width of 1.2 m/z and a normalized collisional energy of 35%.

Dynamic exclusion was enabled (peptide match: preferred; exclude isotopes: on; underfill

ratio: 1%; exclusion duration: 30 sec).

Data analysis. Product ion spectra were searched using SEQUEST and Mascot

implemented on the Proteome Discoverer 2.2 (Thermo Fisher Scientific, Waltham, MA,

USA) in the processing workflow against a curated Uniprot Rattus norwegicus protein

database (AUP000000537; downloaded on June 9, 2017). Search Parameters were as

follows: (1) full trypsin enzymatic activity; (2) mass tolerance at 10 ppm and 0.02 Da for

precursor ions and fragment ions, respectively; (3) dynamic modifications: oxidation on

methionine (+15.995 Da), and phosphorylation on serine, threonine, and tyrosine

(+79.96633 Da); (4) dynamic TMT6plex modification on N-termini and lysine (+229.163

Da); and (5) static carbamidomethylation modification on cysteines (+57.021 Da).

Percolator node was included in the workflow to limit the false positive (FP) rates to less than 1% in the data set. The relative abundances of TMT labeled peptides were quantified with the Reporter Ions Quantifier node in the consensus workflow and parameters were set as follows: (1) both unique and razor peptides were used for quantification; (2) Reject Quan

Results with Missing Channels: False; (3) Apply Quan Value Corrections: False; (4) Co-

Isolation Threshold: 50; (5) Average Reporter S/N Threshold = 10; (6) “Total Peptide

Amount” was used for normalization and (7) Scaling Mode was set “on All Average”. The

ProteinCenter Annotation workflow node was included for Biological Process/Cellular

Component/Molecular Function and Wiki/KEGG Pathway annotations. All the protein identification and quantification information (<1% FP; with protein grouping enabled) was exported from the .msf result files to Excel spreadsheets (Appendix Table 1) for further statistical analyses.

Proteins that were identified in both experiments were kept and all protein abundance ratios

(DHPG/Veh) in each condition were normalized to the corresponding Kv1.2 ratio. Average

fold change (DHPG/Veh at 1h vs. 10 min) was calculated across the two independent

biological replicates with two technical replicates and Student’s t-test was performed

(Supplementary Table 3). For proteins with a p value < 0.05, the normalized ratios were

imported into the JMP Pro 13 (SAS Institute, Cary, North Carolina, US) to construct the

heatmaps.

Immunofluorescence:

40µM slices were obtained from 3-4-week-old rats which were perfused with2% formaldehyde in PBS. The slices were then incubated in 0.3% triton in NGS PBS (at Rt) for epitope unmasking. The slices were then washed and blocked in 3% NGS in PBS solution for 1 hour followed by overnight incubation at room temp with primary antibodies at these dilutions: rabbit anti - mGluR1 (1:50), rabbit anti-PKC γ (thr514) (1:50), rabbit anti calbindin (1:50) mouse anti - Kv1.2 (1:200) in 3% NGS PBS in dark at Rt. The slices were washed in ice cold PBS thrice and were incubated with secondary antibodies goat anti mouse Alexa 647 (1:500), Alexa anti rabbit 568(1:500) overnight and 300nM DAPI nuclear stain for 5 min. The slices were then washed thrice and mounted using prolong

Gold antifade solution. The slices were then observed on a C-2 confocal microscope from

Nikon instruments couple to an Olympus inverted microscope with settings to collect images in the z-stack at a step size of 0.856, pixel dwell time setting at 5.5, the images were captured using 40x oil immersion objective at a resolution of 2048 x 2048. The laser strength and HV setting were adjusted individually for each channel. The 568 and DAPI images were collected together followed by 647 for the same slice with the same Z co-

ordinates. The images were then processed using Fiji image editor. The Z-stacks were

compressed and merged to generate the composite image.

Eyeblink Conditioning:

Subjects. Male Wistar rats were purchased from Charles River (Quebec, Canada)

and housed in pairs upon arrival with access to food and water ad libitum. Rats were single

housed after surgery in an AAALAC-approved facility. The colony room was maintained on a 12-hour light-dark cycle (lights on at 7:00 AM and off at 7:00 PM). Rats weighed 200-

300 g prior to surgery. All behavioral testing took place during the light phase of the cycle and all procedures were approved by the Institutional Animal Care and Use Committee at the University of Vermont. Animal housing and experiments complied with the National

Institutes of Health guide for the care and use of laboratory animals.

Surgery. Surgeries took place 4-6 days after arrival. Surgeries were performed

under aseptic conditions. Rats were anesthetized with 3% isoflurane in oxygen. A midline

scalp incision was made, and four skull screw holes were drilled and skull screws were

placed as anchors for the head stage. A 22G guide cannula (Plastics One, Roanoke VA)

was implanted left of the midline (-11.0 anterior-posterior relative to bregma; -2.7 to -3.0

medial-lateral; -3.2 to -3.7 dorsal-ventral from the skull). A bipolar stimulating electrode

(Plastics One, Roanoke, VA) was positioned subdermally immediately dorsocaudal to the left eye. Two electromyogram (EMG) wires for recording activity of the orbicularis oculi muscle were each constructed of a 75-um Teflon coated stainless steel wire soldered at one end to a gold pin fitted into a plastic threaded pedestal connector (Plastics One). The other end of each wire was passed subdermally to penetrate the skin of the upper eyelid of the left eye and a small amount of insulation was removed so that the bare electrodes made

contact with the orbicularis oculi muscle. A ground wire was wrapped around two skull screws at one end and the other end was also soldered to a gold pin fitted into the pedestal connector. The guide cannula, ground wire, bipolar and EMG electrodes were cemented to the skull with dental cement. Rats were given 6-7 days to recover prior to training.

Apparatus. Eyeblink conditioning took place in one of four identical testing boxes

(30.5 x 24.1 x 29.2 cm; Med-Associates, St. Albans, VT), each with a grid floor. The top of each box was altered so that a 25-channel tether/commutator could be mounted to it.

Each testing box was kept within a separate electrically-shielded, sound attenuating chamber (45/7 x 91.4 x 50.8 cm; BRS-LVE, Laurel, MD). A fan in each sound-attenuating chamber provided background noise of approximately 60 dB sound pressure level. A speaker was mounted in each corner of the rear wall and a light (off during testing) was mounted in the center of the rear wall of each chamber. The sound-attenuating chambers were housed within a walk-in sound-proof room. Stimulus delivery was controlled by a computer running Spike2 software (CED, Cambridge, UK). A 2.8 kHz, 80 dB tone, delivered through the left speaker of the sound-attenuating chamber, served as the CS. The

CS was 615-ms in duration. A 15-ms, 4.0 mA uniphasic periorbital stimulation, delivered from a constant current stimulator (model A365D; World Precision Instruments, Sarasota,

FL), served as the US during conditioning. Recording of the eyelid EMG activity was controlled by a computer interfaced with a Power 1401 high-speed data acquisition unit and running Spike2 software (CED, Cambridge, UK). Eyelid EMG signals were amplified

(10k) and bandpass filtered (100-1000 Hz) prior to being passed to the Power 1401 and from there to the computer running Spike2. Sampling rate was 2 kHz for EMG activity.

The Spike2 software was used to full-wave rectify, smooth (10 ms time constant), and time

shift (10 ms, to compensate for smoothing) the amplified EMG signal to facilitate

behavioral data analysis.

Procedure. Rats received 1 session of adaptation and 6 sessions of EBC. At the

beginning of each session, each rat was connected to the 25-channel tether/commutator which carried leads to and from peripheral equipment and allowed the rat to move freely within the testing box. Adaptation consisted of 100 no-stimulus trials with an average inter-trial interval (ITI) of 30 sec (range = 20-40 sec). EBC consisted of 10 blocks of 10 trials each, with each trial block consisting of trials in the following order: 1 CS-alone trials, 4 CS-US trials, 1 US-alone trial, 4 CS-US trials. The CS was a 615 ms tone and the

US was a 15 ms periorbital stimulation. On CS-US trials, the two stimuli co-terminated

(600-ms delay procedure). The average ITI was 30 sec (range = 20-40 sec). Each session lasted approximately 50-55 minutes.

Immediately prior to sessions 1 and 2 of EBC, rats received an intracerebellar infusion via the guide cannula of 0.5 µL of DHPG (1 uM) or vehicle. This was accomplished by inserting an internal cannula into the guide cannula, with the internal cannula protruding from the tip of the guide cannula by approximately 1.0 mm. Infusions were made with a 10 µl Hamilton syringe loaded onto an infusion pump (KD Scientific,

Holliston MA) set to deliver 0.5 µL of solution over 2 minutes. At the end of the infusion period, the internal cannula remained in place for an additional 1 min to allow diffusion of the infused solution away from the cannula tip. Rats began EBC within approximately 5 minutes after infusion.

Behavior Analysis. CS-US trials were subdivided into three time periods: (1) a

“baseline” period, 280 ms prior to CS onset; (2) a non-associative “startle” period, 0-80 ms

after CS onset; and (3) a “CR” period, 81-600 ms after CS onset. Equivalent scoring

periods were used for no stimulus trials in the adaptation session. In order for a response

to be scored as a CR, eye blinks had to exceed the mean baseline activity for that trial by

0.5 arbitrary units during the CR period. Eye blinks that met this threshold during the startle

period were scored as startle responses and were analyzed separately. The primary

dependent measure of EBC was the percentage of trials with an eye blink response during

the “CR period” across all 80 CS-US trials (Paired group) in the 6 sessions of EBC.

2.4 Results:

2.4.1 DHPG infusion into the cerebellar cortex facilitates EBC

mGluR1 knockout mice show impaired EBC and LTD [17-19]. mGluR1 “rescue” mice,

in which mGluR1 is restored only in PCs, show normal EBC and LTD [18, 19]. Intra-

cerebellar infusion of the mGluR1 agonist DHPG facilitates gain-up vestibulo-ocular reflex

learning [22]. We have previously shown that blocking Kv1.2 by intra-cerebellar infusion of its specific inhibitor TsTX facilitates EBC [2, 23]. As a first step in exploring the role of mGluR1 in Kv1.2 regulation we determined the effect on EBC of DHPG infusion into the lobulus simplex region of the cerebellar cortex. Rats underwent six sessions of 600-ms delay EBC after infusion of DHPG or vehicle solution immediately prior to sessions 1 and

2. A total of 36 rats were tested. Of these, 12 rats were removed due to a bad EMG signal

(4 rats), incorrect or unclear cannula location (6 rats), a damaged bipolar stimulation

electrode (1 rat), and an eye infection (1 rat). This left 11 rats who received DHPG and 13

rats who received vehicle. Intra-cerebellar infusion of DHPG facilitated EBC (Figure

2.1A). This was confirmed by a 2 (Group: DHPG, vehicle) x 6 (Session) repeated-measures

ANOVA on percentage of CRs. This analysis revealed a significant Group effect, F (1,22)

= 9.63, p < 0.01 and a significant Session effect, F(5,110) = 16.62, p < 0.01. The interaction

effect was not significant, p > 0.42. Similar analyses of reflexive responses (the amplitude

of unconditioned eye blinks to the US-alone; the percentage of startle responses to the onset

of the tone CS) failed to reveal any differences across groups or sessions, p’s > 0.14

(Figures 2.2B and 2.2C), indicating that the effects of DHPG were confined to learned eye

blinks.

2.4.2 mGluR1 activation and increases Tyrosine phosphorylation on Kv1.2

Keiko Tanaka et al and Augustine et al have shown that a chemical LTD process involving depolarization with potassium and glutamate initiates the LTD cascade by activating mGluR1 in cerebellar slices[24]. They identify a brief excitatory period of PC excitation lasting 5-10 minutes, followed by LTD initiation after 20-30 minutes [24]. Chae et al [25] have showed that such a stimulus also results in endocytosis of AMPAR2, via phosphorylation of S880 [26, 27]. Surface expression of Kv1.2 is also regulated by phosphorylation dependent mechanisms [2, 12, 14, 23, 28, 29], involving serine/threonine and tyrosine kinases [10, 11, 13]. Since Kv1.2 surface expression both influences and is influenced by EBC, we predicted that chemical LTD stimulus would modulate Kv1.2 phosphorylation levels. Cerebellar slices were treated with a K-Glu stimulus for 5 min (50 mM potassium and 100µM glutamate). Picrotoxin (100µM) was included to reduce inhibitory drive during the stimulus. A subset of slices were pre-treated with the mGluR1 inhibitor YM298198(YM). Slices were collected after 5 minutes and processed for immunoprecipitation and immunoblot as described in Methods. Blots of immunoprecipitated Kv1.2 were probed with anti-phosphotyrosine and anti-PKA substrate antibodies. In the pre-immunoprecipitation lysate we observed an increased ser/thr

phosphorylation of proteins (100 – 130 Kd range) with K-Glu treatment for 5 min

(normalized intensity K-Glu = 2.27± 0.298 vs Vehicle, N=3, **p= 0.0081 Dunnett’s multiple comparison) indicating that the slices actively respond to the stimulus. Inhibition of the response by the mGluR1 inhibitor YM (Figure 2.2A) indicates a role for mGluR1 in this process. Using p-Tyrosine 100 antibody we observed and increase in tyrosine phosphorylated proteins, with the strongest signal in the molecular weight range expected for Kv1.2 (Figure 2.2B) (Relative intensity K-Glu = 1.507 vs Vehicle, *p = 0.021, students

T test, intensity was measured from 55 Kd to around 170KD). mGluR1 inhibitor YM attenuates such increase in binding of tyrosine phosphorylated proteins to Kv1.2.

2.4.2 mGluR1 activation reduces surface Kv1.2

To more directly test the effect of mGluR1 on Kv1.2, we assessed the effect of the mGluR1 agonist DHPG on Kv1.2 expression at the cell surface. Cerebellar slices were subject to a

10 min application of DHPG (100µM) followed by 1-hour incubation in ACSF (Figure

2.3A). Surface proteins were labeled using biotinylation, collected onto nutravidin beads and resolved with SDS-PAGE. The level of surface Kv1.2 and AMPA receptor protein was determined by immunoblot. DHPG caused significant reduction in surface Kv1.2 compared to control slices as measured by the ratio intensity of surface to total Kv1.2 signal relative to Vehicle control (DHPG 0.68 ±0.082 vs control; 1 ±0.12; n = 16 slices, N = 4, p

= 0.045) (Figure 2.3B). Because Kv1.2 was previously shown to be regulated by PKC, we used the PKC activator PMA (1µM) as a positive control for Kv1.2 endocytosis. PMA also caused significant loss of surface Kv1.2 (0.65±0.088 vs Control; n=11slices, N=3, p=0.03)

(Figure 2.3B). Total Kv1.2 did not change in slices treated with DHPG or PMA, suggesting that the reduction in surface Kv1.2 is due to effects on channel trafficking. PC

depolarization and activation of mGluR1 is known to result in decrease of surface AMPA

type ion channel AMPAR2, so we measured the surface levels of AMPAR2 in the same

samples. DHPG did not alter surface AMPAR2 levels (Figure 2.3C) (DHPG 1.001 ±0.168

vs Control; 1 ±0.147; n = 16 slices, N = 4, p = 0.99). In contrast to DHPG, PMA did cause

a reduction in surface AMPAR2 channel (0.60±0.12 vs Control; n=11slices, N=3,

p=0.0504). Therefore, although mGluR1 can regulate surface expression of both Kv1.2 and

AMPAR2 receptors, it can affect each independently.

2.4.3 Kv1.2 interacts with components of mGluR1 signaling pathway

We next wanted to identify the mechanisms by which mGluR1 regulates Kv1.2. As is true

for many proteins, Kv1.2 regulation is in part dictated by its local cellular environment,

and in particular by dynamic interaction with proximal regulatory proteins. We therefore

used a quantitative mass spectrometry-based proteomics approach to identify components of the cerebellar Kv1.2 protein interaction complex, and to assess mGluR1 mediated changes in those interactions. Cerebellar slices were treated with DHPG (100µM) or vehicle control for 10 min or 1 hour, after which Kv1.2 and its associated protein complex were collected by immunoprecipitation and prepared for analysis by mass spectrometry as described in Methods. Briefly, trypsin digested peptides were labeled with isobaric tandem mass tags, with each tag being specific to an experimental condition. Tagged peptides were

then combined prior to mass spectrometry analysis. We have identified 121 proteins that

co-immunoprecipitated with Kv1.2 in both biological replicates. (Appendix) Among the

proteins identified in the Kv1.2, were those shown in other studies to be in complex with

mGluR1 in the cerebellum, including PKC-γ, CaMKII (α, δ isoforms), Gαq, and GRID2

[20, 30].

Interaction with a subset of proteins was increased in slices treated with DHPG for

1h relative to those treated for 10 minutes, PKC-γ, CAMKII (α, δ isoforms), and Gαq, (fold ratio of (DHPG 1hour/DHPG 10min) >1.5) suggesting an increased binding to Kv1.2 upon mGluR1 activation (Figure 2.4, Appendix 1). In contrast, Kv1.2 interacting proteins likely

to be part of the channel itself, inlcuding Kv1.1 and Kv1.6, had fold change ratio < 1.5 with

DHPG treatment, suggesting mGluR1 activation does not alter the channel alpha-subunit

composition/stoichiometry. Western blot analysis concurred with our MS analysis that the

interaction of PKC – γ increases with Kv1.2 in the Post 1-hour DHPG treated samples compared to Vehicle (Figure 2.4C). We have observed Grid2 Co -ip with Kv1.2 (Figure

2.4C) very frequently in our mass spectrometry experiments and in one of the biological

replicate for TMT experiment, similary we observed PKC-β and PKC-ε in our mass

spectromentry experiments infrequently, neither mGluR1, or other mGluR isoforms, were

detected in complex with Kv1.2 either by mass spectrometry or by immunoblot.

2.4.4 Kv1.2, PKC-γ and mGluR1 are expressed in the molecular layer of the

cerebellum

Previous studies have shown that mGluR1 is expressed in the molecular layer of

cerebellum [20, 31-34]. Anton N. Shuvaev et al (Figure 2D in Reference [33]) show that mGluR1 is primarily localized in PCs in the molecular layer and Armburst et al performed cluster analysis of mGluR1 and Calbindin in mouse cerebellum and show that mGluR1 is localized in the Purkinje cell dendrites [32] Figure 2.5). Separate studies showed that Kv1.2 is expressed in the molecular layer of the cerebellar cortex both in the PC dendrites and BC axon terminals [1, 2, 35, 36]. Here, we used immunofluorescence to determine the areas of overlap between Kv1.2, mGluR1 and the mGluR1 signaling protein PKC-γ in parasagittal

sections from adult rat cerebellum. Consistent with our previous observations [13] and with multiple earlier studies[1], Kv1.2 expression was detected in the molecular layer and in the BC axon terminals (Figure 2.6). In agreement with previous studies we observed that the relative abundance of mGluR1 appears higher in the molecular layer than in the BC axon terminal [20, 34]. We also observe that like mGluR1, PKC-γ is highly expressed in the PCs, but not in BC axon terminals (Figure 2.6). We therefore conclude that modulation of Kv1.2 originates from mGluR1 expressed in PCs and most likely involves Kv1.2 expressed in PCs as well.

2.4.5 Broad spectrum PKC inhibitor attenuates mGluR1 mediated loss of surface

Kv1.2

Since mGluR1 activation causes endocytosis of ion channels via PKC, we used a broad- spectrum PKC inhibitor Go6983 which blocks all calcium dependent PKC but not a typical

PKC isoforms. In the presence of the PKC inhibitor (PKCi in Figures) neither DHPG nor

PMA caused further loss of surface Kv1.2 (Figure 2.7) (PKCi DHPG 0.81 ± 0.1276, PKCi

PMA 0.88±0.2038 vs PKCi 1.0 ± 0.17, p = 0.404, n= 8 slices, N = 2). We conclude that mGluR1 modulation of surface Kv1.2 proceeds in part through a PKC dependent mechanism, possibly involving PKC-γ.

2.5 Discussion

One of the most well-known mechanisms for learning-related synaptic plasticity in the cerebellum involves cerebellar LTD. During cerebellar LTD, activation of mGluR1 occurs, which through its canonical signaling pathway activates PKC, which results in phosphorylation and endocytosis of AMPAR2 thus resulting in LTD. Knock out of

mGluR1 impairs EBC, a cerebellar form of learning and memory, and mGluR1 rescue in

PCs restores EBC [18, 19]. Schonewille et al. showed that in mutant mice with impaired

AMPAR2 endocytosis and consequently impaired parallel fiber-PC LTD, EBC is normal,

suggesting that AMPAR trafficking is involved in but not required for EBC. Further,

Yamaguchi et al. were able to induce LTD in cerebellar slices in AMPAR2 endocytosis

deficient mutants via modified stimulation protocols, and that PKCα is required for this

LTD, again suggesting that mGluR1 signaling is essential but might involve mechanisms

beyond AMPAR2 endocytosis [37, 38]. Overall, this suggests that for normal EBC to

occur, mGluR1 activation is required, but AMPAR2 endocytosis is not essential and there

may be other mechanisms that might be involved.

We have previously shown that Kv1.2 is involved in cerebellar learning and

memory. Pharmacological inhibition of Kv1.2 facilitates EBC and EBC itself modulates

surface Kv1.2 expression [13, 14, 23]. Here we show that direct activation of mGluR1 by

infusion of DHPG into the cerebellum improves EBC acquisition. We also show that

application of glutamate and high potassium to cerebellar slices results in increase in

tyrosine phosphorylation of proteins immunoprecipitated with an anti-Kv1.2 antibody, and

an mGluR1 antagonist prevented that increase in tyrosine phosphorylation. Tyrosine phosphorylation of Kv1.2 is associated with channel endocytosis [39] and a resultant decrease in channel function. It is therefore consistent with the finding that DHPG reduces surface Kv1.2 levels in cerebellar slices. Since pharmacological inhibition of Kv1.2 enhances EBC [13], such a reduction in surface Kv1.2 is also consistent with our finding that mGluR1 activation with DHPG enhances EBC. However, Kv1.2 is likely to be regulated by phosphorylation of multiple tyrosine’s [9, 12] and their roles in Kv1.2 regulation in specific cerebellar neurons remains to be determined. Further, Kv1.2 surface

expression and function are regulated by serine/threonine phosphorylation as well [11, 28].

Nevertheless, the findings presented here are consistent with model in which mGluR1

modulates Kv1.2 function in part by affecting the channel’s post-translational

modification.

One of the consequences of Kv1.2 post-translational modification is modulation of

its interaction with other proteins. For example, in HEK293 cells and in GST-pull down studies, tyrosine phosphorylation of Kv1.2 decreases its interaction with the actin regulating protein cortactin [40]. This in turn promotes endocytosis of the channel from the cell surface.

In this study we therefore sought to identify protein interactors of Kv1.2 as a way

to shed light on the potential pathways that might be involved in its regulation by mGluR1.

In the TMT mass spectrometry study summarized in supplementary table 1, several

proteins are particularly significant because they are known to also interact with

mGluR1[41]. These include PKC – γ and calmodulin kinases (multiple isoforms) were

shown physically interact with Kv1.2 [41]. We note that we did not find mGluR1 in

complex with immunoprecipitated Kv1.2 in either immunoblot or mass spectrometry

analyses. Whether this is because mGluR1 does not interact directly with Kv1.2, or because

the interaction is too unstable to detect under the immunoprecipitation conditions used in

this study remains to be determined.

The DHPG induced reduction of surface Kv1.2 levels in cerebellar slices was

mimicked by the PKC activator PMA and reduced by the PKC inhibitor Go6983. This

suggests that the canonical mGluR1 – PKC signaling mechanism might be involved in the

regulation of Kv1.2. Whether this involves PKC-γ or another PKC isoform not detected in

our mass spectrometry analysis and remains to be determined. It is possible, for example,

that the multiple isoforms of PKC that are activated by mGluR1 might have different

targets, with PKC-gamma targeting Kv1.2 and PKC-alpha targeting AMPAR2 [42-44].

Hirono et al have shown that PKCα and PKCβI isoforms translocate to the PC dendrites upon stimulation with high K+ - Glutamate stimulus while PKC γ did not Leitges et al have

shown that LTD is absent in PKC α deficient cerebellum and was rescued only with the

expression of the α isoform indicating that α isoform of PKC is more important for LTD

and there for AMPAR2 endocytosis [42, 43] . Such a model would be consistent with our

finding that in our studies DHPG did not affect AMPAR2 surface levels while global

activation of diacylglycerol sensitive PKC isoforms with PMA did [44].

mGluR1 and PKC-γ are mostly expressed in the molecular layer within the PC cell

body and dendrites. Little evidence exists for their expression on basket cell soma or axon

terminals. Consistent with this, we detected expression of mGluR1 and PKC-gamma in the

molecular layer but not in pinceau’s (Figure 2.6). It is therefore likely that mGluR1

modulates Kv1.2 in PC dendrites and not in BC axon terminals. However, it is possible

that the regulation of Kv1.2 by mGluR1 is indirect. We have previously shown that secretin

receptors regulate Kv1.2, secretin is released from the PCs upon depolarization[45],

therefore it is possible that mGluR1 activation which results in increased Ca levels induces

secretin release from PCs and the released secretin via secretin receptors on PCs and basket

cell axons down regulate Kv1.2. It is essential therefore to study the activation of mGluR1

when secretin receptors are inhibited on Kv1.2 levels and on AMPAR2. Regardless of the

mechanisms by which mGluR1 modulates Kv1.2, Suppression of Kv1.2 channels in the

PC could lead to increased excitability and thereby facilitate LTD [41] by increasing

calcium influx. While endocytosis of Kv1.2 at the BC results in increased GABAergic

input on the AIS of PC there by suppressing PC [3, 13]. The net effect of enhanced LTD

and/or increased GABAergic inhibition would disinhibit the deep cerebellar nuclei and

might help improve EBC.

Acknowledgements: This work is done by UVM internal Grant to AM, JG. The

proteomics work was supported in part by Vermont Genetics Network Proteomics Facility

is supported through NIH grant P20GM103449 from the INBRE Program of the National

Institute of General Medical Sciences.

Author Contributions: SM, AD, JG: Contributed to original ideas, MS, JG contributed to design, execution and data analysis for EBC experiments in Figure1; SM, AD contributed to design, execution and data analysis for Figures 2-6. SM, YL contributed to design,

execution and data analysis for TMT mass spectrometry experiments.

Conflicts of Interest: None

Figures for chapter 2

Figure 2.1. Eye blink conditioning Experiments. (A) Percentage of eyeblink conditioned responses on CS-US trials as a function of session. Each session consisted of 80 CS-US trials intermixed with 10 CS-alone trials and 10-US alone trials. (B) Amplitude of eyeblink unconditioned responses on US-alone trials as a function of session. (C) Percentage of startle responses on CS-US trials as a function of session. Startle responses were eyelid movements that exceeded 0.5 units above pre-trial baseline within the first 80-ms after tone CS onset.

Figure 2.2. Identification of Kv1.2 Tyrosine Phosphorylation: Cerebellar slices were treated with 50mM Potassium+100 µM Glutamate (K-Glu) or vehicle, in absence or presence of mGluR1 inhibitor YM298198 for 5 min and slices were collected after 1-hour post treatment or after 5 min of K-Glu treatment and were subjected to western analysis with anti – PKA ser/thr substrate antibody. A) K-Glu treatment increased Ser/Thr phosphorylation of proteins around 130 KD with in 5 min of treatment even in presence of YM298198. below bar graph quantifying the intensities relative to Vehicle treated slices N=3, **p= 0.0081 vs Vehicle Dunnet’s multiple comparison test. B) Cerebellar slices treated with K-Glu were subjected to Kv1.2 immunoprecipitation and were subjected to western analysis using anti p-Tyr antibody, K-Glu treatment results in increase in tyrosine phosphorylation (bar graph below), YM298198 attenuates such increase. N=2, *p=0.021 vs Vehicle student’s T test, Dunnett’s multiple comparison test p = 0.857 vs Vehicle. C) Western blot analysis of the IP was probed for PKC-γ (top) and Kv1.2 (below).

Figure 2.3. Kv1.2 Endocytosis in the cerebellum. A) A schematic representation of the treatment of Cerebellar slices for biotinylation analysis. B) Cerebellar slices were incubated with 50µM DHPG alone for 10 min or 1µM PMA for 30 min. Then the slices were subjected to biotinylation as mentioned in methods. Western blots (above) and Bar graphs (below) representing changes in the Surface to total ratio of Kv1.2 with various treatments. C) Western blots (above) and Bar graphs (below) representing changes representing changes in the Surface to total ratio of AMPAR2 with various treatments quantified in the same slices as B . * indicates p<0.05 and ** indicates p<0.01 compared to the Control.

Figure 2.4. Quantitative Mass spectrometry. A) Schematic representation of the Immunoprecipitation, Gel processing and TMT labels used. The orange boxes show the areas of gel lanes that were processed for TMT labelling, Igg heavy (55KD) and light chain (~25KD) were excluded from processing. B) Heat map of ratio of proteins (represented by Accession numbers) that Co-ip with Kv1.2 at 10 min and 60 min of DHPG treatment relative to vehicle treated condition. N = 2 biological replicates and 2 technical replicates, pooled from 14 animals.

Figure 2.5. TMT Experiment - western blot validation: A) Western blot of Kv1.2 IP aliquot saved from the TMT mass spectrometry experiment, cerebellar slices treated with DHPG for 10 min or 60 min. The IP was probed for PKC gamma and Kv1.2 (above). The bar graph below shows quantification of PKC gamma relative to Kv1.2. N=2 experiments ** indicates significant difference compared to control. P<0.01 with a one-way ANOVA multiple comparison test. B&C) Independent validation of the Co-ip proteins identified in the MS experiment. Cerebellar lysates were subjected to Kv1.2 immunoprecipitation and were analyzed via western blots with antibodies against CaMKII (PAN-CaMKII antibody), Gq/G11 subunit, PKC- γ, Grid2. Pre – indicates the input, Igg – control Mouse antibody, Post – lysate after immunoprecipitation, IP – immunoprecipitation with anti-Kv1.2 Antibody.

Figure 2.6. Immunofluorescence images of Rat Cerebellum: Showing Kv1.2, PKC-γ, Calbindin and mGluR1 expression. Cerebellar slice stained with anti-Kv1.2 antibody showing highest expression in the Pinceaux and in molecular layer. Cerebellar slice stained with anti-mGluR1, PKC-γ, Calbindin antibody showing high expression in the Purkinje cell body and in the dendrites in the molecular layer. Composite image showing relative expression of Kv1.2 and mGluR1.

Figure 2.7. PKC dependent Kv1.2 Endocytosis in the cerebellum. Cerebellar slices were pre-incubated with incubated with PKC inhibitor G06983 for 1 hour prior to treatment with 50µM DHPG alone for 10 min or 1µM PMA for 30 min. Then the slices were subjected to biotinylation as mentioned in methods. Western blots (above) and Bar graphs (below) representing changes in the Surface to total ratio of Kv1.2 with various treatments. Western blots (above) and Bar graphs (below) representing changes representing changes in the Surface to total ratio of Kv1.2.

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CHAPTER 3: CONSTITUTIVELY ACTIVE MGLUR1 INCREASES SURFACE

EXPRESSION OF KV1.2 VIA PKA SIGNALING PATHWAY

3.1 Abstract

Voltage gated potassium channels are major determinants of excitability of Purkinje cell

(PC) neurons in the cerebellum. We have previously shown that in the cerebellum, activation of mGluR1 with agonist DHPG leads to reduced surface expression of Kv1.2 and that Kv1.2 co-immunoprecipitates with PKC-γ and CaMKII which are known interactors of mGluR1. However, mGluR1 can also signal independently of agonist through a constitutively active, protein kinase A-dependent pathway. Here we show that in HEK293 cells, co-expression of mGluR1 increases the surface expression levels of

Kv1.2. This effect occurs in absence of mGluR1 agonists and in the presence of an mGluR1 inhibitor. Co-expression of known downstream effectors of the agonist driven mGluR1 pathway, including PKC-γ and CaMKII, had no effect on Kv1.2 surface expression or on the ability of mGluR1 agonist to modulate that expression. In contrast, the inverse agonist

BAY 36-7620 significantly reduced the mGluR1 effect on Kv1.2 surface expression, as did pharmacological inhibition of PKA with KT5720.

3.2 Introduction:

The voltage gated potassium channel Kv1.2 plays an important role modulating membrane potential of neurons. A major role of Kv1.2 within the cerebellum is to modulate the excitatory [1] and inhibitory [2] input to Purkinje cells (PC). Excitatory input to PCs is influenced by Kv1.2 expressed PC dendrites, which receive excitatory input from parallel fibers. Khavandagar et al have shown that Kv1.2 prevents random Ca2+ spikes and dendritic hyper excitability of Purkinje cells[1], they also show that blocking Kv1.2 potentiated and prolonged the responses to parallel fiber stimulation. McKay et al showed that Kv1 channels shape the CF firing onto PC, inhibiting Kv1 channels resulted in spontaneous firing of CF suggesting Kv1 channels are expressed in presynaptic sites as well. On PC, blocking Kv1 channels reduced the current threshold to evoke Ca-Na burst and altered the firing pattern of Purkinje cells, from firing trains of action potentials, PC with Kv1 channels blocked fired brief and rapidly inactivating action potentials[3].

Inhibitory input to PCs is influence by Kv1.2 expressed in basket cell axon terminals, which provide strong inhibitory input to Purkinje cells [2]. PCs are the major information processing units of the cerebellum and provide the sole output of the cerebellar cortex, placing Kv1.2 in a central position to influence information processing in the cerebellum[4].

We have previously shown that, in the cerebellum, suppression of Kv1.2 with the specific inhibitor Tityus toxin (TsTX) or by secretin receptor mediated endocytosis of

Kv1.2 results in enhanced EBC [2, 5]. We have also shown that EBC affects surface expression of Kv1.2 [5, 6]. mGluR1 is a G-protein coupled receptor which is essential for normal cerebellar function and for EBC activity[7]. We have previously shown that in the

cerebellum mGluR1 activation leads to loss of surface Kv1.2 (Chapter 2). The cellular

mechanisms by which mGluR1 modulates Kv1.2 are not well understood. In a previous

study we used mass spectrometry to show that that Kv1.2 physically interacts with potential regulatory proteins known to be activated by mGluR1, including PKC gamma, CaMKII,

Grid2, and Gq/11. We also found that mGluR1 regulates Kv1.2 in part through a PKC-

dependent mechanism. In this study we used HEK293 cells and co-expression of mGluR1

and selected components of its downstream signaling pathways as a way to analyze the

molecular mechanism by which mGluR1 regulates Kv1.2 in greater detail. Interestingly,

we found that mGluR1 activation with the agonist DHPG had no effect on Kv1.2 surface

expression in HEK cells. This lack of effect on Kv1.2 persisted even with co-expression of

mGluR1 downstream effectors that are endogenously expressed in cerebellar neurons but

not in HEK293 cells, including PKC-γ, Grid2 or CaMKIIα, either individually or in combination. In contrast, expression of mGluR1 elicited a strong increase in Kv1.2 surface expression. This phenomenon was completely independent of agonist activation of the receptor but was blocked using the inverse agonist BAY-36-7620. Indicating that mGluR1 is constitutively active.

In heterologous expression system it has been shown that mGlur1a and mGluR5 have basal agonist independent activity. mGluR1a is shown to increase both inositol phosphate (IP) and cAMP levels in HEK cells without activation. This constitutive activity was attenuated in N782I and E783Q mutants [8]. It has also been shown that cAMP – PKA prevents desensitization of mGluR1a ,1b by preventing internalization by GRK2 (GPCR kinase 2) and arrestin 2 [9], therefore potentiating the agonist independent signaling of the receptor [10]. Similarly Homer proteins are intracellular scaffolding proteins that interact

with C-terminal PPXXF domains of receptors and act as a scaffold for other signaling

proteins. Homer proteins are classified into long form which include homer 1b, 1c,2 and 3

proteins while Homer 1a is the short form. Homer 3 protein is known to interact with

mGluR1/5 and prevents constitutive activation [11] while Homer1a binds competitively

with mGluR1 replacing Homer3 and activates the receptors. In the cerebellum mGluR1

receptors were shown to be constitutively active via PKA and increase the intrinsic

excitability of PCs by affection HCN ion channels [12].

Here we show that mGluR1 is constitutively active and signals via PKA to

increase surface Kv1.2 because PKA inhibitor KT-5720 prevented mGluR1 mediated increase in surface Kv1.2

3.3 Methods

Materials:

Flag-mGlur1α plasmid are a gift from Dr. Stephen Ferguson, Canada, CaMKII GFP was a gift from Dr. Stephen Tavalin, University of Tennessee, USA. Grid2 plasmid was a gift from Dr. Carol Levenes, Centre de Neurophysique, Physiologie et Pathologie, France.

Cell culture and transfection: HEK – M cell line which stably expresses Kv1.2/Kvβ2 was used for all the experiments. Cells were maintained in DMEM-F12 medium supplemented with 10% FBS, Pen/Strep glutamine, Zeocin, G418 and were maintained in

o 5% CO2 environment at 37 C. On the day of transfection 35 mm dishes were coated with

1µg/ml PEI for 15 min followed by wash with PBS. The HEK cells were plated at a

medium confluence on to the PEI coated dishes and were let grow to achieve 70-80% confluence. Medium was replaced on the day before transfection.

Required amount of DNA (maximum of 5µg) was mixed with 150mM NaCl

incubated by 10 incubation. 18µl (1µg/ml) of PEI was mixed with 112µl of 150mM NaCl

and was incubated for 10 min followed by mixing with the plasmid mixture and another

10 min incubation. Cells were then transfected with the PEI-plasmid mixture and were allowed to grow for 24hours.

Flow cytometry:

Transfected cells were plated on to a PDL coated (1mg/ml) 6 well dish and were serum starved in Neurobasal-A medium overnight. The next day medium was replaced at least

1hour before drug treatment. All drugs were added from stock solution made with

Neurobasal medium and were added for indicated times. At the end of drug incubation endocytosis was stopped by addition of 0.4% sodium azide and cells were incubated for 20 min at 37oC in the incubator. Cells were then scrapped, washed with 0.1% FBS in PBS

solution (flow sauce). The cells were resuspended and incubated in anti-Kv1.2 extracellular

antibody as described in [13] for 1 hour. The cells were then washed once and anti-rabbit

SPRD antibody was added and incubated for 17 min. The cells were then washed twice in

flow sauce spun down and resuspended in 300µl of flow sauce with 2µg of DAPI for

live/dead cell staining. The stained cells were then enumerated on MACS VYB benchtop

flow cytometer (Milteny biotech). Initially 10,000 events were counted, as the number of

transfections increased 100,000 events were counted as indicated.

Western Blots:

Cells were transfected, and serum starved as above and were lysed in RIPA buffer with the

following formulation: 50mM Tris, 150mM NaCl, 2mM EDTA, 0.25% deoxycholate, 1%

NP40 alternative, 10%glycerol, 1mM DTT, supplemented with 1x HALT protease

inhibitor, 100mM Na3VO4, 1mM NaF, 5mM BAPTA, BPvphen, mg132. The lysates were

subjected to centrifugation to separate nuclear pellet and were separated on SDS – PAGE

and was transferred on to nitrocellulose paper and subjected to western blot analysis. Total

Kv1.2 was detected via anti-Kv1.2 antibody from Neuromab, anti-mGluR1, anti PKA

ser/Thr substrate antibodies from CST.

Immunofluorescence microscopy: Immunofluorescence imaging was performed as described previously[14] using anti flag anti body (sigma). Images were acquired and processed with the DeltaVision deconvolution restoration microscopy systems (applied

Precision, Issaquah, WA).

3.3 Results:

3.4.1 Expression of functional mGluR1 receptors in HEK293 cells

HEK- M cells were transfected with (Flag-mGluR1), encoding mGluR1.

Expression of mGluR1 protein was verified by immunoblot and immunofluorescence.

Western blot analysis revealed expression of FLAG-mGluR1 protein was stable between

24 to 72 hours post-transfection (Figure 3.1A). Immunofluorescence analysis showed no detectable mGluR1 in un-transfected cells, i.e. those not expressing GFP (Figure 3.1B). mGluR1 expression in transfected cells was detectable at the cell surface by using an antibody directed at the FLAG-tag in the extracellular N-terminus of the expressed mGluR1 (Figure 3.1C).

3.4.2 mGluR1 agonist DHPG activates calcium signaling and increases ERK

phosphorylation

We next asked if the expressed receptors were functional. Augustine et al have

shown that mGluR1 increases ERK phosphorylation [15]. Several researcher have shown

that mGluR1 activation leads to release of intracellular calcium [16, 17]. In HEK-M cells

expressing mGluR1, application of DHPG increased ERK phosphorylation only in cells

transfected with mGluR1 (Figure 3.2A), while in cells pre-incubated with mGluR1

noncompetitive antagonist YM 298198, the inhibitor blocked the increase in

phosphorylation). Similarly, DHPG (100µM) and glutamate (200µM) caused a transient

increase in intracellular calcium measured in individual cells using the calcium indicator dye Fluo4-AM (Figure 3.2B). Preincubation with the competitive antagonist YM 298198 completely blocked the calcium signaling events in HEK cells transfected with mGluR1

(Figure 3.2B) YM 298198 did not affect calcium signal detected by application of the calcium ionophore A23187. We also observed that neither glutamate nor DHPG were able to alter intracellular calcium after an initial application, consistent with desensitization of the mGluR1 receptor response[9, 18]. Collectively, these findings confirm the expression of functional, agonist sensitive mGluR1 receptors in HEK-M cells.

3.4.3 Detection of Surface Kv1.2 by Flow Cytometry

In our previous studies we have shown that Kv1.2 is regulated by trafficking of

Kv1.2 from cell surface via endocytosis and that such endocytosis results in reduced Kv1.2 ionic current. In previous studies flow cytometry was used to study the surface expression of Kv1.2 in HEK cells[13, 14]. Those studies have identified changes in surface expression in response to activation of another G-protein coupled receptor, the M1 muscarinic receptor

[19, 20]. Therefore, in this study we also used flow cytometry to detect mGluR1 effects on

Kv1.2 surface trafficking. To accomplish this, HEK cells were co-transfected with Flag- mGluR1, eGFP, and control vector PRK5 plasmids. Live cells were distinguished by exclusion of DAPI nuclear stain, and GFP expression was used as positive marker for

transfection. Only cells that were live and had GFP expression were selected for analysis of surface Kv1.2. Surface Kv1.2 was detected using a fluorescently labeled antibody directed against an extracellular epitope within Kv1.2 ([13], Methods). Cells that received

PRK5 only, received 20 Ab only and i.e. do not express Kv1.2, were negative controls for antibody staining (Figure 3.3).

3.4.4 mGluR1 activation by agonist does not affect Kv1.2 surface expression

In a previous study we found that mGluR1 activation decreases the net expression of Kv1.2 at the cell surface in cerebellar slices (Chapter 2). We therefore expected mGluR1 activation in HEK-M cells to have a similar effect on Kv1.2 surface levels. As shown in

Figure 3.5A, the mGluR1 agonist DHPG did not reduce the surface expression of Kv1.2 as we observed in the cerebellum (Chapter 2). This lack of effect was likely not caused by desensitization of mGluR1 for several reasons. First, as shown in Figure 3.2, both MAP kinase and calcium signals were observed in response to DHPG treatment in HEK cells expressing mGluR1. Second, DHPG was also without effect in cells that had been pre- treated with Sodium pyruvate and glutamate pyruvate transaminase (GPT) to scavenge of any glutamate that might have accumulated in the bath from the cells themselves (Figure

3.5b).

3.4.5 Over expression of PKC-γ, Grid2, CaMKIIα does not affect surface expression

of Kv1.2

HEK cells do not express many of the proteins known to be involved in mGluR1 signaling in the cerebellum. We have identified mGluR1 interacting proteins that bind to

Kv1.2 such as PKC γ, CaMKIIα, and Grid2. None of these proteins are expressed endogenously in HEK cells. We therefore transfected Kv1.2/β2 stable M1 cell lines with

PKC-γ, Grid2 or CAMKII, alone or in combination, in an attempt to reconstitute the

mGluR1 signaling complex in HEK cells. We did not observe any change in level of

surface expression of Kv1.2 in the cells transfected with Grid2 and CaMKII compared to

control transfected cells Figure 3.4, while cells transfected with PKC-γ had a slight increase

in surface Kv1.2 (p=0.0031 vs GFP PBS cells). In no instance did expression of these

proteins, alone or in combination, result in Kv1.2 modulation by DHPG (Figure 3.4).

3.4.6 Modulation of Kv1.2 via constitutively active mGluR1

Although DHPG had no effect on Kv1.2 surface expression despite the presence of

functional, agonist sensitive mGluR1 receptors, we consistently observed that mGluR1

expression alone elicited a significant increase in surface Kv1.2 levels relative to HEK cells

expressing Kv1.2 but not mGluR1. Like many G-protein coupled receptors, mGluR1 is capable of signaling in the absence of agonist activation [8]. We therefore hypothesized

that such constitutive activation of mGluR1 was responsible for the elevation in surface

Kv1.2 observed in cells co-expressing mGluR1. To test this idea, we used an inverse

agonist, BAY 36-7620, which blocks constitutive activity of mGluR1[21, 22]. We found

that incubation with Bay 36-7620 overnight significantly attenuated the mGluR1 mediated

decrease in surface Kv1.2 (Figure 3.6A).

3.4.7 mGluR1 increases surface Kv1.2 via PKA mediated mechanism and not via

PKC

Several lines of research showed that mGluR1 also couples to and signals via Gs –

PKA pathway in heterologous expression systems and in the cerebellum [8, 12, 23],

intracellular loops 2 and 3 play an important role in binding of mGluR1 to Gs G – proteins

[8]. The same study also showed that mGluR1 transfection results in an increase in basal

levels of cAMP and inositol phosphate production[8]. Further it has been shown that PKA activation prevents mGluR1 receptors from regular constitutive internalization therefore potentiating the constitutive activity and signaling inositol pathways [9]. In the cerebellum it was shown that mGluR1 via PKA increases intrinsic excitability by modulating HCN potassium channels [12]. We hypothesized that mGluR1 regulates Kv1.2 in HEK cells via

PKA when constitutively active. To test this, we incubated the cells with PKA inhibitor

KT 5720 and PKC inhibitor Go6980 for 1 hour prior to flow. In cells expressing Kv1.2 but not mGluR1, KT 5720 did not affect surface Kv1.2, while PKC inhibitor increased surface expression of Kv1.2 (1.124±0.085 vs GFP veh, p=0.0043). However, in cells co-expressing mGluR1 along with Kv1.2, KT5720 decreased the surface expression (1.26±0.13 mGluR1 vs 1.096±0.071 p<0.0001,) (Figure 3.6B). Collectively, these findings indicate that in

HEK293 cells, constitutive activation of PKA signaling by mGluR1 elevates surface Kv1.2 levels.

3.5 Discussion

Here we identify a new mechanism for the regulation of Kv1.2 homomeric ion channels in a heterologous expression system. We have also shown that in the cerebellum

Kv1.2 interacts with several proteins including PKC-γ, CaMKIIα, Gq/11 etc. which are also known to interact with Kv1.2[24]. In that study, agonist activation of mGluR1 reduced the level of Kv1.2 at the cell surface in cerebellar slices (Chapter 2). In depth analysis of the molecular mechanisms of that modulation in native tissue is complicated by the presence of an array of active signaling systems, each potentially able to modulate Kv1.2. This natural complexity also makes it difficult to determine whether the effect of mGluR1 on

Kv1.2 is direct or is perhaps secondary to mGluR1 effects on availability of other

neurotransmitters capable of modulating the channel. Numerous studies use HEK293 cells

as an experimental system for studying Kv1.2 regulation in isolation of such confounding

factors. We therefore in this study we attempted to reconstitute in HEK293 cells the

mGluR1 agonist-driven modulation of Kv1.2 that we observed in cerebellar tissue.

In HEK cells, mGluR1 activation with the specific agonist DHPG or with natural

agonist glutamate, did not affect surface Kv1.2 levels. This is in contrast to the effect we

observed in cerebellar slices where DHPG application reduced surface Kv1.2 (Chapter 2).

This lack of effect on surface Kv1.2 in HEK cells was not because the mGluR1 receptors

were non-functional since DHPG did elicit an increase in ERK phosphorylation and could

elevate intracellular calcium (Figure 3.2A).

It is possible that although mGluR1 can couple to some signaling pathways in

HEK cells, the pathways linking the receptor to Kv1.2 might be missing. In our previous work we identified several proteins that interact with Kv1.2 in the cerebellum but that are not endogenously expressed in HEK cells. These include Grid2 [24, 25] an ionotropic

glutamate ion channel which does not respond to glutamate directly but does so when

mGluR1 is activated via DHPG or glutamate, as well as (22,23) PKC -γ and CaMKIIα, also known interactors of mGluR1 [24, 26]. We anticipated that co-expression of these proteins might reconstitute an ability of mGluR1 to regulate Kv1.2 in an agonist-dependent way. As shown in Figure 3.4, this did not occur. However, although Grid2, PKC-γ,

CaMKIIα are good candidates for reconstituting mGluR1 signaling to Kv1.2 in HEK cells, they are not the only proteins that interact with cerebellar Kv1.2, proteins such as 14-3-3,

WNK/STK39, spectrin class proteins, adapter protein 2 and many others were shown to interact with Kv1.2 as shown in Chapter 2. It is therefore possible that co-expression of

other proteins, in particular scaffold proteins such as Disk large proteins (also known as

post synaptic density, PSD) interact with both mGluR1 and Kv1.2 or isoforms of Homer proteins that are known to regulate constitutive activation, might lead to successful reconstitution mGluR1 signaling to Kv1.2.

Although mGluR1 agonist-dependent signaling did not affect Kv1.2, we were surprised to find that expression of mGluR1 alone was able to affect surface Kv1.2 levels.

Agonist-dependent mGluR1 signaling proceeds, in part, through Gq/11 [27]. However,

like many G-protein coupled receptors, mGluR1 can signal in the absence of agonist. Such

constitutive activation is, in the case of mGluR1, mediated by coupling of the receptor to

Gs and a resultant increase basal levels of cAMP and PKA [8, 10, 11, 23]. Connors et al

[13] had previously shown that both cAMP and PKA can modulate surface Kv1.2 levels in

HEK cells. Hence to determine if mGluR1 increases surface expression via a cAMP/PKA- dependent pathway we inhibited both PKA and PKC. In our current study only PKA inhibitor KT5720 reduced the mGluR1 mediated increase in surface Kv1.2, suggesting that

PKA is largely responsible for the increase. It is important to note that the effect of PKA on Kv1.2 is not straightforward. In the study by Connors et al, indirect evidence suggested

that PKA could, depending on its level of activity, reduce surface Kv1.2 levels. However

constitutive mGluR1 signaling involves not only PKA but involve other components, including Homer proteins, arrestins and phospholipase C[28]. Thus, although the effect of mGluR1 on Kv1.2 appears to require PKA activity, the specific outcome of an increase in surface Kv1.2 levels likely involves these other signaling pathways as well.

Although mGluR1 constitutive activity has been shown in the cerebellum, whether or it affects Kv1.2 there remains to be determined. In this respect it is important to note

that in this study we examined Kv1.2 homomultimeric channels. However, in the

cerebellum Kv1.2 is thought to exist as homomultimers, but also as heteromultimers with

Kv1.2 and possibly other Kv1 alpha subunits [9, 29]. These subunits could confer different

sensitivities to mGluR1 constitutive activity. Conversely, they might be the missing

component required to couple Kv1.2 to agonist-dependent mGluR1 signaling. For example, the Kv1.1 subunit is dephosphorylated when mGluR1 receptors are activated [30]

and the extent of inactivation of the Kv1.1/β1.1 ion channels was reduced in HEK cells.

Direct activation of PKA phosphorylates intracellular Kv1.1 and promotes the surface

Kv1.1, while direct activation of PKC stimulated protein synthesis of Kv1.1 in HEK cells

[31] and in cerebellar granule cells, inhibition of PKC by bisindolylmaleimide reduced expression of Kv1.1 [32] but there was no evidence of direct phosphorylation [31]. In contrast Kv1.3 and Kv1.4 are downregulated by PKC activation [33, 34]. Therefore, while the current study provides enticing new insight into the mechanisms by which mGluR1 might regulate Kv1.2, the actual mechanisms for regulating the channel are likely to be strongly influenced by the signaling systems at play in specific neurons.

Figures for chapter 3

Figure 3.1. Expression of mGluR1 in HEK cells. A) Western blot of cell lysates

transfected with control vector (PRK5) in lanes C, mGluR1 and lysates collected 24 hours

(24H), 48 hours (48H), 72 hours (72H) post transfection. Arrows indicate the monomer

(around 150 Kd and dimers 250 Kd). B Immunofluorescence confocal microscopy images

of HEK cells co-transfected with Flag-mGluR1 and GFP, counterstained with DAPI nuclear stain, only cells transfected with GFP (green) showed surface mGluR1(red) expression.

Figure 3.2. Functional expression of mGluR1 in HEK cells. A) Western blot of cell lysates transfected with control vector (PRK5), mGluR1. Cells were in glutamate free medium and were incubated in HBSS for 1 hour followed by treatment with DHPG

(100µM) for 10 min. Cell were incubated with YM 298198 (YM in figure) prior to DHPG.

The lysates were then collected. B) HEK cells co-transfected with Flag-mGluR1 and were in glutamate free overnight and were incubated with Flou-4AM Cells transfected as indicated were treated with DHPG or 200 µM glutamate. Only cells transfected with mGluR1 and treated with DHPG or glutamate (DHPG trace is highlighted) show increase in calcium levels (Ca-Peak red line). mGluR1 inhibitor YM 298198 (YM in figure legend) blocks Calcium increase.

100

Figure 3.3. Gating strategy for estimating surface Kv1.2 via flow cytometry. A) Side scatter (SSC) and forward scatter (FSC) profile of HEK cells. B) Live cell gating. DAPI was used as Live dead marker, PRK5 only transfected cells are DAPI -ve (red shaded

population) was used as negative control. mGluR1 transfected cells (blue shaded

population). C) PRK5 only cells (red shaded population) was used to select transfected

cells which are GFP +ve (blue shaded population.). D PRK5 (blue shaded) only cells which

are -ve for Kv1.2 antibody was used to gate Kv1.2 +ve cells (red shaded cells).

101

Figure 3.4. mGluR1 increases surface expression of Kv1.2: Hek 293 M1 cells that stably express Kv1.2 and β2 sub units were transfected as mGluR1, PKCγ, Grid2, CaMKIIα (GFP tagged) alone or in combinations indicated in figure. The cells were analyzed via flow.

Median fluorescence intensity was normalized to GFP transfected PBS treated cells. ****

indicates P<0.0001 vs GFP PBS cells, ** indicates p=0.0031 vs GFP PBS cells. P value

calculated via Tukey’s multiple comparison test 2-way Annova with transfections and

Drug treatments as variants. CaMKIIα were compared via one-way annova and Sidak’s

multiple comparison test as the transfections were performed separately.

102

Figure 3.5. mGluR1 increases surface expression of Kv1.2 independent of Agonist:

Hek 293 M1 cells that stably express Kv1.2 and β2 sub units were transfected as mGluR1.

The transfected cells were then serum starved in neurobasal medium without glutamate (A) and Cells in (B) were treated with glutamate pyruvate transaminase and 100mM sodium pyruvate (GPT in B) for 2 hours followed by treated with 100µM DHPG or Glutamate for

10 min. MFI was analyzed via flowcytometry and normalized to GFP transfected cells.

PRK5 cells are mock transfected and are Kv1.2 Ab (-) ve, GFP (-) ve and were used to gate transfected cells. * indicates p<0.05 vs GFP, p value calculated by students T test with

Welch correction.

103

Figure 3.6. Constitutively active mGluR1 increases surface expression of Kv1.2 via

PKA: Hek 293 M1 cells that stably express Kv1.2 and β2 sub units were transfected with mGluR1. The transfected cells were then serum starved in neurobasal medium without glutamate. A) Cells were incubated with mGluR1 inverse agonist BAY 36-7620 (BAY) for 1 hour or overnight (OV), with noncompetitive antagonist 100µM YM298198 for 1 hour prior to flow cytometry B) Cells were treated with PKC inhibition Go-6987 (1µM) and PKA inhibitor KT-5720 (5µM) 1 hour prior to flow analysis. * indicates p<0.05 vs

GFP, p value calculated by students T test with Welch correction # indicates p<0.05 compared to mGluR1 Veh.

104

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25. Kakegawa, W., et al., D-serine regulates cerebellar LTD and motor coordination

through the delta2 glutamate receptor. Nat Neurosci, 2011. 14(5): p. 603-11.

26. Jin, D.Z., et al., Phosphorylation and Feedback Regulation of Metabotropic

Glutamate Receptor 1 by CaMKII. J Neurosci, 2013. 33(8): p. 3402-12.

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27. Valenti, O., P.J. Conn, and M.J. Marino, Distinct physiological roles of the Gq-

coupled metabotropic glutamate receptors co-expressed in the same neuronal

populations. 2002. 191(2): p. 125-137.

28. Chung, G. and S.J. Kim, Sustained Activity of Metabotropic Glutamate Receptor:

Homer, Arrestin, and Beyond %J Neural Plasticity. 2017. 2017: p. 9.

29. Tateyama, M. and Y. Kubo, Coupling profile of the metabotropic glutamate

receptor 1alpha is regulated by the C-terminal domain. Mol Cell Neurosci, 2007.

34(3): p. 445-52.

30. Levy, M., et al., Activation of a metabotropic glutamate receptor and protein kinase

C reduce the extent of inactivation of the K+ channel Kv1.1/Kvbeta1.1 via

dephosphorylation of Kv1.1. J Biol Chem, 1998. 273(11): p. 6495-502.

31. Winklhofer, M., et al., Analysis of phosphorylation-dependent modulation of Kv1.1

potassium channels. Neuropharmacology, 2003. 44(6): p. 829-42.

32. Hu, C.L., et al., Kv 1.1 is associated with neuronal apoptosis and modulated by

protein kinase C in the rat cerebellar granule cell. J Neurochem, 2008. 106(3): p.

1125-37.

33. Andersen, M.N., et al., Regulation of Kv1.4 potassium channels by PKC and AMPK

kinases. Channels (Austin), 2018. 12(1): p. 34-44.

34. Martinez-Marmol, R., et al., Ubiquitination mediates Kv1.3 endocytosis as a

mechanism for protein kinase C-dependent modulation. Sci Rep, 2017. 7: p. 42395.

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CHAPTER 4: Discussion

4.1. Overview

Previous studies have shown that regulation of Kv1.2 in the cerebellum via secretin receptors and the cAMP – PKA pathway leads to dynamin dependent endocytosis. Previous studies have also shown that Kv1.2 regulation impacts cerebellar function since it improved acquisition of cerebellum-dependent EBC. One of the accepted mechanisms for EBC is long term depression (LTD) of the parallel fiber to

Purkinje cell (PF-PC) synapse, which depends on activation of mGluR1 receptors and endocytosis of AMPAR2 ion channels. However, recent evidence showed that animals which lack AMPAR2 endocytosis have normal EBC and are able to elicit LTD under modified stimulation protocols. Since mGluR1 is required for EBC whether or not

AMPAR2 endocytosis occurs, there might be other mechanisms by which mGluR1 contributes to EBC. Since data from our labs shows that pharmacological inhibition of

Kv1.2 enhances the acquisition of EBC, it is possible that mGluR1 contributes to EBC in part by modulating Kv1.2. In Chapter 2 we tested the hypothesis that mGluR1 regulates the surface expression of Kv1.2 in the cerebellum. We show that direct mGluR1 activation reduces surface Kv1.2 via PKC dependent mechanism. We also show that several interaction partners of mGluR1 including PKC gamma and CaMKIIα are in complex with Kv1.2 in the cerebellum as well. In chapter 3 we used HEK293 cells to examine the effect of these interactors individually and in combination with mGluR1 signaling to Kv1.2. Surprisingly, the main effect on Kv1.2 in these studies was by mGluR1 expression itself through a PKA-dependent constitutive activation mechanism.

This finding suggests constitutive activity of mGluR1 could be mechanism for

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modulating Kv1.2 in the cerebellum as well. Collectively, these findings add new dimension to our understanding of mechanisms of how Kv1.2 is regulated in the brain, and to how ligand-dependent and possibly ligand-independent mGluR1 regulation of

Kv1.2 might contribute to cerebellar learning.

4.2 mGluR1 activation improves EBC and reduces surface Kv1.2 Channels in

cerebellum

Blocking Kv1.2 results in enhanced EBC responses [1] and EBC conditioning affects surface expression of Kv1.2 in the cerebellar cortex [2, 3]. mGluR1 dependent cerebellar LTD is thought to be the main mechanism for EBC, but there was no evidence that direct stimulation of mGluR1 would result in increase in EBC. There was only one study that showed direct stimulation of mGluR1 improves Vestibluo ocular reflex (VOR) another form of cerebellar dependent learning [4]. In Chapter 2 we addressed this gap in literature by infusing 1µM DHPG an mGluR1 agonist into the cerebellum of rats prior to

EBC training. DHPG infused animals showed enhanced EBC responses compared to Veh

(PBS) infused animals. This provided the evidence that direct mGluR1 stimulation improves EBC. Since mGluR1 activation improved EBC and Kv1.2 inhibition via direct blocking with Tityustoxin or via secretin receptors induced endocytosis and enhanced

EBC, we hypothesized that mGluR1 activation also affects surface Kv1.2 in the cerebellum. In chapter 2 we tested this hypothesis and we show that mGluR1 activation via DHPG reduces surface Kv1.2 in the cerebellum. However, our approach for identifying surface expression of Kv1.2 using biotinylation of whole slices does not differentiate between Kv1.2 expressed in basket cells or in Purkinje cells. This is an important limitation since mGluR1 is expressed in the Purkinje cell but also to a small

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extent in basket cell dendrites [5]. It is therefore possible that Kv1.2 is regulated in both these cell types. If mGluR1 activation reduces Kv1.2 in the basket cell pinceaux, the result would be increased GABAergic inhibition of Purkinje cells (PC). Since PCs provide inhibitory input to deep cerebellar nuclei, including the interpositus nucleus which is required for EBC, the expected result would be decreased inhibition of the interpositus nucleus and facilitation of EBC. It is also unknown if surface AMPA receptors in the basket cells are affected upon mGluR1 stimulation. It was shown that mGluR1 activation results in excitability of cerebellar GABAergic interneurons which includes stellate and basket cells[6], it also has been shown that mGluR1 activation leads to spontaneous firing of basket cells and these effects were blocked by mGluR1 inhibitors

[7]. Thibault et al shown that mGluR1 activation results in repetitive slow calcium transients in molecular layer interneurons of the cerebellum [8]. Together, these findings show that mGluR1 receptor activation could affect the inhibitory signals provided by basket cells on to PC through several mechanisms, including the regulation of Kv1.2. It will be worthwhile in future studies to examine the role of mGluR1 in pinceaux more directly, possibly through direct patch clamp analysis of Kv1.2 activity in pinceaux, or microscopy-based analysis of Kv1.2 trafficking in pinceaux (3). In contrast, reduction of

Kv1.2 activity in PC dendrites would lead to an increase in intrinsic excitability of

Purkinje cells. Therefore, smaller inputs form the climbing fiber and parallel fibers might result in increased Ca2+ entry. That increase in PC dendrite excitability and might then facilitate the development of LTD. It will be worthwhile to test the hypothesis that blocking Kv1.2 channels affect LTD. It could be possible that rather than facilitate LTD,

Kv1.2 inhibition might occlude LTD in the PC and tip the excitability towards LTP. John

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H Freeman proposed that PC influence the EBC responses through its connections with different sub sets of interpositus neurons. For example, it is postulated that LTD at PC-

PF synapses decrease inhibition of type – A neurons in the interpositus nucleus resulting in increased excitatory input on the red nucleus and facilitate eyelid closure by increasing activity of the orbicularis oculi motor neurons. While LTP at the PF-PC is postulated to increase inhibition of Type - B neurons in the anterior interpositus nucleus and leads to decreased output to red nucleus and downstream levator palpebrae motor neurons that facilitate eyelid closure [9].

4.3 Potential mGluR1 regulation of Kv1.2 in other parts of the brain and its effect

on learning.

In the brain, Kv1.2 often heteromultimerizes with Kv1.1 to form a functional channel. Inhibition of Kv1.1 proteins in the CA1 and the dentate gyrus region of the hippocampus resulted in loss of spatial memory in rats and passive avoidance conditioning in mouse respectively [10]. In these studies acquisition of memory during training was similar to the control animals suggesting that Kv1.1 is important for memory retention rather than formation [10]. Furthermore, inhibiting Kv1.1 did not affect LTP in the CA1 region or in the dentate gyrus, where LTP was considered to be responsible for memory formation. Campanac et al showed that in hippocampus a high frequency stimulation of Schaffer collateral neurons resulted in increased excitation of CA1 basket cells neurons. They show that blocking Kv1 channels with 4-AP or with Dendrotoxin

(DTx-I) which block Kv1.1/Kv1.2 containing potassium channels mimicked LTP and increased intrinsic excitability of the basket cells. They also show that the suppression is mediated my mGluR5 receptors in a calcium dependent manner [11]. DHPG is known

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to induce LTD in the hippocampus, hence it would be beneficial to test for the surface expression of Kv1.2/Kv1.1 in the hippocampal slices upon DHPG treatment, since inhibition of Kv1.1 protein using oligodeoxynucleotides resulted in LTP in the CA1 area of the hippocampus and mGluR5 activation results in loss of Kv1.1 currents in the hippocampal basket cells [11, 12] we expect that similar to our observations in the cerebellum and in line with literature we might observe loss of surface Kv1.2/1.1 channels.

4.4 Kv1.2 protein interactions

Previous studies have shown that several protein interactions affect the function and expression of Kv1.2 such as cortactin, RhoA, Trim 32 [13-15]. Connors et al show that Kv1.2 could be regulated via cAMP possibly by phosphorylation of serine 440/441 and threonine 49 [16, 17] in HEK cells. In the cerebellum Williams et al showed that secretin receptors via adenylate cyclase – cAMP – PKA causes endocytosis of Kv1.2 in a dynamin dependent process [1]. In chapter 2 we aimed to identify potential signaling molecules that might be involved in regulation of Kv1.2 upon mGluR1 stimulation.

Recent advances in the field of quantitative proteomics enabled us to identify protein – protein interactions via shot gun proteomic approaches. Eugene Cilento, a previous graduate student in the lab (Cilento et al unpublished data) identified several protein interactors of Kv1.2 from the cerebellum from naïve rats, of those, proteins such as

Grid2, PKC (α, γ isoforms), CaMKII (α, β, γ, δ isoforms) caught our attention as these proteins are known to interact with mGluR1. But it was not known which of these proteins were responsible for reduction of Kv1.2 in the mGluR1 pathway. If these proteins increased or decreased their interactions with Kv1.2 in the cerebellum when

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mGluR1 is activated with agonist DHPG, it could indicate a role for those proteins in

Kv1.2 regulation. In order to identify theses changes in protein – protein interactions we use quantitative mass spectrometry using Tandem Mass tags. Using this approach, we identified several proteins that increased their binding in response to mGluR1 activation with DHPG. Of these identified proteins PKC- γ and CaMKII proteins increased their binding to Kv1.2. We also observed Grid2 increasing its binding in one of our biological replicate. In these immunoprecipitation and mass spectrometry experiments we did not observe mGluR1 co-immunoprecipitate with Kv1.2. This might be because the proteins might be expressed in different dendritic locations of the PC cell. For example, it has been reported that mGluR1 is expressed peri synaptically in the dendrites while Kv1.2 is diffusely expressed throughout the molecular layer. It is also possible that the amount of

Kv1.2 – mGluR1 interaction is very low and is below the detection limits to be picked up in Western blotting or mass spectrometry techniques. It will be important in future studies to examine the physical proximity of mGluR1 and Kv1.2 using high-resolution microscopy techniques as a way of resolving this issue.

Apart from mGluR1 interacting proteins we have identified several other proteins which are involved in kinase regulation such as 14-3-3 kinases, Wnk/STK 39 kinase,

PKA regulatory subunits, cytoskeletal proteins such as Syntaxin, Actinin4, spectrin beta chain. The role of these proteins in Kv1.2 regulation is not well understood and examining their roles in channel regulation in detail could add much value to the field of ion channel regulation especially in relation to learning.

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4.5 Grid2, PKC-γ, CaMKII and the regulation of Kv1.2

Grid2 is a unique ionotropic glutamate receptor which was classified as an orphan receptor until very recently. Although it is classified as an ionotropic glutamate receptor based on sequence homology with others of that group, it was considered to be unique because it initially was thought to not be activated by glutamate. Instead, its agonists were found to include cerebellin (cbln1) and D – serine [18-21]. Grid2 KO mouse showed impaired learning of delayed EBC, impaired LTD which was restored by virus mediated expression of WT protein and not by the C-Terminal mutant [22-24]. These mutant mouse displayed intact trace EBC suggesting that their role is limited to cerebellar dependent forms of learning. Both the Grid2 and Cbln1 mutants had abnormal Purkinje cells and climbing fiber synapses indicating that these proteins also play an important role in normal development of the cerebellum. Mutations or deletions of Grid2 resulted severe ataxia in humans and some cases brain atrophy and retinal dystrophy [25-30].

Since KO of Grid2 or cbln1 did not disrupt PC LTP it was proposed that delay EBC is

LTD dependent and not LTP dependent. D-serine was shown to regulate endocytosis of

AMPA receptors via its interactions of Grid2 [21]. Grid2 is known to facilitate LTD via coordinating phosphorylation of Y876 and S880 on AMPAR2 via a phosphatase identified as PTPMEG that is bound to the c-terminal of Grid2 [31, 32] which dephosphorylates Y876 which is then postulated to facilitate phosphorylation of S880 on

AMPAR2 receptors [31]. Later on, Ady V et al from Carol Levenes group showed Grid2 responds to glutamate and DHPG only when mGluR1 is present and activated [33]. They also identified the molecular mechanism of Grid2 gating via mGluR1, in both HEK cells and in cerebellar Purkinje cells, they showed mGluR1 specific agonist DHPG induced

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Grid2 current and it is PKC dependent. Both PLC inhibitor U73122 and PKC inhibitor

GF109203X both reduced DHPG induced Grid2 currents [34]. Therefore, it is possible that Grid2 coordinates signaling between mGluR1 and Kv1.2, just as it does between

Grid2 and the AMPAR2 receptors [35]. In chapter3 we co- expressed Grid2, mGluR1 and Kv1.2 in HEK293cells, we observed an increase in surface Kv1.2 with mGluR1 transfection, but neither Grid2 nor mGluR1 activation by agonist had a significant effect on Kv1.2.

PKC – γ is another protein that we have identified as an interacting protein of

Kv1.2 in multiple mass spectrometry experiments. PKC – γ is specifically expressed in the Purkinje cells of the cerebellum and is not known to be expressed in other cells types in the cerebellum. KO of PKC – γ in mouse was shown to result in impaired VOR reflexes and the cerebellar Purkinje cells have multiple climbing fiber innervations [36].

Mutations of PKC – γ is known to cause Spinocerebellar ataxia type 14 [37, 38] via an unknown mechanisms [39], it was also shown that in a mutant mouse model PKC γ

H101Y has loss of PKC – γ activity , loss of connexin 57 phosphorylation and loss of

Purkinje Cells. While its role in LTD and EBC is still unclear, PKC – γ null mouse show impaired climbing fiber elimination without affecting LTD [40, 41]. It is mostly thought that PKC-α isoform is important for mGluR1 mediated LTD [42] and PKC – γ might play important role in synapse maintenance along with Grid2. This is in line with our observations from chapter 2 where blocking PKC using Go6983 which blocks both PKC

α and γ isoforms attenuated the loss of surface Kv1.2. However, in Chapter 3 over expression of PKC – γ resulted in slightly elevated surface Kv1.2 and when co- transfected with mGluR1 and Grid2 we observed that PKC – γ caused further increase in

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surface Kv1.2 compared to mGluR1 alone and mGluR1+Grid2 co – expression. These results might indicate that PKC – γ might be involved in modulating Kv1.2 in some way.

PKC – γ has been recently shown to directly phosphorylate CaMKIIβ at Ser 315 when activated by PMA or by mGluR1 stimulation and regulates Purkinje cell dendritic spines[43]. In the cerebellum we have consistently show that different isoforms of

CaMKII co-immunoprecipitate with Kv1.2. We have identified α, δ and γ isoforms in our mass spectrometry studies. It has been shown that in CaMKIIα null mouse both LTD cerebellum dependent learning such as VOR and optokinetic reflex are impaired while

LTP is intact [44]. In the forebrain CaMKII was found to co-immunoprecipitate with

Kvβ1.1 [45] and its role in Kv1.2 regulation is unknown we tested if CaMKIIα is involved in mGluR1 mediated regulation of Kv1.2. In Chapter 3 we show that over expression of CaMKIIα isoform does not affect surface Kv1.2, when co-transfected with mGluR1 we still observed an increase in surface Kv1.2, application of DHPG resulted in slight decrease of surface Kv1.2 compared to vehicle treated cells. Further research to identify the role of CaMKIIα in Kv1.2 regulation is necessary to identify how this calcium dependent kinase regulates potassium channels. An invitro assay of constitutively active CaMKII mutant could be used to identify the sites of phosphorylation on Kv1.2’s N or C terminal or Kv1.2 serine/threonine mutants could be used to narrow down the interaction sites and potential phosphorylation sites that could help provide first evidence about how CaMKII regulates Kv1.2.

In summary, the question of whether or how Grid2, PKC – γ and CaMKII affects

Kv1.2 function remains open and might have to be addressed using gene manipulation

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in PCs and BCs, rather than in model cell systems which could lack the full complement of signaling components required.

4.6 mGluR1 mediates PKA dependent increase in surface Kv1.2.

In chapter 3 we observed that transfection of mGluR1 results in increase in surface expression of Kv1.2. Activation of mGluR1 receptors with specific agonist

DHPG did not result in reduced surface expression of Kv1.2 as observed in cerebellum.

We hypothesized that the Purkinje cells and HEK cells are not representative of each other and that HEK cells lack several proteins that are expressed in Purkinje cells that are important to mGluR1 signaling. Our immunoprecipitation and mass spectrometry experiments suggested that Purkinje cell specific proteins such as Grid2, PKC – γ,

CaMKIIα might be important. When these proteins were co-expressed with mGluR1 we have always observed a strong increase surface Kv1.2 which was agonist independent.

The agonist independent nature of the increase in surface Kv1.2 suggests that mGluR1 is constitutively active in heterologously expresses cells. mGluR1 inhibitor YM298198 which is a noncompetitive inhibitor and PKC inhibitor Go6983 were not able to block the increase in surface Kv1.2. These data indicated that mGluR1’s constitutive activity is not PKC dependent. As a control we use PKA inhibitor KT-5720 in mGluR1 transfected cells, in cells without mGluR1, PKA inhibition did not affect the surface expression of Kv1.2, but to our surprise, in mGluR1 transfected cells PKA inhibition reduced the surface expression of Kv1.2. Tateyama et al showed that mGluR1 also signals via cAMP and that activation of mGluR1 increases both cAMP and IP3 formation in cells [46]. But in our study, we did not observe an agonist dependent effect on surface

Kv1.2. It could be possible that in the dimeric form mGluR1 could be constitutively

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active and could signal via PKA and inhibit the desensitization and internalization of

mGluR1a, forming a positive feedback loop which could responsible for increase in

surface Kv1.2. It was also shown that only an inverse agonist prevented the constitutive

internalization of mGluR1a [47]. Here in this project we show that overnight incubation

with an inverse agonist BAY 36-7620 attenuated the increase in surface Kv1.2

expression. These data suggest that mGluR1 is constitutively active via PKA mediated

mechanisms to regulate the basal levels of Kv1.2 in the transfected cells.

A question that arose from above observations is about the role of PKA plays in

EBC. Literature search identified work from Claire Cartford.M et al that showed

inhibition PKA using Rp-cAMPs completely blocks the acquisition of conditioned

responses but not the established conditioned responses post learning [48]. Shim et al

have shown that in the cerebellum, mGluR1 receptors regulate HCN channels via PKA

to regulate the intrinsic excitability of the Purkinje cells [49]. Therefore, it is possible

that in the cerebellum mGluR1’s constitutive activity maintains basal levels of active

PKA and maintain a basal level of Kv1.2 for acquisition of memory, latter on upon

learning stimulus when both mGluR1 is activated via agonist glutamate, and increased

Ca2+ entry into purkinje cell, the now highly active PKC might phosphorylate and reduce

the surface Kv1.2.

4.7 Conclusion: Kv1.2 interactors potential targets for Ataxia and role for

Kv1.2 in Alzheimer’s, Epilepsy and Chronic pain.

Overall in the current project identifies two novel mechanisms of Kv1.2 regulation by mGluR1. We also identify the first evidence in support of a link between mGluR1 signaling and Kv1.2 regulation in learning, although at this point demonstration

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of that idea requires further study. We also identified several protein – protein interactors

of Kv1.2 in cerebellum that are also known to interact with mGluR1. These proteins are

also important for normal health of cerebellum as any deletion or mutations in these

proteins were found cause cerebellar ataxia. Hence Kv1.2 and its interactome could be a

set of potential druggable targets to treat ataxia or other cerebellar disorders. Another area

of research for Kv1.2 regulation could be in the hippocampus dependent memory tasks.

There is a lack of evidence about the role Kv1.2 plays in memory formation or in retention of a formed memory in hippocampus and how the identified mutations affect hippocampal physiology and learning.

Another avenue of interest to us is that in patients suffering from Alzheimer’s, patients show increased activity of another group1 metabotropic receptor mGluR5 in the hippocampus. In Alzheimer’s the prion receptor PrPC and mGluR5 act as co-receptors for

Aβ oligomers and inhibits memory formation by disrupting the normal mGluR5 –

glutamate signaling, activation of PYK2, and CaMKII [50, 51]. Given the role that mGluR5

activation inhibits Kv1.1 in the hippocampal basket cells, it is essential to identify the role

Kv1.1/1.2 containing potassium channels play in the diseased condition. Hence further

research about Kv1.2 ion channels in both hippocampus and the cerebellum in mouse

models of Alzheimer’s is necessary to identify potential targets to prevent loss of memory

in these disorders.

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Appendix

TMT MS Table for Chapter 2

A short list of selected proteins identified in TMT Mass Spectrometry from two biological replicates, the quantitation values are normalized to Kv1.2 (KCNA2 gene). Of the 151 proteins identified we selected the proteins based on their involvement in the mGluR1 signaling pathway, endocytic pathway, known interactors of Kv1.2 (KCNA2 gene) identified in previous Mass spectrometry assays. The fold change values are generated from the TMT tags as described in Chapter 2 methods.

Average Average Fold change fold change fold change Accession Description 10 min 1 h 1h/10 min p Value Tubulin beta-2A chain OS=Rattus 0.85204213 2.69657338 3.16483572 0.00282 P85108 norvegicus 3 2 6 2 GN=Tubb2a PE=1 SV=1 Actin-related protein 3 OS=Rattus 0.90321115 2.50556494 0.01411 A0A0G2K1C0 norvegicus 2.77406334 1 2 3 GN=Actr3 PE=1 SV=1 Inositol 1,4,5- trisphosphate receptor type 1 0.86165184 2.30621638 2.67650606 0.09483 F1LNT1 OS=Rattus 5 8 3 4 norvegicus GN=Itpr1 PE=1 SV=3 Calcium/calmodulin -dependent protein kinase type II subunit delta 0.92944107 2.45177420 2.63790172 0.00549 P15791 OS=Rattus 2 6 4 1 norvegicus GN=Camk2d PE=1 SV=1

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Spectrin beta chain OS=Rattus 0.87896978 2.30034445 0.00804 F1MA36 norvegicus 2.61709162 4 6 1 GN=Sptbn2 PE=1 SV=2 Isoform Gamma-2 of Serine/threonine- protein 2.36614877 2.45428804 0.00861 P63088-2 phosphatase PP1- 0.96408764 2 6 9 gamma catalytic subunit OS=Rattus norvegicus GN=Ppp1cc Calcium/calmodulin -dependent protein kinase type II subunit alpha 0.95543885 2.34290956 0.02407 P11275 2.45218159 OS=Rattus 3 5 8 norvegicus GN=Camk2a PE=1 SV=1 Calcium/calmodulin -dependent protein kinase type II subunit gamma 1.05382793 2.57382460 2.44235754 0.05024 P11730 OS=Rattus 6 7 1 2 norvegicus GN=Camk2g PE=1 SV=1 Spectrin beta chain OS=Rattus 0.99410945 2.33735519 2.35120506 0.00482 A0A140UHX6 norvegicus 6 1 9 6 GN=Sptb PE=1 SV=1 Calcium/calmodulin -dependent protein kinase II, beta, isoform CRA_c 1.14583400 2.69271210 2.35000191 0.06500 F1LUE2 OS=Rattus 7 7 2 5 norvegicus GN=Camk2b PE=1 SV=2 Clathrin heavy chain OS=Rattus 1.12912355 2.50716889 2.22045574 0.05627 F1M779 norvegicus GN=Cltc 6 1 8 9 PE=1 SV=1

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Adaptor protein complex AP-2, alpha 1 subunit (Predicted) 1.05328654 2.33045614 2.21255664 0.04696 D3ZUY8 OS=Rattus 8 7 1 7 norvegicus GN=Ap2a1 PE=1 SV=1 Protein kinase C gamma type OS=Rattus 2.38385513 2.16764443 0.04836 P63319 1.09974454 norvegicus 6 9 8 GN=Prkcg PE=1 SV=1 Isoform 2 of Syntaxin-binding 1.04987303 2.20273259 2.09809426 0.00287 P61765-2 protein 1 OS=Rattus 7 7 4 7 norvegicus GN=Stxbp1 Heat shock 70kDa protein 12A (Predicted), isoform 0.99253796 2.06748539 D3ZC55 CRA_a OS=Rattus 2.08302903 0.01311 6 7 norvegicus GN=Hspa12a PE=1 SV=1 Rho guanine nucleotide exchange factor 33 0.87389323 1.76443645 2.01905264 0.13801 D3Z8W6 OS=Rattus 8 6 8 9 norvegicus GN=Arhgef33 PE=1 SV=3 Guanine nucleotide binding protein, alpha q polypeptide, 0.91112184 1.82071303 1.99832002 0.01737 D4AE68 isoform CRA_a 5 1 9 3 OS=Rattus norvegicus GN=Gnaq PE=1 SV=2 2',3'-cyclic- nucleotide 3'- 1.03217615 2.04709814 1.98328370 0.00094 P13233 phosphodiesterase 4 5 2 7 OS=Rattus

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norvegicus GN=Cnp PE=1 SV=2

40S ribosomal protein S2 OS=Rattus 1.11856744 2.18095651 1.94977649 P27952 0.00486 norvegicus 3 1 8 GN=Rps2 PE=1 SV=1 Serine/threonine- protein phosphatase 2B catalytic subunit 1.03852250 2.02306800 1.94802520 P63329 alpha isoform 0.02343 1 9 6 OS=Rattus norvegicus GN=Ppp3ca PE=1 SV=1 Guanine nucleotide-binding protein G(z) subunit 1.22336658 1.91825342 0.10124 P19627 alpha OS=Rattus 2.34672715 7 8 9 norvegicus GN=Gnaz PE=2 SV=3 Guanine nucleotide-binding protein G(i) subunit 0.93814239 1.77209942 1.88894504 0.01617 P10824 alpha-1 OS=Rattus 4 8 7 7 norvegicus GN=Gnai1 PE=1 SV=3 Guanine nucleotide-binding protein G(I)/G(S)/G(T) 1.14605362 2.13333666 1.86146321 0.00052 P54311 subunit beta-1 1 1 7 3 OS=Rattus norvegicus GN=Gnb1 PE=1 SV=4 Ras-related protein Rap-1A OS=Rattus 0.90459076 1.85151341 0.00422 P62836 norvegicus 1.67486193 2 4 3 GN=Rap1a PE=1 SV=1

143

Cullin-associated NEDD8-dissociated protein 1 OS=Rattus 1.26474703 1.82285718 0.13219 P97536 2.30545322 norvegicus 7 2 6 GN=Cand1 PE=1 SV=1 Ras-related C3 botulinum toxin substrate 1 1.08295863 1.65598017 1.52912597 0.00635 A0A0G2K0X4 OS=Rattus 1 3 5 1 norvegicus GN=Rac1 PE=1 SV=1 Voltage-gated potassium channel subunit beta-1 1.10213140 1.37725850 1.24963184 0.00372 P63144 OS=Rattus 8 7 7 8 norvegicus GN=Kcnab1 PE=1 SV=1 Potassium voltage- gated channel subfamily A member 6 1.17913055 1.30151775 1.10379444 0.47916 G3V8L6 OS=Rattus 1 2 5 4 norvegicus GN=Kcna6 PE=3 SV=1 Potassium voltage- gated channel subfamily A member 1 0.97218926 1.03560442 1.06522922 0.07684 P10499 OS=Rattus 8 4 9 7 norvegicus GN=Kcna1 PE=1 SV=1 Keratin, type I cytoskeletal 15 OS=Rattus 1.11501056 1.11822363 1.00288165 0.98579 Q6IFV3 norvegicus 6 7 1 4 GN=Krt15 PE=1 SV=1 Potassium voltage- gated channel P63142 subfamily A 1 1 1 N/A member 2 OS=Rattus 144

norvegicus GN=Kcna2 PE=1 SV=1

Anionic trypsin-1 OS=Rattus 1.29054675 1.20967242 0.93733328 P00762 norvegicus 0.15749 4 5 2 GN=Prss1 PE=1 SV=1

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