Alternative Splicing Controls G Protein Inhibition of Cav2.2 Calcium

Alternative Splicing Controls G Protein Inhibition of

CaV2.2 Calcium Channels

Cecilia Goldsmith Phillips BA, Reed College, 2003

THESIS

Submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in the Department of Neuroscience at Brown University

Providence, Rhode Island May 2012 © 2012 Cecilia Goldsmith Phillips This dissertation by Cecilia Goldsmith Phillips is accepted in its present form by the Department of Neuroscience as satisfying the dissertation requirement for the degree of Doctor of Philosophy.

Date Dr. Diane Lipscombe, Advisor

Recommended to the Graduate Council

Date Dr. Gilad Barnea, Reader

Date Dr. Julie Kauer, Reader

Date Dr. Stephen Ikeda, Outside Reader

Approved by the Graduate Council

Date Dr. Peter M. Weber, Dean of the Graduate School

iii CURRICULUM VITAE

23 Elton St, Providence, RI 02906 (503) 705-7387 [email protected]

EDUCATION

Brown University, Providence, RI PhD, Neuroscience, May 2012

Reed College, Portland, OR BA, Biology, May 2003 Senior Thesis: Processing of GFP-tagged ELH Prohormone in PC12 Cells Advisor: Dr. Stephen Arch

RESEARCH POSITIONS

Research Assistant to Dr. Stephen M Smith, Division of Molecular Medicine, Oregon Health Sciences University, Portland, OR. September 2003 – August 2005

Internship with Dr. Peter Gillespie, Vollum Institute, Oregon Health Sciences University, Portland, OR. June 2003 – August 2003

Research Assistant to Dr. Maryanne McClellan, Department of Biology, Reed College, Portland, OR. June 2002 – August 2002

Research Assistant to Dr. David McKinnon, Department of Neuroscience, SUNY Stony Brook. June 2001 – August 2001 and June 2000 – August 2000

PUBLICATIONS

Allen SE*, Phillips CG*, Raingo J, and D Lipscombe. The neuronal splicing factor Fox-

2 controls Gs protein inhibition of CaV2.2 calcium channels. In submission. *Equal contributions by these authors.

Phillips CG, Harnett MT, Chen W, and SM Smith (2008) Calcium-sensing receptor activation depresses synaptic transmission. Journal of Neuroscience 28(46): 12062- 12070.

iv ABSTRACTS

Phillips CG, Allen SE, and D Lipscombe (2010) The neuronal splicing factor Fox-2 regulates an exon in CaV2.2 that controls the sensitivity of N-type calcium channels to inhibition by Gs proteins. Society for Neuroscience Annual Meeting, San Diego, CA.

Phillips CG, Allen, SE, and D Lipscombe (2010) Neuronal splicing factor Fox-2 regulates an exon in CaV2.2 that controls sensitivity of N-type calcium channels to inhibition by Gs G proteins. New Horizons in Calcium Signaling, China.

Allen SE, Phillips CG, Lipscombe D (2010) The splicing factor Fox-2 controls N-type calcium channel activity in sympathetic neurons. Second Annual International Calcium Channel meeting, Belize.

Phillips CG and D Lipscombe (2009) G protein-coupled receptor inhibition of N-type calcium channel splice variants. Society for Neuroscience Annual Meeting, Chicago, IL.

Vyleta NP, Chen W, Phillips CG, Harnett MT, and SM Smith (2009) Regulation of nerve terminal function by the extracellular calcium-sensing receptor. Federation of European Physiological Societies Annual Meeting,Slovenia.

Phillips C and D Lipscombe (2008) Modulation of N-type calcium channels associated with β2a subunits. Society for Neuroscience Annual Meeting, Washington, DC.

Raingo J, Phillips C, Denome S, and D Lipscombe (2007) Cell-specific alternative splicing specifies G protein signaling to the N-type calcium channel. Society for Neuroscience Annual Meeting, San Diego, CA.

Smith SM, Phillips C, Harnett MT, and W Chen (2006) Decreases in extracellular Ca2+ activate a non-selective cation channel and facilitate cortical synaptic transmission via the Ca2+ receptor. Society for Neuroscience Annual Meeting, Atlanta, GA.

Phillips C, Harnett MT, and SM Smith (2005) Decreases in extracellular Ca2+ activate a NSCC and facilitate cortical synaptic transmission via the CaR.Spring Brain Annual Meeting,Sedona, AZ.

GRANTS & FELLOWSHIPS

National Institute of Neurological Disorders and Stroke Predoctoral Fellowship 1F31NS066712-01A1, Does alternative splicing regulate G protein inhibition of calcium channels? 1/2010-12/2012

Sidney E. Frank Graduate Fellowship for Biology and Medicine, Brown University, Providence, RI, 2006-7. Awarded to a single first-year graduate student from the Brown

v Division of Biology and Medicine.

HHMI Undergraduate Biological Science Education Program Grant #71100-529603, 2002- 3. Used to fund undergraduate thesis research.

HONORS & AWARDS

Best Brown graduate student research poster, Brown-NIH Graduate Partnership Program retreat, 2009 Reed College Commendation for Academic Excellence, 2002-3

PRESENTATIONS

Research talk at Physiology, Molecular Biology and Neuroscience Institute (IFIBYNCE), Buenos Aires, Argentina, 2011 Research talk at Multidisciplinary Institute of Cell Biology (IMBICE), La Plata, Argentina, 2011 Yearly research talk at neuroscience department in-house seminar, Brown University, 2007-2010 Invited speaker, Rockwell Levy Foundation Board meeting, Brown University, 2007 Invited speaker, graduate student recognition luncheon, Brown University, 2007 Research talk at Molecular Medicine department seminar, Oregon Health Sciences University, 2004 Research talk at Pulmonary & Critical Care Grand Rounds, Oregon Health Sciences University, 2004

TEACHING

Instructor, The Neuron: Form and Function, Summer@Brown, Brown University, June 2009 I independently designed and taught the curriculum for this introduction to molecular neuroscience. The class had a 15 student enrollment and included lectures, videos, group and independent problem solving, research papers and class presentations.

Teaching Assistant to Dr. Carlos Aizenman, Principles of Neurobiology, Brown University, 2008 I taught a weekly review section for 20-40 students covering the previous week’s lectures and led review sessions for exams.

Teaching Assistant to Dr. Maryanne McClellan, Cellular Biology, Reed College, 2003 I prepared experimental tools for class lab, including reagents and cell cultures. I provided direction and guidance to students during a weekly laboratory period as well as during completion of independent research projects.

UNIVERSITY SERVICE

vi Graduate student representative to the Division of Biology and Medicine Faculty Council, 2010-11 Graduate student representative to the Neuroscience Faculty, 2009-10 Member of Division of Biology and Medicine Grad Student Advisory Panel to Dean, 2007-8

OUTREACH

Science Fair Consultant, St. Mary’s Middle School, R.I., 2007 Brain Awareness Week presentation to Girl Scout Troops, Baylor College, R.I. 2007 Brain Awareness Week presentation to Cranston East High School, R.I. 2007

PROFESSIONAL SOCIETY MEMBERSHIP

Society for Neuroscience, 2006-present

vii PREFACE

CaV2.2 voltage-gated calcium channels (CaVs) are expressed at presynaptic terminals in most central and peripheral neurons where they control the calcium (Ca2+) entry that triggers exocytosis. CaV2.2 channels are under tight regulatory control by many intra- and extracellular molecules. One of the best-documented forms of regulation is inhibition by presynaptic G protein- coupled receptors (GPCRs). In the past three decades since this was first discovered we have learned much about inhibition of CaV2.2 by neurotransmitters, neurohormones, and drugs, and also about the structure, function, and binding partners of CaVs, G proteins, and GPCRs. GPCR inhibition of CaV2.2 channels varies by GPCR-type and by tissue. In this thesis I present data that suggest a new perspective in understanding the diversity in coupling between GPCRs and

CaV2.2 channels: that alternative splicing of the target channel determines the ability of specific receptors to inhibit Ca2+ currents. I build off of previous work from our lab showing that alternative splicing of a set of mutually exclusive exons in CaV2.2 pre-mRNA controls the ability of Gi/o protein-coupled receptors (Gi/oPCRs) to inhibit CaV2.2 (Raingo et al., 2007). I first show differential coupling of GPCRs to splice variants of CaV2.2 (Chapter 2), then I show that alternative exon 18a (e18a) modulates inhibition by Gs protein-coupled receptors (GsPCRs; Chapter 3), and finally I present preliminary data gathered to identify the signaling cascade between GsPCRs and e18a- containing CaV2.2 channels (Chapter 4).

viii Thank You

To the mentors who have taught, supported, and encouraged me: I have been incredibly fortunate to have all of you in my life. Especially, and in chronological order:

Harriet Sheridan Anthony Phillips David McKinnon Barbara Rosati Stephen Arch Jesica Raingo

(my committee) Gilad Barnea Julie Kauer Stephen Ikeda

and Diane Lipscombe: my exceptional, creative, and inspiring advisor.

To the past and present members of the Lipscombe Lab:

Arturo Andrade Summer Allen Sylvia Denome Tom Helton Rachel Jiang Kiauntee Murray Andrew Pintea Jesica Raingo Johnathan Tran Kristin Webster Valerie Yorgan

To the Sidney E. Frank Foundation and National Institute of Neurological Disorders and Stroke for supporting my education and research.

To the entire Brown University Neuroscience Graduate Program and Department.

ix TABLE OF CONTENTS

1. Introduction |1

I. CaV2.2 channels are presynaptic and control neurotransmitter release |2

II. CaV channel family |4

III. Alternative Splicing of CaV2.2 pre-mRNA |5 IV. Alternative exon 18a (e18a) |6

V. Regulation of CaV2.2 channels by G proteins|10 VI. E18a sequence suggests posttranslational processing |17 VII. Accessory subunits |19

2. Gq- and Gs-coupled receptor inhibition of CaV2.2 splice isoforms |22

I. Introduction |23 II. Results |26 III. Discussion |35

3. G protein-specific coupling to alternatively spliced exons in Cacna1b controls inhibition of neuronal CaV2.2 calcium channels |39

I. Introduction |40 II. Results |44 III. Discussion |52

4. Threonine and lysine residues in e18a modulate expression and G protein inhibition of CaV2.2 channels |55

I. Introduction |56 II. Results |58 III. Discussion |60

5. Discussion |67

6. Materials and Methods |77

7. References |83

x LIST OF FIGURES + TABLES

FIGURE PAGE

1 Known exocytotic and endocytotic binding partners for CaV2.2 3 channels.

2 Known alternative exons of neuronal CaV channels. 5

3 Alignment of II-III linkers and surrounding transmembrane do- 7

mains of CaV2.2 and CaV2.1

4 Splicing Factors regulate alternative exons. 8

5 Schematic of basic signaling pathways for 3 Gα subunits: Gs, 15 Gi/o, and Gq.

6 Inclusion of e18a renders CaV2.2 susceptible to G protein-medi- 25 ated voltage-independent inhibition when co-expressed with β3 or β2a

7 β2a subunits confer distinct properties to CaV2.2 channels: 27 slowly-inactivating currents and prepulse facilitation (PPF).

8 Gq-coupled M1 mAChR inhibits CaV2.2 splice isoforms in a 29 mostly VI fashion.

9 Gq-coupled B2 bradykinin receptor inhibits CaV2.2 splice iso- 30 forms in a mostly VD fashion.

10 Gq-coupled angiotensin II receptor type 1 (AT1R) inhibits CaV2.2 31 splice isoforms in a mostly VI fashion.

11 Wortmannin prevents B2R inhibition of CaV2.2 channels. 32

12 D1 Dopamine Receptor Inhibits e18a-containing channels less 34 that e18a-lacking channels.

13 D1R inhibition is PTX-insensitive and unrelated to current den- 36 sity.

14 Inclusion of e18a renders CaV2.2 susceptible to G protein-medi- 41 ated voltage-independent inhibition.

xi 15 E18a-dependent voltage-independent inhibition is mediated by 43 Gs and Gβγ

16 The splicing factor Fox-2 represses e18a inclusion. 45

17 Calcium currents from SCG neurons injected with Fox-2 siRNA 47 and uninjected cells are inhibited by the Gs agonist VIP

18 Fox-2 siRNA injection in SCG neurons switches the voltage- 50 dependence of VIP inhibition of the N-type current to mostly voltage independent inhibition

19 β3, but not β2a subunits facilitate e18a-mediated current den- 57 sity increase.

20 Posttranslational modification prediction software predicts 59 phosphorylation of e18a

21 Voltage-dependence of G protein-mediated inhibition and 60

expression of CaV2.2 channels are modulated by specific e18a residues

22 Schematic representing location of e18a-encoded residues and 61 channel mutants

23 Current density and voltage-dependence of GTPγS-mediated 62 inhibition of e18a mutants

24 Schematic of convergent and parallel models of GPCR-mediated 69

inhibition of CaV2.2.

25 Synaptic binding partners for CaV channels 73

TABLE PAGE

1 Voltage-gated calcium channel nomenclature 4

2 Heterotrimeric G proteins 11

xii 1| INTRODUCTION

Portions of this chapter were taken from Lipscombe D, Allen S, and C Phillips (2011) Modulation of voltage-gated calcium channels, In Preparation Trends In Neuroscience. SA created Figure 4. I. CaV2.2 channels are presynaptic and control neurotransmitter release The resting extracellular [Ca2+] in the brain is approximately 1.1 mM (Nicholson et al., 1977), many orders of magnitude greater than the intracellular concentration of 50-100 nM (Dubinsky

2+ and Rothman, 1991). Thus, when CaV2.2 channels open, Ca ions flow down a large electro- chemical gradient into the cell. CaV2.2 proteins are precisely positioned near release machinery to respond to the depolarization of a propagating action potential to trigger exocytosis through the influx of Ca2+ (Dodge and Rahamimoff, 1967; Adler et al., 1991; Wadel et al., 2007; Young and Neher, 2009). Precision in the Ca2+ signal is achieved through control of the open and closed states of the channel in response to membrane voltage (Vm). The open-probability of CaV2.2 is close to zero at resting membrane potential (RMP) but increases with depolarization; CaV2.2 channels begin to activate around -20 mV. At approximately +10 mV, the potential that coincides with the peak of an action potential, the net inward current peaks. Once VCa 2.2 channels open, vesicle fusion with the plasma membrane and exocytosis occur with a delay of less than 1 mil- lisecond, possibly less than 100 microseconds (Borst and Sakmann, 1996; Jahn et al., 2003). The core complex that mediates fusion of vesicles with the plasma membrane is called the SNARE (soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptor) complex (Söll- ner et al., 1993). The SNARE complex consists of three intertwined (coiled-coil), transmembrane, helical SNARE proteins: VAMP (aka synaptobrevin), syntaxin, and SNAP-25 (Chapman et al., 1994). VAMP is a vesicular SNARE (Trimble et al., 1988; Baumert et al., 1989), while syntaxin and SNAP-25 are both target (plasma membrane) SNAREs (Oyler et al., 1989; Bennett et al., 1992). A

Ca2+ binding protein links the influx of Ca2+ to the facilitation of membrane fusion by the SNARE complex. There are at least eight identified Ca2+ sensor proteins, including five synaptotagmins, which may work in concert, in competition, or independently on different vesicle pools (Walter et al., 2011). The mechanism of Ca2+ sensor-mediated fusion is not completely understood, but most likely involves lowering the energy barrier to membrane fusion in the presence of Ca2+, and/or inhibiting membrane fusion in the absence of Ca2+ (Walter et al., 2011).

The complementary pathway to exocytosis is endocytosis, the process of membrane retrieval. Classically, endocytosis occurs via the formation of clathrin-coated membrane pits. Generally, 2 adaptor protein AP-2 binds to the mem-

brane lipid PIP2 , and recruits the vesicle coat protein clathrin vesicle CaN to the membrane V-ATPase vesicle munc13 (McMahon and Bou- SV2 NSF VAMP synaptojanin EPS15 RIM1 dynactin clathrin crot, 2011). Clathrin syntaxin synapsin AP-2 SNAP-25 rab3a dynamin molecules then form munc18 endophilin a scaffolding around CaV 2.2 a pit of membrane, Figure 1. Known exocytotic and endocytotic binding partners for CaV2.2 which buds off into channels. Select binding partners for CaV2.2 are shown in a presynaptic bouton. Proteins shown have been identified by more than one proteomics screen and/ the cytoplasm via or a functional assay. The proteins are generally grouped by function, with exocytotic proteins in green colors on left and endocytotic proteins in purple on the the actions of the right. Each protein was identified by (Muller et al. 2010) and either (Khanna et al 2007a) or (Khanna et al. 2007b). Location in synapse is schematic and is not enzyme dynamin meant to imply binding between any other proteins. (McMahon and Bou- crot, 2011).

2+ Given the integral role of Ca influx in exocytosis, presynaptic CaV2.2 channels must colocalize with fusion machinery to achieve spatial and temporal precision in excitation-secretion coupling

(Adler et al., 1991; Young and Neher, 2009). CaV2.2 channels also bind to many endocytotic proteins: recent mass spectrometry-based proteomics studies have identified proteins that associate with presynaptic calcium channels in vivo, and many key proteins governing exocytosis and endocytosis are CaV2.2 binding partners (Khanna et al., 2007a; Muller et al., 2010). Khanna et al. identified 104 unique proteins that associate with CaV2.2 in rat brain synaptosomes, many of which are part of the fusion pathways including dynamin, adaptin 2, NSF, vaculoar H+ ATPase, syntaxin 1B, VAMP-A, clathrin, munc 18, and rab3a. Subsequently Müller et al. identified over

200 proteins that interact with CaV2 proteins from rodent brain, at least 21 of which were previously identified by Stanley and colleagues (Khanna et al., 2007b; Khanna et al., 2007a). In 3 addition to many cytoskeletal, modulatory, and chaperone proteins, a complex of novel proteins involved in exocytosis and/or endocytosis were isolated, including: SNAP-25, SV2A, syntaxin 1A, synaptotagmins 2 and 7, AP2s-1, dynamin 2, EPS15, munc 13, neurexin-1, calcineurin, synap-

Table 1: Voltage-gated Calcium Channel Nomenclature Protein name Current Gene name old type Human Mouse,Rat Zebrafish Pufferfish Drosophila C.elegans

CaV1.1 a1S L CACNA1S Cacna1s cacna1sa,b 1S-a,b,c Ca-a1D eql-19

CaV1.2 a1C CACNA1C Cacna1c cacna1c 1C

CaV1.3 a1D CACNA1D Cacna1d cacna1da,b 1D-a,b,c,d

CaV1.4 a1F CACNA1F Cacna1f cacna1f 1F-a,b,c

CaV2.1 a1A P/Q CACNA1A Cacna1a cacna1aa,b 1A-a,b cac unc-2

CaV2.2 a1B N CACNA1B Cacna1b cacna1ba,b 1B

CaV2.3 a1E R CACNA1E Cacna1e -- 1E-a,b

CaV3.1 a1G T CACNA1G Cacna1g cacna1g 1G-a,b Ca-a1T cca-1

CaV3.2 a1H CACNA1H Cacna1h -- 1H-a,b

CaV3.3 a1I CACNA1I Cacna1i cacna1i 1I

tojanin, and rab-connecting 3. In Figure 1, select proteins that have been shown to bind CaV2.2 and are involved in either the exocytotic pathway (left; green) or the endocytotic pathway (right; purple) are displayed in a purely schematic fashion to illustrate that CaV2.2 channels themselves can be thought of as scaffolding proteins because they bind to a large variety of integral synaptic proteins.

II. CaV channel family

CaV2.2 channels are encoded by one of the 10 genes that code for CaVα subunit proteins. The voltage-gated calcium channel (CaV) nomenclature was recently adopted; previously these proteins were referred to by an a1X designation or by the characteristics of the current that they underlie (Ertel et al., 2000) (Table 1). Although all CaV proteins open in response to changes in

2+ membrane potential and are permeable to Ca , there is great diversity in the CaV family.

The ten genes that encode the pore-forming, so-called α1, subunits are divided into 5 major families: the L-, N-, P/Q-, R-, and T-type channels (Ertel et al., 2000). T-type channels are low- voltage activated (LVA). The L-, N-, P/Q- and R-type CaVs are mostly high-voltage activated (HVA), 4 except for CaV1.3 and perhaps CaV1.4, and can be distinguished by their drug sensitivity. L-type channels (CaV1.1-1.4) are inhibited by dihydropyridines; N-type channels (CaV2.2) are inhibited by ω-conotoxon GVIA; P/Q-type channels (CaV2.1) are inhibited by ω-agatoxin IVA; and R-type channels are not sensitive to the aforementioned toxins, and therefore are responsible for the residual current remaining when all other channels have been inhibited (hence “R” type) (Trig- gle, 1999). In this thesis I focus on regulation of CaV2.2 channels.

III. Alternative Splicing of CaV2.2 pre-mRNA I need to con rm the locations of all the exons, especially the TM ones

All of the CaVa1 pre-mRNAs are alternatively spliced (Lipscombe and Castiglioni, 2004; Liao et al., 2009). Alternative splicing is a ubiquitous cellular process that results in the selective inclusion or exclusion of particular exons in the final mRNA product. Alternative splicing has emerged as 8/8a 24a 31a 8a/8b 12 21/22 24a 3233 31a out CaV 1.2 31a CaV 1.3 14 25a 31a/b 16 31/32 CaV 1.4 in 17 37a/b CaV 2.1 34 9* 35 CaV 2.2 18a 26 2/2a 9/9a 11 18a 37a/b 35a CaV 2.3 25c 10* 17 26 40b 9 CaV 3.1 14 42 10/10a CaV 3.2 9b 10a 20a 46 42a 43 1/1a 44 CaV 3.3 1a/b/c 47 45a 1 37 45 45

Figure 2. Known alternative exons of neuronal CaV channels. A. Known alternative cassette and mutually exclusive exons (circles) are shown in their approximate locations on a general calcium channel schematic. Numbers nearest to exons indicate exon name. Mutually exclusive exons are designated by a slash (ie X/Y).

Exons were taken from the following references: CaV1.2 (Tang et al., 2009); CaV1.3 (Lipscombe and Casti- glioni, 2004); CaV1.4 (Boycott et al., 2001); CaV2.1 (Soong et al., 2002); CaV2.2 (Lipscombe and Castiglioni,

2004); CaV2.3 (Lipscombe and Castiglioni, 2004) and (Pereverzev et al., 2002); CaV3.1 (Ernst and Noebels,

2009); CaV3.2 (Zhong et al., 2006); CaV3.3 (Gomora et al., 2002).

an important mechanism for cell- and developmental-specific specialization, effectively increasing the number of functionally different mRNAs that can be generated from a fixed number of genes. Alternative splicing is especially prevalent in the nervous system (Lipscombe, 2005; Li et al., 2007), where patterns of alternative splicing vary with neuronal-subtype, development, and neuronal activity (Grabowski and Black, 2001; Nilsen and Graveley, 2010). This variety is consistent with the idea that alternative splicing is a way for individual cells to optimize protein activity 5 as needed. Although large, multi-exon voltage-gated calcium channel genes undergo extensive alternative splicing, the function of only a few alternative exons is currently known (Lipscombe et al., 2002; Chaudhuri et al., 2004; Shen et al., 2006; Andrade et al., 2010).

Interestingly, most alternatively spliced exons encode intracellular and extracellular portions of CaV channels, as is manifest in a collective view of all alternative exons of the neuronal CaV channels together (Fig. 2); their locations are consistent with the idea that alternative exons encode discrete modules that define protein-protein interactions. Splicing can then provide cell- and developmental-specific optimization of channel functions. When I aligned the amino acid sequences of the intracellular II-III linkers and surrounding transmembrane domains of the closely related CaV2.1 and CaV2.2 channels, the sequence homology in the transmembrane domains stood in stark contrast to the intracellular linkers, where few residues are conserved (Fig. 3). Figure 3 also shows the locations of alternative exons, sequence deletions, and sites of posttranslational modifications. This sequence alignment emphasizes that the unique variations arising from alternative splicing in intracellular and extracellular regions provide clues about the structural bases of cellular functions that distinguish closely related two closely related channels

Elegant cellular mechanisms have evolved to regulate inclusion of specific exons depending on cell-type and stage of development (Grabowski and Black, 2001; Li et al., 2007; Chen and Man- ley, 2009; Licatalosi and Darnell, 2010). RNA binding proteins called splicing factors bind to pre- mRNA and can increase or decrease the likelihood of inclusion of an alternative exon (Fu, 1995). Splicing factor enhancers promote exon inclusion; repressors inhibit exon inclusion (Fig. 4A). The splicing factors that control several alternative exons in CaV2.2 are known (Ule et al., 2006; Allen et al., 2010), but most have remained elusive (Fig 4B). In Chapter 3 I present data that I collected in collaboration with Summer Allen which show that splicing factor Fox-2 acts as an repressor for e18a, thus controlling CaV2.2 inhibition by Gs proteins.

IV. Alternative exon 18a (e18a)

In this thesis I focus on a single alternative exon in CaV2.2 pre-mRNA: exon 18a (e18a). E18a is a 6 channel amino acid # e16 S I667 channel amino acid # CaV2.2 583 e16 S I667 CaV2.1 590 CaV2.2 583 CaV2.1 590 e16 E N704 synprint S L718 e17 E K727 Δ1 del. S L754 Δ1 del. S R756 e17 S Y705 synprint S A722 e17a I K727 Δ2 del. S A738 e18a I A755 PKC P S774 e16 E N704 synprint S L718 e17 E K727 Δ1 del. S L754 Δ1 del. S R756 CaV2.2 683 PKC P S774 CaV2.1 e17 S Y705 synprint S A722 e17a I K727 Δ2 del. S A738 e18a I A755 CaV2.2 690683 CaV2.1 690 CaMKII P S784 (2.2) IIS6 II-III linker 2 del. S R793 (2.1) CaMKIIΔ P S784 (2.2) IIS6 II-III linker 781 CaV2.2 Δ2 del. S R793 (2.1) CaV2.1 790 CaV2.2 781 CaV2.1 790 CaMKII P S896,S898 Δ1 del. E P947 PKC P S898 2 del. E P947 CaMKII P S896,S898 Δ1 del. E P947 CaV2.2 871 Δ PKC P S898 2 del. E P947 CaV2.1 884 Δ CaV2.2 871 CaV2.1 884 synprint E H963 synprint E R984 Δ2 del. E P1001 CaV2.2 963 synprint E H963 CaV2.1 968 synprint E R984 Δ2 del. E P1001 CaV2.2 963 CaV2.1 968

CaV2.2 1048 CaV2.11062 CaV2.2 1048 CaV2.11062 Δ1 del. E L1139 CaV2.2 1114 Δ1 del. E L1139 CaV2.11162 CaV2.2 1114 CaV2.11162 II-III linker IIIS1 Figure 3. AlignmentII-III of linker II-III IIIS1linkers and surrounding transmembrane domains of acidic Ca 2.2 and Ca 2.1. Ca 2.1 and Ca 2.2 show little homology in the intracellular II- basic V V V V acidic III linker and strong homology in transmembrane domains. Alignment of Ca 2.1 hydrophobicbasic V polarhydrophobic (NM_012918) and CaV2.2 (AF055477) amino acids covering the intracellular II-III linker di erentpolar and surrounding IIS6 and IIIS1 transmembrane domains adapted from alignment using S di erent Start Clustal Omega software (Sievers et al., 2011). Amino acids are color-coded according E End S Start to their biochemical properties: acidic (green) D, E; basic (purple) K, R, H; hydrophobic EI InsertionEnd PI PhosphorylationInsertion (orange) A, F, I, L, M, P, V, W; polar (blue) C, G, N, Q, S, T, Y. Black bars below alignment P Phosphorylation indicate when the CaV2.1 and CaV2.2 amino acids are in different categories. White circles indicate residues of interest, with description shown above and residue indicated, in categories: (S) alternative exon, deletion (del.), or synprint area start; (E) alternative exon or del., or synprint area end; (I) alternative exon insertion; (P) phosphorylation. References: Alternative exons 16,

17, and 17a of CaV2.1 (Soong et al., 2002); CaV2.2 alternative e18a (Coppola et al., 1994); CaV2.2 synprint

(Sheng et al., 1996); CaV2.1 synprint (Rettig et al., 1996); PKC and CaMKII phosphorylation (Yokoyama et al., 2005); CaV2.1 Δ1 and Δ2 (Rajapaksha et al., 2008); CaV2.2 Δ1 and Δ2 (Kaneko et al., 2002).

63 nucleotide exon that encodes 21 amino acids in the intracellular II-III loop of CaV2.2 (Fig. 2). It is a cassette exon, and is therefore either included or excluded in the final CaV2.2 mRNA, resulting in CaV2.2[e18a] or CaV2.2[D18a] channels, respectively. Below I discuss four characteristics of e18a: channel location; cellular expression; physiological effects; and sequence.

Location: the II-III linker

The intracellular II-III loop of CaV2.2 is encoded by exons 18-21 and a large portion of it is re- 7 Figure 4 ferred to as the synprint (synaptic protein interaction) site, because it includes areas important for in vitro binding of the channel to synaptic proteins like syntaxin and SNAP-25 (Sheng et al., 1996; Jarvis et al., 2000; Hurley et al., 2004; Keith et al., 2007; Watanabe et al., 2010; Fig. 3).

A. Example splicing factor mechanisms B. Known exons regulated by splicing factors

Channel Exon Location Splicing CaV 2.1 24 24a 25 31 31a 32 pre-mRNA factor + – ↑ Nova Nova CaV2.1 24a IIIS3-4 Nova + – ↓31a IVS3-4 Nova CaV 2.2 24 24a 25 31 31a 32 pre-mRNA CaV2.2 ↑24a IIIS3-4 Nova ↓31a IVS3-4 Nova ↓18a II-III Fox Fox PTB Fox CaV1.2 ↓8a IS6 PTB – – + CaV 1.2 7 8a 8 9 9* 10 31/ 33 34 35 ↓9* I-II Fox pre-mRNA 32 ↑33 IVS3-4 Fox E12 cortical neurons adult cortical neurons CaV1.3 ↓8b IS6 Fox PTB Fox ↑11 I-II Fox

CaV 1.2 7 8 9 9* 10 31/ 34 35 7 8a 9 10 31/ 33 34 35 ↑ mRNA 32 32 CaV1.1 29 IVS3-4 Fox

Figure 4. Splicing Factors regulate alternative exons. A. Example splicing factor mechanisms. The first example shows three important principles about splicing regulation: (1) the same splicing factor can act as both a splicing enhancer and a repressor; (2) a single splicing factor—here Nova—can regulate more than one exon—here exons 24a and 31a—in the same transcript; and (3) the same splicing factor can coregulate exons in multiple pre-mRNAs—here CaV2.1 and 2.2 (Allen et al., 2010). The second schematic shows how the expression of splicing factors in a given cell-type and/or at a specific developmental time point determines which splice isoforms are expressed. In this example, PTB is expressed in E12 cortical neurons and represses the inclusion of exon 8a in CaV1.2 mRNA, allowing mutually exclusive exon 8 to be included. Adult cortical neurons do not express PTB but do express Fox, which represses exon 9* and enhances exon 33 (Tang et al., 2011; Tang et al., 2009). This means that the dominate CaV1.2 splice isoform in these cells includes exon 8a, lacks 9*, and contains exon 33. B. Table shows splicing factors known to regulate alternative exons in CaV genes. References: CaV2.1 and CaV2.2e31a (Allen et al.,

2010); CaV2.2e24a (Ule et al., 2006); CaV2.2e18a (Minovitsky et al., 2005); CaV1.2e8a (Tang et al., 2011);

CaV1.2e9* and e33 (Tang et al., 2009); CaV1.3 and CaV1.1 (Gehman et al., 2011).

Because of these interactions, it has been suggested that the II-III linker is important for local- izing channels with the presynaptic release complex (Zamponi, 2003). In support of this, splice variants of CaV2.2 lacking a large portion of the synprint region (D1 variant; Fig. 3) show altered presynaptic clustering in cultured hippocampal neurons (Szabo et al., 2006). Yet, targeting of

CaV2.2 to synaptic terminals is not affected by this deletion and insertion of the synprint region from CaV2.2 into the CaV1.2 II-III linker does not change their trafficking from somatodendritic compartments to axonal (Szabo et al., 2006). Others have shown that transplantation of the 8 homologous synprint region in CaV2.1 to the CaV1.2 II-III loop conferred on CaV1.2 channels the ability to support synaptic transmission (Mochida et al., 2003). These findings provide strong support for a particular role for the II-III linker in localization with presynaptic machinery, but not trafficking to specific subcellular compartments. However, new work suggests that presynaptic CaV channels are also complexed with endocytotic proteins: the synprint region of CaV2.2 and CaV2.1 but not CaV1.2 interacts with the endocytotic component AP-2μ (adaptor protein for clathrin-mediated endocytosis) (Watanabe et al., 2010). In addition, injection of a CaV2.2 synprint peptide into presynaptic calyx boutons specifically interferes with endocytosic capacitative changes, suggesting the novel hypothesis that interactions via the II-III linker of VCa 2.2 control the endocytotic, not the exocytotic, process (Watanabe et al., 2010). E18a is inserted into the II-III linker relatively close to the IIS6 transmembrane domain, and soon after the start of the synprint region (Fig. 3). Because the amino acids chosen to be part of the synprint region were done so on a gross scale (as peptides of ~90+ amino acids (Sheng et al., 1994)) and the exact sites for synaptic protein binding to CaV2.2 are unknown, it is difficult to know if the site of e18a insertion is, in fact, close to where synaptic proteins bind. Additionally, all of the binding studies with

CaV2.2 or CaV2.1 and synaptic proteins have only used a small peptide encoding portions of the II-III linker (Sheng et al., 1996; Jarvis et al., 2000; Hurley et al., 2004; Keith et al., 2007; Watanabe et al., 2010). No study has shown an interaction of a synaptic protein with a full-length channel. Therefore, although it is an attractive hypothesis that e18a could affect SNARE protein interactions with CaV2.2, we do not yet have evidence to support this theory.

E18a expression

CaV2.2 channels containing e18a are found in central (CNS) and peripheral (PNS) nervous systems. The expression pattern of CaV2.2[e18a] channels suggests enrichment in monoaminergic cells: in rat CNS, the highest levels of e18a sequences were found in dopaminergic and sero- tonergic cells of the ventral tegmental area, substantial nigra, dorsal raphe nucleus, and locus coeruleous (Ghasemzadeh et al., 1999). In the PNS, postganglionic sympathetic neurons of SCG, which secrete norepinephrine, are rich in e18a-containing CaV2.2 mRNAs (Pan and Lipscombe, 2000). In addition to tissue-specificity, the expression of e18a is controlled developmentally. 9 While only ~15% of P1 rat SCG neurons contain e18a, ~70% of adult neurons do (Gray et al., 2007; Raingo, Lipscombe 2007) suggesting an important role for e18a in mature synapses.

Physiological effects of e18a Diversity in the kinetics and voltage-dependence of ion channel gating resulting from alternative

splicing are extensively docu- Sympathetic and sensory neurons: model cells for CaV2.2 research. mented (Auld et al., 1988; Most early studies and the majority of contemporary studies on calcium channel physiology are conducted on two main cell-types: sensory neurons Dietrich et al., 1998; Bourinet of the dorsal root ganglion (DRG) and sympathetic neurons of the superior cervical ganglion (SCG). What is special about these two cell-types? First, et al., 1999; Hans et al., 1999; these cells are both in easily accessible peripheral ganglia. Researchers can therefore study, with high reproducibility and limited diversity, the function Pan et al., 2001). Earlier stud- of specific proteins in relatively homogeneous cell populations. Second, both cell-types show a predominance of CaV2.2 channels, which makes for ies from our lab showed that ease of study. For instance, 65-95% of the calcium current in rat SCG is car- ried by CaV2.2 channels (Lin et al., 1996), while in the central nervous system, Ca 2.2 channels only carry 20-30% of the calcium current (Ikeda and e18a protects CaV2.2 channels V Dunlap, 1999). from closed-state and cumu- lative inactivation (Pan and Lipscombe, 2000). These observations are consistent with its presence in the II-III linker, a region of CaV2.2 channels strongly implicated in control of inactivation. This thesis explores two additional effects of e18a on CaV2.2 channels: sensitivity to G protein-mediated voltage-independent inhibition and increased current density.

V. Regulation of CaV2.2 channels by G proteins.

Given their central role in controlling neurotransmission, it is unsurprising the CaV2.2 channels are highly regulated. One of the best-documented forms of inhibition is via presynaptic G protein-coupled receptors (GPCRs). There are more than 1000 GPCRs in mammalian genomes, around 200 of which have identified endogenous ligands. GPCRs selectively bind to and activate intracellular heterotrimeric G proteins Gα, Gβ and Gγ (Gβ and Gγ function as a dimer, Gβγ). In their basal state, GPCRs are bound to Gα and Gβγ, with Gα bound to GDP. When a GPCR agonist binds its receptor, the receptor undergoes a conformational change that catalyzes Gα to exchange one molecule of GDP for GTP. Both Gα-GTP and Gβγ detach from the receptor and activate downstream effector molecules. Signaling by G proteins is terminated when Gα hydrolyzes 10 GTP to GDP and reassociates with Gβγ. G protein Expression α subunits Gs class There are 17 known Gαs, 5 Gβs, and 12 Gγs, many Gs ubiquitous GsXL neuroendocrine fewer than the number of G protein-coupled re- Golf olfactory epithelium, brain ceptors (Table 2; (Wettschureck and Offermanns, Gi/o class Gi1 widely distributed 2005)). This suggests interesting questions about Gi2 ubiquitous the ability of any one GPCR to transduce a specific Gi3 widely distributed Go neuronal, neuroendocrine intracellular signal. GPCRs are grossly classified Gz neuronal, platelets based on which Gα subunit they bind; there are Ggust taste cells, brush cells Gt-r retinal rods, taste cells four main groups of Gα proteins: Gs, Gi/o, Gq, and Gt-c retinal cones Gq/11 class G12; and although GPCRs have a preferred Gα Gq ubiquitous partner, most have the ability to couple to more G11 almost ubiquitous G14 kidney, lung, spleen than one pathway (Hermans, 2003). The specificity G15/16 hematopoietic cells of Gβγ isoform coupling to receptors is an area of G12/13 class G12 ubiquitous active investigation, and some Gβγ pairs have been G13 ubiquitous shown to bind with higher affinity to certain recep- β subunits β1 widely, retinal rods tors (Oldham and Hamm, 2008). However, more β2 widely, distributed experiments are needed in this area. β3 widely, retinal cones β4 widely distributed β5 mostly brain G proteins undergo lipid modifications that help γ subunits γ1, γrod retinal rods, brain target them to the plasma membrane. Gγ is prenyl- γ14, γcone retinal cones, brain ated—the covalent attachment of isoprenoids to γ2, γ6 widely γ3 brain, blood C-terminal cysteine residues—and therefore Gβγ γ4 brain, other tissues γ5 widely travels in the membrane to exert its effects (Chen γ7 widely and Manning, 2001). Gα proteins are palmitoylated, γ8, γ9 olfactory, vomeronasal epithelium γ10 widely which is a dynamic process so their anchoring to γ11 widely Table 2. Heterotrimeric G proteins. Each of the G protein γ12 widely types, α, β, and γ, is encoded by multiple genes with γ13 brain, taste buds variable expression patterns. AC (adenylyl cyclase); inh. (inhibits); act. (activates). Table was adapted from Wet- tschureck and Offermanns (2005). 11 the plasma membrane is a mechanism that is subject to regulation (Chen and Manning, 2001). Interestingly, Gi/o proteins are uniquely susceptible to N-myristoylation, the irreversible attachment of a hydrophobic myristoyl group. This modification is thought to help transiently target Gi/o to the plasma membrane where the membrane’s intrinsic palmitoyl-CoA can palmitoylate the subunit, causing more stable anchoring (Chen and Manning, 2001). The relevance of this to Gi/o vs Gq or Gs membrane targeting is not yet known.

Three classes of Gα proteins inhibit CaV2.2 channels: Gs, Gi/o, and Gq. Pathways activated by these G proteins are of great physiological significance: some modulate the short-term dynam- ics of presynaptic calcium signaling and attenuate synaptic transmission (Pfrieger et al., 1994; Wu and Saggau, 1994; Wu and Saggau, 1995; Dittman and Regehr, 1996; Brown et al., 2003; Gundlfinger et al., 2007; Zhang and Linden, 2009). In this thesis I present data on the coupling between Gs-coupled receptors and e18a-containing CaV2.2 channels.

GPCR inhibition of CaV2.2 channels: early findings. In 1978, at a time when the whole-cell patch clamp recording technique was nascent (see (Hamill et al., 1981) a paper currently with >16,000 citations), Kathleen Dunlap and Gerald Fischbach showed that the neurotransmitters serotonin, GABA, and norepinephrine (NE) all decreased the calcium component of the somatic action potential when applied to cultured dorsal root ganglion (DRG) neurons (Dunlap and Fischbach, 1978). Previous work had shown that neurotransmitters were capable of inhibiting presynaptic transmitter release, but the mechanism was unclear. Dunlap and Fischbach correctly speculated that “if the same phenomenon occurs at nerve terminals this might effectively reduce transmitter release.” Soon after, with whole-cell DRG recordings they showed that serotonin, GABA, and NE all directly decrease calcium channel conductance (Dunlap and Fischbach, 1981). A few years later, Dunlap followed up with another seminal study showing that pertussis toxin (PTX)-sensitive G proteins (the Gi/o class) were responsible for the neurotransmitter-mediated calcium channel inhibition (Holz et al., 1986). G proteins were found to be responsible for inhibiting calcium currents, but by what mechanism? One possibility, that G proteins could promote removal of calcium channels from the plasma 12 membrane, was a hypothesis favored by some (Dunlap and Fischbach, 1981). However, this was not the mechanism. In 1989 Bruce Bean showed that NE decreases whole-cell calcium current in DRG by shifting the voltage-dependence of channel activation in a positive direction and by- slow ing channel activation, without altering cell-surface expression of channels (Bean, 1989). Soon after Diane Lipscombe et al. showed the effects of NE inhibition at the single channel level in

SCG neurons (Lipscombe et al., 1989): NE, acting via Gi/o-coupled a2-adrenergic receptors, had no effect on the unitary conductance of N-type channels, but decreased the mean open time of channels by more than 50%. Thus the inhibition of the whole-cell current that Bean described could be explained by a decrease in single channel open probability via an increase in the rate of channel closing (Lipscombe et al., 1989). This type of inhibition is the prototypical voltage- dependent inhibition (VDI), which has since been extensively documented (Boehm and Kubista, 2002; Tedford and Zamponi, 2006).

Having established that neurotransmitters inhibit presynaptic calcium entry via G protein activation, the question of mechanism needed to be answered. Some in the field noted that completely different neurotransmitters could induce “indistinguishable” inhibition of calcium channels, despite binding to completely different receptors that couple to different Gα proteins (Ehrlich and Elmslie, 1995). Ehrlich and Elmslie speculated that distinct Gα proteins that associate with distinct GPCRs (in particular Gs and Gi/o) activate a common downstream signaling molecule to inhibit N-type currents. However, others speculated that Gβγ may mediate VDI (Hille, 1994). A definitive explanation came in the form of a pair of back-to-back Nature papers by Stephen Ikeda and William Catterall’s group in 1996 (Herlitze et al., 1996; Ikeda, 1996b). Stephen Ikeda’s elegant study showed that when Gβγ subunits were injected into SCG neurons the inhibition recapitulated the observed VDI of N-type currents by NE, and occluded further inhibition by NE (Ikeda, 1996). He also showed that overexpression of Go had little effect on basal currents but through its action as a Gβγ sink, it too occluded inhibition by NE. Herlitzeet al. also demonstrated that Gβγ subunits injected into tsA201 cells expressing CaV2.1 channels mimicked VDI seen with Gi/o-coupled receptors. Soon after, multiple groups demonstrated that Gβγ binds directly to the CaV2.2 channel, and although binding sites in the N-terminus, C-terminus, and I-II linker 13 have been found the primary site mediating VDI is thought to reside in the I-II linker (DeWaard et al., 1997; Qin et al., 1997; Zamponi et al., 1997; Furukawa et al., 1998; Stephens et al., 1998; Canti et al., 1999).

GPCR inhibition of CaV2.2 channels: voltage-independent inhibition. Although the focus of much early research was on VDI of calcium channels, there was also evidence that neurotransmitters could mediate a different, voltage-independent form of inhibition (VII). For instance, Bertil Hilleet al. found that the same muscarinic acetylcholine receptor (mAChR) agonist could induce both VDI and VII of N-type currents in SCG neurons (Hille et al., 1995). Researchers began to parse inhibitory pathways into two groups, VDI and VII, based on particular characteristics. A pathway was VD if it was membrane-delimited, induced a right-shift in the voltage-dependence of channel activation, and slowed channel activation kinetics. VII was not membrane-delimited, generally required a diffusible second-messenger, and was observed independent of membrane potential. Electrophysiologically, these two pathways can be grossly separated by a simple protocol in which current is measured by a single voltage step, then compared to the current observed when this voltage-step is proceeded within a few milliseconds, by a strong depolarizing prepulse (pp) to 80 mV. G protein-mediated inhibition relieved by this pp is VDI, while the remaining inhibition is VII. Physiologically, the pp might be analogous to a train of action potentials that induce longer, sustained, presynaptic depolarization. Consistent with this theory, VD inhibition of N-type currents recorded from neurons of chick ciliary ganglia is robust when currents are activated by a single action potential waveform (Artim and Meriney, 2000) whereas VD inhibition of N-type currents in chick DRG is relieved during AP waveform trains (Park and Dunlap, 1998).

GPCR inhibition of CaV2.2 channels: diverse mechanisms mediate VII.

VDI of CaV2.2 channels by GPCRs is wide-spread in neurons in different parts of the nervous system. At least 17 different neurotransmitters activate a VD inhibitory pathway to the CaV2.2 channel (Elmslie, 2003). In contrast, VII of CaV2.2 is more cell-type and neurotransmitter-type specific; only 7 neurotransmitters have been shown to couple to a VI pathway (Elmslie, 2003; Lechner et 14 adenylyl

cyclase PIP2 DAG PKC Gs Gi/o Gq + P PLCβ IP3 protein protein ATP cAMP ChTX PTX IPR 3 Ca2+ PKA P protein protein

Figure 5. Schematic of basic signaling pathways for 3 Gα subunits: Gs, Gi/o, and Gq. See text for details. al., 2005; Kisilevsky et al., 2008). While the mechanism of VDI seems to involve direct binding of Gβγ to CaV2.2 regardless of which GPCR is activated, multiple Gα-specific mechanisms mediate VII. Although similar in functional output, these different VII pathways use distinct signaling molecules and are cell-specific (Surmeier et al., 1995; Ikeda and Dunlap, 1999; Jeong and Ikeda, 2000a; Kammermeier et al., 2000; Suh and Hille, 2002). Few of these mechanisms are completely understood. Each Gα has a specific set of downstream activators that can give clues as to how VII could be Gα-specific (Fig. 5). Both Gi/o and Gs proteins modulate adenylyl cyclase (AC), the enzyme responsible for catalyzing the conversion of ATP to cAMP. Gi/o proteins inhibit AC, thus leading to a decrease in cellular cAMP concentrations, and all of the resultant cAMP-dependent signaling cascades, including activation of Ca2+-dependent protein kinase (PKA). Gi/oPCRs can be pharmacologically inhibited by application of pertussis toxin (PTX), which uncouples Gi/o proteins from their receptor via ADP-ribosylation. Gs proteins function in opposition to Gi/o proteins as they activate AC, leading to an increase in cAMP levels, which can then activate multiple intracellular proteins including PKA. GsPCRs can be pharmacologically inhibited by application of cholera toxin (ChTX), which prevents Gs from being able to hydrolyze GTP, eventually leading to downregulation of the Gs subunit. Gq proteins activate phospholipase C-b (PLCβ), which then cleaves membrane phosphatidylinositol 4,5-bisphophate (PI(4,5)P2 or PIP2) into inositol 1,4,5-tri- sphosphate (IP3) and diacylglycerol (DAG). IP3 activates receptors on the endoplasmic reticulum, leading to an increase in cytosolic [Ca2+]. Protein kinase C (PKC) can then be activated though a combination of DAG activity and Ca2+ binding.

15 PIP2 depletion underlies GqPCR-mediated VII of CaV2.2.

Several GqPCRs inhibit CaV2.2 channels via a VII pathway, including the M1 mAChR (M1R), angiotensin II type 1 receptor (AT1R) and bradykinin receptor type 2 (B2R) (Boehm and Kubista,

2002). Multiple labs now agree on the hypothesis that it is the depletion of membrane PIP2 by Gq-activated PLCβ that mediates Gq-induced VII (Suh and Hille, 2002; Liu and Rittenhouse, 2003;

Gamper et al., 2004). The specific mechanism of PIP2-mediated inhibition of CaV2.2 channels is unknown. I discuss this pathway in greater detail In Chapter 2.

Alternative exon 37a controls Gi/o-mediated VII of CaV2.2.

Although Gi/o proteins had been shown to mediate VDI of CaV2.2 in multiple tissues (see GPCR inhibition of CaV2.2 channels above), in chick DRG it was found that GABA and NE (both of which signal through Gi/oPCRs) could inhibit CaV2.2 currents through both VDI and VII pathways

(Luebke and Dunlap, 1994). Our lab recently showed that VII of CaV2.2 channels via Gi/oPCRs is controlled by the presence of the alternative exon e37a (Raingo et al., 2007; Andrade et al.,

2010). Jesica Raingo et al. showed that alternative exon 37 in CaV2.2 controls inhibition by Gi/oP- CRs. A pair of mutually exclusive exons, e37a and e37b, encode 32 amino acids in the C-terminus of CaV2.2. Channels including e37b are widely expressed, and are inhibited in a VDI manner by Gi/oPCRs. Channels containing e37a have a much more restricted expression pattern: they are highly enriched in small nociceptive neurons of the DRG, but expression in other tissues is more limited (Bell et al., 2004). Raingo found that CaV2.2 channels containing e37a instead of e37b, are susceptible to a Gi/o-mediated inhibition that is voltage-independent. This inhibitory pathway was abolished when a single tyrosine in e37a was mutated to a phenylalanine, and when src-tyrosine kinases were inhibited. In our lab, Sylvia Denome recently created lines of knock-in mice that only express channels containing e37b by replacing e37a in the genome with a second copy of e37b (Andrade et al., 2010). When Andrade recorded from the small nociceptors in DRG from these mice, he found that cells from mice without e37a showed less VII via Gi/o-coupled μ-opioid receptors (μORs). Further, when Yu-Qiu Jiang tested the peripheral analgesic effects of morphine (a μOR agonist) in the mouse lines she found that mice without e37a were significantly less sensitive to morphine analgesia in response to noxious thermal stimuli. These results 16 elegantly demonstrate that a single cell-specific splicing event can control a behaviorally relevant physiological process. The mechanism by which Gi/oPCRs activate a tyrosine kinase is not yet known, nor is the mechanism of VII. However, recent data from our lab point to differential ubiquitination as a potential mechanism to control cell-surface expression of CaV2.2[e37b] and

CaV2.2[e37a] channels. Ubiquitination, current density and G protein-mediated inhibition are somehow related (Spiro Marangoudakis; unpublished).

The work I present in this thesis demonstrates that another alternative exon controls G protein inhibition of CaV2.2, showing that this phenomenon is not limited to e37a. I show that e18a, an alternatively expressed exon that differs from e37a in amino acid sequence, tissue distribution, and its location on the channel, also controls G protein inhibition, but via Gs proteins. The e18a-dependent inhibition is also voltage-independent, and, like e37a, e18a inclusion results in increased current density.

VI. E18a sequence and possible posttranslational processing

CaV2.2 channels undergo posttranslational modification (PTM) events that can effect their expression, activity, and modulation. Here I discuss four PTMs: phosphorylation, ubiquitination, SUMOylation, and acetylation.

Phosphorylation

When mouse CaV2.2 cDNA was first cloned in the early 1990’s (Coppola et al., 1994) one of the clones contained a sequence variation that corresponds to e18a. The authors noted that e18a is rich in serines and contains putative CaMKII and PKC binding sites. The authors concluded their report with the following statement, “it is tempting to speculate that [e18a] might be involved in processes like excitation to secretion coupling.” Since then, studies have shown that CaV2.2 channels are upregulated by many kinases including Ras, ERK, MAPK, CaMKII and PKC (Ahlijan- ian et al., 1991; Martin et al., 2006). Given the importance and ubiquity of regulation of proteins through phosphorylation, it seemed possible, if not probable, that one or several amino acids in e18a might be subject to phosphorylation. In Chapter 4 I present data on the behavior of a series 17 of e18a mutants, including those lacking strategic serines and threonines, to test their potential role in e18a-dependent CaV2.2 behavior.

Lysine modifications: ubiquitination, SUMOylation and acetylation. Ubiquitination, SUMOylation, and acetylation, all involve the covalent attachment of a molecule—ubiquitin, SUMO (small ubiquitin-related modifier), or acetyl—to lysine residues. They are all known to affect a large variety of cellular processes. Ubiquitin attachment is a frequently utilized signal to modify synaptic proteins (DiAntonio and Hicke, 2004; Yi and Ehlers, 2007; Tai and Schuman, 2008; Rotin and Staub, 2010). For instance, activity-dependent ubiquitination of postsynaptic AMPA receptors regulates synaptic plasticity (Colledge et al., 2003; Patrick et al., 2003). Despite functional evidence that ubiquitin-dependent changes in synaptic efficacy involve presynaptic components (Speese et al., 2003; Bingol and Schuman, 2005 ; Rinetti and Schweizer,

2010), CaV channels have been conspicuously absent from lists of ubiquitin targets (Yi and Ehlers,

2007). However, evidence is accruing to suggest that CaV channels are in fact ubiquitinated and that this process is likely important for controlling activity-dependent calcium entry. Two recent studies have shown that ubiquitination of both CaV1.2 and CaV2.2 channels is strongly influenced by auxiliary CaVβ subunits (also see Accessory subunits below) (Altier et al., 2011; Waithe et al.,

2011). Both studies converge on a model in which CaVβ subunit binding protects channels from proteasomal degradation. CaVβ coexpression prevents CaV1.2 channels from ubiquitination by RFP2 ubiquitin ligase, and from entering the endoplasmic reticulum-associated protein degradation (ERAD) complex, a check-point on the way to proteasomal degredation (Altier et al., 2011).

Likewise, a CaV2.2 mutant which cannot bind CaVβ subunits is normally expressed at much lower levels than wild-type CaV2.2, and proteasomal inhibition led to a much larger increase in ubiquitinated mutant protein (360%) versus wild-type (58%), although not increased surface expression

(Waithe et al., 2011). The nature of the CaVβ subunit might be important in predicting its interaction with the ubiquitin-proteasome system. The SH3 domain of CaVβ2a has been reported to down-regulate CaV1.2 channel expression in a dynamin-dependent fashion (Gonzalez-Gutierrez et al., 2007). Furthermore, this internalization is dependent on oligomerization of the β subunits (Miranda-Laferte et al., 2011), although others have shown that oligomerization augments ex- 18 pression of CaV1.2 (Tareilus et al., 1997; Lao et al., 2010).

SUMO proteins can affect protein localization, protein-protein interactions, or activity (Geiss- Friedlander and Melchior, 2007). Although first thought of as a modifier of nuclear proteins, multiple neuronal transmembrane proteins have now been identified as substrates for SUMO including potassium channels and GPCRs (Wilkinson et al., 2010). For instance, SUMOylation shifts the voltage-dependence of activation of KV2.1 and steady-state inactivation of KV1.5 (Benson et al., 2007; Plant et al., 2011).

Acetylation regulates diverse and critical cellular processes (Choudhary et al., 2009). For instance, tau protein is acetylated, which prevents degradation of phospho-tau and prevents tau association with microtubules, leading to tau aggregation, a hallmark of multiple neurodegenerative diseases including Alzheimer’s Disease (Min et al., 2010; Cohen et al., 2011).

Phosphorylation, SUMOylation, acetylation, and ubiquitination processes can modify the same target and influence the likelihood of other PTMs. Transcription factors RUNX3 and smad7 are acetylated, and this acetylation prevents their ubiquitin-mediated degradation (Gronroos et al., 2002; Jin et al., 2004). Phosphorylation of the huntingtin protein enhances its ubiquitination, SUMOylation, and acetylation, all leading to increased proteasomal degradation (Thompson et al., 2009). Dephosphoryation of a serine residue in transcription factor MEF2A leads to a switch from SUMOylation to acetylation of a nearby lysine residue, which significantly affects postsynaptic differentiation (Shalizi et al., 2006).

In Chapter 4 I present data that suggest that both lysine and threonine posttranslational modifications contribute to the e18a-dependent CaV2.2 channel behaviors.

VII. Accessory subunits Besides alternative splicing, calcium channels also achieve great functional diversity through the combination of subunits that form functional channels within cells. CaV channels exist in the cell 19 as a complex of at least 3 proteins: the pore-forming CaVα1 subunit, and the accessory CaVβ and

CaVα2δ subunits. The β and α2δ subunits are encoded by 4 genes each (Arikkath and Campbell,

2003). Although CaV channels are referred to by their α1 subunit identity, each channel subunit imparts a unique constellation of characteristics, and therefore great heterogeneity in calcium channel activity is achieved through various combinations of pore-forming and accessory subunits.

Cavβ accessory subunits: which is the binding partner for CaV2.2? There are four neuronal accessory β subunits: β1, β2a, β3, and β4. β1, β3, and β4 are cytosolic proteins, while β2a can be palmitoylated, and is thereby membrane-anchored. β subunits traffic α subunits to the membrane (E. Takahashi and T. Nagasu, 2005) and modulate many important characteristics of the pore-forming subunit, including current density, activation and inactivation kinetics (Pan and Lipscombe, 2000), sensitivity to G proteins (Sandoz et al., 2004; Tedford and Zamponi, 2006; Heneghan et al., 2009), and protection from proteasomal degradation (Waithe et al., 2011). In this thesis I study e18a, the inclusion of which is enriched in monoaminergic neurons in general (Ghasemzadeh et al., 1999) and sympathetic neurons of the superior cervical ganglion (SCG) in particular (Pan and Lipscombe, 2000). I focus on the channels expressed in SCG, and therefore have particular interest in which b subunit binds to CaV2.2 in SCG. All four β subunits are expressed in rat SCG tissue (Lin et al., 1996), and it is unclear which b subunit is the in vivo binding partner for CaV2.2, although multiple pair-wise associations are likely. When

CaV2.2 is expressed with β3, the channel rapidly inactivates in response to depolarization.

However, when CaV2.2 is expressed with β2a, two characteristic features are observed: (1) the channels are essentially non-inactivating (Yasuda et al., 2004), and (2) there is basal prepulse facilitation in response to a strong depolarization. In one of the first studies of calcium currents in SCGs, Stephen Ikeda showed many clear examples of non-inactivating calcium currents that display a “double-pulse facilitation” reminiscent of what is found in expression systems (Ikeda, 1991). Many studies since then have shown similar electrophysiological profiles (e.g., (Wollmuth et al., 1995; Lin et al., 1997)). Monoaminergic chromaffin neurons show prepulse facilitation and non-inactivating currents (Artalejo et al., 1992), and when bovine CaV2.2, α2δ, and β2a clones 20 are expressed in Xenopus oocytes they mimic these non-inactivating currents. Direct presynaptic nerve terminal recordings from another monoaminergic tissue, the cholinergic chick ciliary ganglion, show Ca2+ currents with the same non-inactivating profile, once again consistent with the presence of β2a (Stanley and Goping, 1991). Given the distinctive characteristics of CaV2.2 channels containing β3 compared to β2a, I hypothesized that CaV2.2 channels in SCG neurons containing e18a bind β2a. Therefore in Chapter 3 I show recordings of CaV2.2 channels from cells expressing either b3 or b2a. I show that the β subunit does not affect e18a-dependent G protein mediated inhibition of CaV2.2 channels.

21 Gq- and Gs-coupled receptor

2 inhibition of CaV2.2 splice isoforms

Portions of this chapter were taken from the manuscript presented as Chapter 3 by Allen SE*, Phillips CG*, Raingo J, and D Lipscombe In Submission (*equal contributions). J Raingo performed the experiments shown in Figs. 1A, B, and some of Fig. 8. I. INTRODUCTION There are four main GqPCRs in SCG that have been extensively studied and shown to inhibit

CaV2.2 channels: muscarinic acetylcholine receptor type 1 (M1R), bradykinin receptor type 2 (B2R), angiotensin II receptor type 1 (AT1R), and purinergic receptor type 2 (P2YR). GqPCRs classically activate PLCβ, which hydrolyses PIP2 into IP3 and DAG, two cytosolic second messengers

(Fig. 5). This leads to depletion of the membrane lipid PIP2. For years it was known that these GqPCRs inhibit N-type currents in SCG, but the mechanism remained unclear. Work from Mark

Shapiro’s lab was the first to show that the mechanism of M1R inhibition of CaV2.2 channels in SCG is depletion of PIP2 (Gamper et al., 2004), as had already been shown for CaV2.1 (Wu et al., 2002). This work has now been confirmed by many, including Bertil Hille’s group (Suh et al.,

2010). However, Gamper et al. found that while the B2R induced PIP2 hydrolysis, it did not inhibit Ca2+ current like the M1R. Their data suggested that the B2R-mediated increase in intracellular

2+ Ca stimulated PIP2 synthesis, compensating for the hydrolysis, and therefore not inhibiting the calcium current. They recently followed up on this idea by defining two subgroups of GqPCRs: the M1R and the AT1R vs the B2R and the P2YR (Zaika et al., 2011). While the M1R and AT1R

2+ produce small increases in intracellular Ca , deplete PIP2, and inhibit N-type currents, the B2R

2+ and P2YR produce large increases in intracellular Ca , don’t deplete PIP2, and do not inhibit N-type currents (but see Boehm’s work below). They propose that B2R and P2YR colocalize with intracellular IP3Rs, thus the hydrolysis of PLCβ into IP3 leads to IP3R activation and a spike in

2+ 2+ intracellular Ca . This intracellular Ca then activates NCS1, which activates PI4K, which synthe- sizes PIP2, thus leading to no net depletion of PIP2. They suggest that the M1R and AT1R are not located close enough to IP3Rs to activate them, thus there is no compensatory synthesis of PIP2,

and CaV2.2 channels are inhibited by PIP2 depletion. Others have shown that different phospho- lipases and their products are also necessary for M1R inhibition: Liu et al. from Anne Ritten-

house’s group found that PLA2, AA, and DAGL are necessary for CaV2.2 inhibition by M1Rs in SCG (Liu et al., 2004; Liu et al., 2008).

Interestingly, both the inability of B2R to inhibit CaV2.2 and the necessity of PLA2 for inhibition

have been disputed. For instance, Stefan Boehm’s lab consistently shows inhibition of CaV2.2 23 by the B2R (Edelbauer et al., 2005; Lechner et al., 2005). Additionally, they found no effect of

2+ depletion of intracellular Ca stores on the inhibition (Lechner et al., 2005) and inhibition of PLA2 had no effect on B2R-mediated inhibition (Lechner et al., 2005). It is difficult to reconcile these results since the labs use very similar preparations and solutions. More work needs to be done to resolve these discrepancies.

While some have focused on the role of PIP2, others have challenged the assumption that Gq

activation of PLCβ is the only G protein pathway necessary for M1R inhibition of CaV2.2. In fact,

both Gα and Gβγ subunits released from M1R are involved in the VII of CaV2.2 in SCG (Kam- mermeier et al., 2000). When the Gq subunit was specifically inhibited, inhibition by M1R was converted from almost completely VII to mostly VDI (the overall amount of inhibition was unchanged), suggesting that the VII and VDI pathways initiated by M1R activation are in opposition, with the VI pathway normally predominating. However, surprisingly, when Gβγ proteins were sequestered by MAS GRK2, all forms of M1R inhibition were almost completely abolished,

demonstrating that Gβγ is also necessary for VII of CaV2.2.

Other recent work on GPCR inhibition of CaV2.2 has purported a role for the β subunit in determining the efficacy of inhibition. Some have shown β2a, as opposed to β3 or β4, when expressed

with CaV2.2, dramatically enhances the ability of the Gβ G protein to inhibit the channels (Feng

et al., 2001). Others have shown that the M1R cannot inhibit CaV2.2 when coexpressed with β2a (Heneghan et al., 2009). And still others have shown no difference in the characteristics of Gβγ modulation of N-type channels when they were expressed with β1b or β2a (Meir and Dolphin, 2002). These data, although potentially conflicting, provided us with the impetus to identify

if both β3 and β2a could facilitate inhibition of CaV2.2 splice isoforms by GTPγS. We generally

use CaVβ3, which is found co-localized with CaV2.2 proteins in several brain regions (Scott et al.,

1996). But CaV2.2 proteins can associate with different CaVβ subunits, and all four CaVβ subunits

are expressed in sympathetic ganglia where CaV2.2[e18a] transcripts are at their highest levels

(Lin et al., 1996). Because CaVβ2a supports slowly inactivating N-type currents, with kinetics similar to native N-type currents recorded from sympathetic neurons (Ikeda, 1991; Lin et al., 1996), I 24 A. D18a + b3 B. e18a + b3 test potential (mV) test potential (mV) -60 -40 -20 20 40 60 -60 -40 -20 20 40 60

-100 -100 (pA/pF) (pA/pF) 2+ Con -pp 2+ Ca Ca

I I -150 GTPγS -pp -200 GTPγS +pp

C. D18a + b2a D. e18a + b2a test potential (mV) test potential (mV) -60 -40 -20 20 40 60 -60 -40 -20 20 40 60

-40 -40

(pA/pF) (pA/pF) 2+ 2+ Ca Con +pp Ca

I I GTPγS -pp -80 GTPγS +pp -80

Figure 6. Inclusion of e18a renders CaV2.2 susceptible to G protein-mediated voltage-independent inhibition when co-expressed with β3 or β2a. Calcium currents from cells expressing CaV2.2[D18a] (A,C), or CaV2.2[18a] (B,D) along with b3 (A,B) or b2a (C,D) and α2δ1. Cells were recorded from with internal solution without (Con) or with (GTPγS) 0.4mM GTPgS. Currents were evoked by 50 ms test pulses to voltages shown on X-axis without (-pp) or with (+pp) a 25 ms prepulse to 80 mV. (A,C) In the presence of GTPγS, currents from cells without e18a (D18a) showed only voltage-dependent inhibition: they recovered fully to the Con levels after prepulse (+pp). (B,D) Channels expressing e18a showed both voltage- dependent and voltage-independent inhibition: they recovered partially after prepulse (+pp). N values: (A) Con –pp = 14, GTPγS = 12 , GTPγS +pp = 10 ; (B) Con –pp = 19, GTPγS = 15, GTPγS +pp = 11; (C) Con +pp = 10, GTPγS = 7, GTPγS +pp = 6 (D) Con +pp = 16, GTPγS = 6, GTPγS +pp = 6. studied N-type currents in cells co-expressing β2a or β3.

Recent work from our lab has shown that GTPγS-mediated VII of CaV2.2[e18a] channels in tsA201 cells is both dependent on Gβγ and inhibited by RGS2, similar to what has been found for M1R inhibition in SCG ((Kammermeier et al., 2000) and see Chapter 3). Additionally, e18a-containing channels are expressed at their highest levels in SCG tissue (Pan and Lipscombe, 2000).

Similarities to Kammermeieret al.’s findings, in addition to the fact that VII of CaV2.2 has been shown for not only the Gq-coupled M1R and B2R but also the AT1R (Shapiro et al., 1994; Yamada et al., 2002), prompted us to investigate the hypothesis that e18a controls VII Gq signaling to. 25 II. RESULTS

β3 and β2a both support e18a-mediated VII of CaV2.2. In order to investigate the effect of alternative exon 18a on G protein-mediated inhibition of

CaV2.2, cloned channels lacking e18a (CaV2.2[Δ18a]) or containing e18a (CaV2.2[e18a]) were coexpressed with β3 and α2δ1 in tsA201 cells (Fig. 6A,B). Calcium currents were then recorded under conditions designed to stimulate G proteins. Endogenous G proteins were activated by the inclusion of a non-hydrolysable GTP analogue, GTPγS, in the recording pipette (0.4 mM). GTPγS bypasses GPCRs by globally and non-specifically activating all G proteins, inducing maximal G protein inhibition. G proteins inhibit CaV2.2 channels by two well-described, functionally distinct pathways that are voltage-dependent (VD) or voltage-independent (VI). VD inhibition is reversed by a brief, strong depolarizing prepulse (pp) to 80 mV. VDI depends on Gβγ binding to the I-II intracellular CaV2.2 linker, is membrane delimited, and is widely observed. VII is resistant to strong depolarization, uses at least three different Gα proteins (Gi/o, Gs, and Gq) that each activate distinct signaling cascades, and is cell-specific (Surmeier et al., 1995; Ikeda and Dunlap, 1999; Jeong and Ikeda, 2000b; Kammermeier et al., 2000; Suh and Hille, 2002). In tsA201 cells,

CaV2.2[Δ18a] channels are inhibited by internal GTPγS via a VD mechanism exclusively: in the presence of GTPγS, strong depolarizing prepulses (+pp) normalize the voltage-dependence of activation and current amplitudes to control levels consistent with VD inhibition (Fig. 6A and see Chapter 2; Raingo et al., 2007). By contrast, in cells expressing CaV2.2[e18a] channels, we observed VDI as well as prominent VII in the presence of internal GTPγS (Fig. 6B). Currents were strongly reduced compared to control recordings over a range of test potentials, and significant inhibition remained following prepulses to +80 mV (Con –pp vs. GTPγS +pp at 5 mV, p = 0.034).

Additionally, current densities in cells expressing CaV2.2[e18a] channels are significantly larger than in cells expressing CaV2.2[Δ18a] channels (at 0 mV, p = 0.048).

Others have reported that the efficacy and properties of G protein inhibition of CaV2.2 channels depend on the identity of the auxiliary CaVβ. The above experiments were therefore replicated with β2a substituted for β3. Consistent with β3 recordings, internal GTPγS only triggered VII of 26 Figure 7

A CaV 2.2[∆18a] B CaV 2.2[e18a] C test potential (mV) test potential (mV) ∆18a -60 -40 -20 20 40 60 -60 -40 -20 20 40 60 e18a 150

125 -40

-40 100 % pp facilitation (pA/pF) (pA/pF) 2+

2+ Ca Con -pp I Ca Con +pp I -40 -20 0 20 40 -80 -80 testpulse potential (mV) D CaV 2.2[∆18a] E CaV 2.2[e18a] F *

80 mV 50 100 80 mV 5 mV 5 mV 1.5 -80 mV -80 mV

-pp -pp 200 pA 200 pA +pp +pp 1.0 25 ms 25 ms normalized current area current normalized PTX PTX+pp

80 mV 120 mV 80 mV G H I 0 mV 0 mV 0 mV 0 mV 0 mV 0 mV -100 mV -100 mV -100 mV 200 pA 200 pA 20 ms 10 ms 500 pA 10 ms

2.0 2.0 1.0

1.5 1.5

0.8

1.0 1.0

normalized current amplitude 0 10 20 30 40 50 60 normalized current amplitude 0 20 40 60 80 100 120 normalized current amplitude 0 20 40 60 80 100 prepulse duration (ms) prepulse potential (mV) interpulse interval (ms)

27 Figure 7. β2a subunits confer distinct properties to VCa 2.2 channels: slowly-inactivating currents and prepulse facilitation (PPF). (A,B) PPF is present in both Δ18a and e18a splice isoforms (Δ18a n=10 ; e18a n=16). C) PPF is voltage-dependent, reaching a maximum of 155% of control at ~ 0mV. (D,E) Example recordings demonstrating that β2a creates CaV2.2 currents that exhibit PPF. (F) PPF is PTX-insensitive. Cells were treated with PTX (500ng/ml, 16hrs, n=5), and recordings were done as in (A); individual points are shown in circles over average bar graph (star indicates 2 overlapping data points). (G-H) β2a-dependent PPF is voltage- and time-dependent. Magnitude of PPF was compared under different stimulation protocols. (G) Cells were held at -100 mV and challenged with a +80mV PP 12.5 ms before a 25 ms step to 0mV. Duration of PP was varied from 1 to 55 ms. (H) Cells were held at -100mV and challenged with a 25 ms PP 12.5 ms before a 25ms step to 0 mV. Magnitude of PP was varied from 0 to 120 mV. (I) Cells were held at -100mV and subjected to a 25 ms PP before a step to 0 mV. Interpulse interval was varied from 1 to 96 ms. Yellow bar represents value used in all other experiments; traces above show example recordings with first (black) and last (red) traces from each protocol; individual cells are shown in gray, and average +/- std error in black.

CaV2.2[e18a], not CaV2.2[Δ18a] channels expressed with β2a (Fig. 6C,D). Therefore, at least in the case of β3 and β2a, the e18a-mediated VII is β subunit independent.

There were, however, differences between recordings with β3 and β2a. CaV2.2 currents in cells co-expressing β2a inactivated more slowly compared to β3 and showed robust prepulse facilitation in control recordings in the absence of GTPγS (Fig. 7), as reported by others (Dresviannikov et al., 2009). Basal facilitation of CaV2.2 currents by prepulses was voltage-dependent, peaked at ~150% at 0 mV (Fig. 7C), and was PTX-insensitive (Fig. 7F). Basal prepulse facilitation of

CaV2.2[e18a] channels was statistically greater than VCa 2.2[Δ18a] channels (at 10 mV, p = 0.035). The basal prepulse facilitation was also voltage- and timing-dependent (Fig. 7G-I).

GqPCRs inhibit CaV2.2 with different efficacies.

Our data show that e18a imparts a unique sensitivity to VII on CaV2.2 channels. We also found that this inhibition is dependent on Gβγ and is sensitive to RGS2 (see Chapter 3). These data correspond remarkably well to what Stephen Ikeda’s group found when studying Gq-coupled M1R inhibition of N-type currents in SCG (Kammermeier et al., 2000), the tissue that happens to express the greatest concentration of e18a-containing transcripts (Pan and Lipscombe, 2000).

Therefore, we hypothesized that e18a was controlling Gq signaling to CaV2.2. The Gq-coupled

M1R should therefore inhibit CaV2.2[e18a] channels in a more voltage-independent manner than 28 CaV2.2[Δ18a] channels. In order to test this hypothesis, we co-transfected tsA201 cells with the

M1R and CaV2.2+/Δ18a, β3, and α2δ1. We then stimulated the receptor with the agonist oxo- tremorine-M (oxo-M), and looked at the pattern of calcium current inhibition by the M1R. Be- cause GPCRs undergo desensitization, I recorded IVs with only 8 points to minimize any change in receptor function or expression. Surprisingly, the M1R inhibited both isoforms in a mostly VII B A CaV2.2[e18a] or oxo-M

CaV2.2[D18a] oxo-M 200

CR GP G P M1R C R 100

peak current (-pA) peak current

R C P

G 0 1 2 3 time (min.) C D E Δ18a +18a test potential (mV) test potential (mV) -20 20 40 60 -20 20 40 60

oxo-M -100 oxo-M +pp -50

con +pp control -200 oxo-M oxo-M +pp current density (pA/pF) current current density (pA/pF) current -100 -300

100 F G 100 H 100

80 75 75

60 50 50

% of control % of control 40 % of control

25 20 25 +18a Δ18a +18a Δ18a +18a Δ18a +18a Δ18a % voltage-independent inhibition 4 8 oxo-M oxo-M +pp Δ18a +18a oxo-M oxo-M +pp

Figure 8. Gq-coupled M1 mAChR inhibits CaV2.2 splice isoforms in a mostly VI fashion. (A) Schematic indicates tsA201 cells expressing either CaV2.2[e18a] or CaV2.2[Δ18a] were co-expressed with the M1R and inhibited by application of oxo-M (50μM). (B) Example diary plot showing oxo-M inhibition of calcium currents. (C) Example currents demonstrate the voltage-independent nature of M1R inhibition. (D,E) IVs are shown for cells held at -70mV and stepped to the voltages indicated on the X-axis for 50ms. oxo-M was rapidly applied onto the cell with a microperfusion pipe, and after inhibition had reached a steady state level, voltage-steps were repeated with and without a preceding prepulse (+pp). Values are shown as average +/- std error, points fit with Boltzman-linear function. (F,G) Individual IVs were integrated then averaged to calculate % inhibition and voltage-dependence of inhibition.(H) Single steps from -100 mV to 0 mV showed similar pattern to steps from -70 mV (F). N values shown on histogram bars. 29 fashion, and there was no difference in the overall amount of inhibition of CaV2.2[Δ18a] and

CaV2.2[e18a] channels (Fig. 8).

This was unexpected given the similarity of our results with GTPγS to those found by others in SCG. However, as discussed above, there is diversity in the behavior of GqPCRs. Therefore

I tested the inhibition of CaV2.2 by another Gq-coupled receptor, the B2 bradkyinin receptor (B2R), which has been shown to inhibit N-type channels in SCG neurons in a VI manner (Lechner et al., 2005). I used the β2a subunit instead of β3 in these recordings; although it has not been definitively shown which b subunit couples to CaV2.2 in symapthetic neurons, I hypothesized

A B 500 nM bradykinin Ca 2.2[e18a] or V 600 bradykinin CaV2.2[D18a]

CR GP G P B2R C R 400

Current amplitude (-pA)

R C P G 1 2 3 4 5 6 Time (min.) C D E 100100 100100 D18a +18a

80 mV 80 mV 10 mV 10 mV 7575 7575 -80 mV -80 mV

5050 5050

bk bk % of control

con bk +pp 25 25 200 pA 25 25 20 ms con 150 pA 20 ms con+pp bk +pp con+pp +18a D18a +18a D18a % voltage-independent inhibition 4 5 0 BK BK +pp +18a D18a

Figure 9. Gq-coupled B2 bradykinin receptor inhibits CaV2.2 splice isoforms in a mostly VD fashion. (A)

Schematic indicates tsA201 cells expressing either CaV2.2[e18a] or CaV2.2[Δ18a] were co-expressed with the B2R and inhibited by application of bradykinin (0.5 μM). (B) Example diary plot showing bradkyinin inhibition of calcium currents. (C) Example currents demonstrate the voltage-dependent nature of B2R inhibition of both CaV2.2[Δ18a] and CaV2.2[e18a] channels. (D,E) As shown in C, cells were held at -80mV and stepped to +10mV for 50 ms with and without a preceding prepulse (+pp) to +80mV. Currents were integrated then averaged to calculate inhibition. N values shown on histogram bars. 30 CaV2.2[e18a] or angiotensin II A B -600 X1 mM CaV2.2[D18a] Ang II

CR GP G P -400 AT1R C R

-200 current amplitude (pA) current

R C P

G 0 1 2 3 time (min.)

C D 100 E 100

D18a +18a 80

80 mV 80 mV 75 10 mV 10 mV -80 mV -80 mV 60

50 40 ang II ang II+pp ang II % of control ang II+pp 20 25 100 pA 200 pA 20 ms con 20 ms con+pp con +18a 18a +18a % voltage-independent inhibition 0 con+pp Δ Δ18a 1 2

Ang II Ang II +pp +18a Δ18a

Figure 10. Gq-coupled angiotensin II receptor type 1 (AT1R) inhibits CaV2.2 splice isoforms in a mostly

VI fashion. (A) Schematic indicates tsA201 cells expressing either CaV2.2[e18a] or CaV2.2[Δ18a] were co-expressed with the AT1R and inhibited by application of angiotensin II (1 μM). (B) Example diary plot showing angiotensin II inhibition of calcium currents. (C) Example currents demonstrate the voltage- independent nature of AT1R inhibition of both CaV2.2[Δ18a] and CaV2.2[e18a] channels. (D,E) As shown in C, cells were held at -80mV and stepped to +10mV for 50 ms with and without a preceding prepulse (+pp) to +80mV. Currents were integrated then averaged to calculate inhibition. N values shown on histogram bars. that it was β2a as discussed in the Introduction chapter. In my recordings when I coexpressed

β2a with CaV2.2, shown in Fig. 7, I saw robust basal prepulse facilitation and slowed inactivation kinetics, similar to native SCG currents (Ikeda, 1991). Therefore to replicate as closely as possible the native situation I coexpressed VCa 2.2 +/Δ18a with β2a, α2δ1, and the B2R. I stimulated the B2R with bradykinin (500 nM) and recorded the inhibition and voltage-dependence of inhibition of the channels (Fig. 9). The B2R showed a surprising and strikingly different inhibitory pattern to what I saw with the M1R: the inhibition was almost completely voltage-dependent (Fig. 9E).

However, the receptor did not differentiate between the two splice isoforms.

31 A 100 B

100 75 75

50 50 25

25 % inhibition by bradykinin % VD inhibition 0

% volatge-independent inhibition 0 2 4 6 8 10 12 12 3 9 time of wortmannin treatment M1R AT1R B2R before bradykinin (min) C D wortmannin bradykinin wortmannin bradykinin bradykinin 4.9 min 2.3 min 10.4 min 3.8 min 1.0 1.0

0.8 0.8

0.6 0.6 bk bk con con 0.4 bk+pp 0.4

100 pA bk+pp 100 pA con+pp con+pp 10 ms 10 ms 0.2 0.2 Normalized current area Normalized current area 0.0 0.0 0 2 4 6 8 10 12 0 5 10 15 20 25 time (min) time (min) Figure 11. Wortmannin prevents B2R inhibition of CaV2.2 channels.( A) The voltage-dependence of inhibition of all cells (both Δ18a and e18a) shown in Figs. 8, 9, and 10 were averaged together demonstrating that the M1R and AT1R induce very voltage-independent inhibtion of CaV2.2 while the B2R inhibition is very voltage-dependent. N values shown on bars. (B) Four cells were incubated with 50 uM wortmannin in DMSO for various times (0 - 10.4 min.) then challenged with 200 nM bradykinin. The relationship between time of wortmannin incubation and inhibition of CaV2.2 currents by bradykinin is represented. (C,D) Two example diary plots of cells where wortmannin has no effect C( ) and where it prevents bradykinin-mediated inhibition (D) are shown. Inset are example current trances from control currents or bradykinin treated currents +/- prepulse to +80 mV (pp).

Finally, I tried a third Gq-coupled receptor, the AT1R, which has also been shown to inhibit CaV2.2 in a VII fashion (Shapiro et al., 1994). I co-expressed the +/Δ18a isoforms with β3 and α2δ1 and stimulated the receptor with angiotensin II (1 mM). As shown in Figure 10, although the receptor did not differentiate between the e18a isoforms, it did show a behavior that was unique as compared to M1R and B2R: the inhibition was almost completely VII (Fig. 10E).

Recently, Shapiro and colleagues published work that showed that the M1R and AT1R deplete 32 plasma membrane PIP2, while the B2R does not (Zaika et al., 2011). Given that in my experiments M1R and AT1R inhibit CaV2.2 in a VI fashion, while B2R inhibits in a VD fashion, I hypothesized that I wasn’t seeing B2R-mediated VII because PIP2 wasn’t being depleted. Shapiro and colleagues have shown that B2R activation simultaneously stimulates the synthesis of PIP2 and the depletion of PIP2, thereby leading to no net change in membrane PIP2 concentration (Zaika et al., 2011). I therefore tested the hypothesis that if VII is due to PIP2 depletion, I could increase the amount of VII via B2R by inhibiting the PIP2 synthesis pathway. The PIP2 synthesis pathway involves the conversion of PI to PI4P by PI4K, which is then converted to PIP2 by PI5K. I therefore treated cells with the PI4K inhibitor wortmannin (50 µM), which should prevent PIP2 synthesis. I used a variety of acute incubation times to find the ideal time for wortmannin efficacy and- mea sured the inhibition of CaV2.2 currents by B2R (Fig. 11). I found that wortmannin did not induce B2R-mediated VII, but, surprisingly, it occluded all inhibition by the receptor. I have not done further work on this line of inquiry.

After finding that none of three GqPCRs differentiate betweenV Ca 2.2[D18a] and CaV2.2[e18a] channels, I revisited the evidence that we had based the Gq hypothesis on. Jesica Raingo had shown that co-expressing RGS2 (regulator of G protein signaling) with CaV2.2 occluded the

GTPgS-mediated VII, but left the VDI seemingly unchanged (see Chapter 3). RGS2 is ubiquitously expressed in the nervous system and was first cloned and presented as a Gq-specific inhibitor (Heximer et al., 1997; Ingi et al., 1998; Han et al., 2006). RGS2 has therefore been used many times to show Gq specific events. However, more recent studies have shown that RGS2 may be a non-specific G protein inhibitor (Hendriks-Balk et al., 2008) as it can inhibit Gi proteins (Ingi et al., 1998; Han et al., 2006) as well as Gs proteins (Roy et al., 2006a). Because Raingo also found that the e18a-dependent VII is PTX-insensitive, I ruled out Gi proteins as the mediator of VII. This left Gs proteins.

There are many GsPCRs expressed in the nervous system, but I wanted to find one relevant to tissues that express CaV2.2[e18a] channels. In addition to SCG, e18a is expressed centrally in multiple areas that include the substantia nigra, ventral tegmental area, dorsal raphe nucleus, 33 A Cav2.2[∆18a] B dopamine or Cav2.2[e18a] dopamine -1400

D1R GPCR GPCR -1200

-1000 GPCR

GPCR -800 Current amplitude (pA)

GPCR -600 0 50 100 150 +18a ∆18a time (sec)

C +18a test potential (mV) D Δ18a test potential (mV) 80mV 0mV 0 0 PP Ca 2.2[∆18a] -40 -20 0 20 40 -40TP -20 0v 20 40 or Ca 2.2[e18a] -80mV dopamine v -50 -10

GPCR D1R GPCR dopamine

-100 pA/pF

pA/pF dopamine +pp

-20 DA GPCR control

DA+ GPCR P P control-150 control +pp 200 pA control + P P

current density (pA/pF) current density (pA/pF) current GPCR -30 10 ms 2+ 100 pA 2+ Ca Ca +18a 10 ms ∆18a E F G H +18a Δ18a 80mVD1R inhibits0mV both e18a splice-variants. 80PP mV 80 mV 0 TPmV 0 mV -80mV-80 mV -80 mV

DA DA+ P P control 200 pA control + P P 10 ms 100 pA 10 ms

Figure 12. D1D1R Dopamine inhibits both Receptor e18a splice-variants. Inhibits e18a-containing channels less that e18a-lacking channels.

(A) Schematic indicates tsA201 cells expressing either CaV2.2[e18a] or CaV2.2[Δ18a] were co-expressed with the D1R and inhibited by application of dopamine (20 μM). (B) Example diary plot showing dopamine inhibition of calcium currents. (C,D) IVs are shown for cells held at -80mV and stepped to the voltages indicated on the X-axis for 50ms. Dopamine was rapidly applied onto the cell with a microperfusion pipe, and after inhibition had reached a steady state level, voltage-steps were repeated with and without a preceding prepulse (+pp). Values are shown as average +/- std error, points fit with Boltzman-linear function. (E) Example currents from both isoforms. (F,G) Currents were integrated then averaged to calculate the % inhibition and voltage-dependence of inhibition. N values: +18a (24); Δ 18a (16).

locus coeruleous (Lin et al., 1997; Ghasemzadeh et al., 1999). One striking commonality is that all these tissues contain monoaminergic cells. The dopamine receptor type 1 (D1R) is an im-

portant Gs-coupled receptor expressed presynaptically on midbrain neurons that project to the striatum (Yoland Smith, 2008). There is strong evidence that the D1R inhibits the N-type channel: 34 in the striatum the D1R inhibits the N-type channel through phosphatase activity (Surmeier et al., 1995), and in cells of the prefrontal cortex the D1R inhibits the N-type channel in a voltage- independent manner (Kisilevsky et al., 2008).

I therefore co-expressed CaV2.2[D18a] and CaV2.2[e18a] channels with the D1R as well as α2δ1 and β3 (which shows a high level of expression in mouse midbrain (Lein et al., 2007)). I tested the hypothesis that the Gs-coupled D1R inhibits e18a-containing channels in a more VII fashion than e18a-lacking channels. I found that the D1R robustly inhibited expressed CaV2.2 channels

(Fig. 12), but the voltage-dependence of the inhibition was not different for D18a and +18a channels (Fig. 12H; p = .87). However, there was a surprising and significant difference in the overall amount of inhibition: channels containing e18a are inhibited less than those without e18a (Fig 12G; p = .0009). Because PTX-sensitive proteins have been shown to strongly couple to VDI pathways, I tested the effect of PTX incubation on the VD D1R pathway, and found that it PTX-insensitive, consistent with Gs, and not Gi/o, mediated signaling (Fig. 13A,B). Additionally, because cells with e18a channels expressed a much higher current density than D18a channels (Fig. 13C and see Chapter 4), I tested for a potential correlation between current density and inhibition that could account for the isoform-dependent difference. I found no significant trends

(Fig. 13D). Overall, the D1R is the only GPCR which differentiated between the VCa 2.2 channel isoforms with and without e18a.

III. DISCUSSION

I show in this chapter that inclusion of e18a mediates GTPgS-induced voltage-independent inhibition of CaV2.2. This inhibition occurs in the presence ofb 3 or b2a. However, I was unable to recapitulate this finding using a GPCR instead of GTPgS. The only receptor that differentiated between CaV2.2[D18a] and CaV2.2[e18a] channels is the Gs-coupled D1R, but the difference was is the overall amount of inhibition, not the voltage-dependence of inhibition.

PIP2-mediated inhibition of CaV2.2 by GqPCRs

Here I show robust inhibition of CaV2.2 currents with three GqPCRs: the M1R, AT1R, and B2R. 35 A B 1001.0 +18a PTX (7) D1R cells with PTX 100 ∆18a PTX (6) 0.880 l

o 75 0.660 r t n o c

0.440 f o 50

t n

e 0.220 c r percent voltage-independent e 25 p 0.00

plus delta % voltage-independent inhibition (+ PTX) +18a Δ18a dopamine dopamine p = 0.039 +PP C D +18a 6000 Δ18a 1.0

4000

0.5

-(pA*ms/pF) 2000 peak charge densitypeak charge (-pA*ms/pF) +dopamine fraction of control +dopamine -5000 -10000 -15000 0 +18a Δ18a control pA*ms/pF p18a d18a Figure 13. D1R inhibition is PTX-insensitive and unrelated to current density. (A,B) Cells were treated exactly as in Fig. 12, except they were preincubated with pertussis toxin (PTX; 500ng/ml, 16hs) before recordings then analyzed for % inhibition by dopamine and voltage-dependence of inhibition. (C)

CaV2.2[e18a] channels (n=24) co-expressed with D1R show greater current density than CaV2.2[Δ18a] channels (n=16). (D) Magnitude of dopamine inhibition is plotted versus current density, showing no significant trend.

These receptors have been shown to inhibit CaV2.2 activity via depletion of membrane PIP2 .

But what is the mechanism? PIP2 comprises approximately 1% of the membrane phospholipids (Gamper and Shapiro, 2007a). Among its known functions include serving as a “lipid anchor” that attaches the cytoskeleton to the membrane and thus can effect both exocytosis and endocytosis (Itoh et al., 2001). Hypotheses concerning how PIP2 depletion could differentially affect different ion channels were discussed in Gamper and Shapiro (2007). One common hypothesis is that there are local microdomains of concentrated PIP2 , so that a GqPCR in such a microdomain

36 would deplete PIP2 only locally, however there are many different theories as to the mechanism of such clustering. Additionally, because this inhibition can be recorded in a cell-attached patch when the agonist is applied outside of the pipette, the second messenger, here PIP2 , must be diffusible. Therefore, there may be microdomains, but they cannot be static. Thus, the mechanism still remains elusive, and PIP2 binding sites have yet to be found in CaV channels (Gamper and Shapiro, 2007b). Hints at mechanism maybe be found in studies of other ion channels; PIP2 modulates many other channels, and recent work in cortical neurons showed that PIP2 inhibits NMDA receptors via clathrin- and dynamin-dependent internalization (Mandal and Yan, 2009).

Variability in voltage-dependence of inhibition by GqPCRs My recordings with Gq-coupled M1Rs, AT1Rs, and B2Rs, though they did not prove valuable for differentiating between e18a-containing and e18a-lacking CaV2.2 channels, did show striking differences in the voltage-dependence of their inhibition of calcium currents. M1Rs and AT1Rs inhibit in a mostly VII fashion, while B2Rs show very VDI. This was surprising, and lead me to hypothesize that Mark Shapiro and colleagues’ observation that B2Rs, unlike M1Rs and AT1Rs, do not cause net depletion of membrane PIP2 (Zaika et al., 2011), may underlie the differences in my recordings. However, when I prevented the resynthesis of PIP2 (which would normally com- pensate for PLCβ-induced depletion of PIP2) using wortmannin, instead of changing the voltage- dependence of B2R-mediated inhibition I saw a complete ablation of inhibition. Wortmannin targets both PI3K and PI4K. Like PLCβ, PI3K acts on PIP2, except it converts it to PIP3 instead of to IP3 and DAG. PI4K is involved in the PIP2 synthesis pathway, converting PI to PIP, which is then converted to PIP2 by PIP5K. When Boehm and colleagues treated SCG with wortmannin, they found that bradykinin still inhibited Ca2+ currents, but there was little recovery from inhibition (Lechner et al., 2005). They showed this effect was independent of PI3K activity, and concluded that PIP2 resynthesis is necessary for recovery from B2R-induced inhibition. In my experiments I did not see inhibition by wortmannin on its own, suggesting that wortmannin was not acting on PIP2 depletion via PI3K. However, some component of the B2R-mediated inhibitory pathway was occluded by wortmannin. This is particularly intriguing because of the strong voltage- dependence of B2R inhibition, which suggests the involvement of Gβγ subunits, which are not 37 generally incorporated into models of GqPCR-mediated PIP2 depletion (but see (Kammermeier et al., 2000)). More experiments need to be done to understand why B2Rs cannot inhibit CaV2.2 channels after wortmannin treatment; it would be interesting to see what effect buffering Gβγ had on the pathway, as well as if wortmannin affects M1R or AT1R inhibitory pathways.

No difference in GTPγS-mediated e18a-dependent inhibition of CaV2.2with β2a and β3.

Multiple labs have shown substantial differences in susceptibility to G proteins when CaV2.2 is expressed with different accessory β subunits (Feng et al., 2001; Sandoz et al., 2004; Heneghan et al., 2009). However, at least for GTPγS-mediated inhibition of CaV2.2 channels, I see no difference in the pattern of inhibition. There are, however, substantial differences in the kinetics of activation and inactivation between the two subunits, but this has been reported by others in similar preparations (Stanley and Goping, 1991). Therefore, although β subunits may affect other

G protein pathways, the one that couples G proteins to e18a-including CaV2.2 channels is unaf- fected by the differences in β isoform.

D1R differentially inhibits CaV2.2[Δ18a] and CaV2.2[e18a] channels I hypothesized that the Gs-coupled D1R would inhibit channels containing e18a in a more voltage-independent fashion than those lacking e18a. However, I saw no difference in the voltage- dependence of inhibition and rather saw a difference in the overall magnitude of inhibition by D1R. Surprisingly, channels without e18a are inhibited more than channels with e18a. It would be interesting to understand why that is – perhaps e18a insertion interrupts a binding site on the

II-III linker that faciliatates inhibition of CaV2.2.

Overall the data I present in this Chapter shows that there is great diversity in the signaling pathways to which GPCRs can couple, and diversity in the sensitivity of different CaV2.2 splice isoforms to inhibition by particular GPCRs.

38 G protein-specific coupling to alternatively spliced exons in Cacna1b 3 controls inhibition of neuronal Ca 2.2 V calcium channels

This chapter was taken from the manuscript of the same name by Allen SE*, Phillips CG*, Raingo J, and D Lipscombe In Submission (*equal contributions). ABSTRACT

Numerous Gi/o, Gq, and Gs protein-coupled receptors (GPCRs) converge on and inhibit presynaptic

CaV2.2 channels. Such broad sensitivity to G protein-dependent pathways explains how CaV2.2 channels convey signals from many neurotransmitters and drugs to modify synaptic transmission. However, this widely accepted convergence model, cannot explain the different sensitivi- ties of CaV2.2 channels to GPCRs in different neurons. We showed previously that alternatively spliced exon e37a of the Cacna1b gene controls Gi/o protein inhibition of CaV2.2 channels. Here we show that a second alternatively spliced exon, e18a, confers sensitivity to inhibition by Gs proteins. We also link the cell-specific splicing factor Fox-2 to e18a repression and control of

Gs-dependent inhibition of native CaV2.2 channels in individual neurons. We suggest that alternative splicing sets the sensitivity of CaV2.2 channels to at least two specific G proteins that inhibit splice isoforms via parallel independent, rather than convergent, signaling pathways.

I. INTRODUCTION

An impressive number of G protein-coupled receptors (GPCRs) that signal through Gi/o, Gq, or

Gs proteins converge on CaV2.2 channels to inhibit presynaptic calcium entry and transmitter release (Boehm and Kubista, 2002). GPCR inhibition of CaV2.2 channels underlies short-term depression of synaptic transmission induced by neurotransmitters and drugs at several synapses (Wu and Saggau, 1994; Wu and Saggau, 1995; Dittman and Regehr, 1996; Brown et al., 2003; Gundlfinger et al., 2007; Zhang and Linden, 2009). Consequently, there is tremendous interest in defining the signaling pathways that mediate GPCR inhibition of CaV2.2 channels. Addition- ally, given the high degree of GPCR convergence on a single class of calcium channels that is expressed widely in the nervous system, the question of how cell-specificity of neurotransmitter and drug action is achieved remains unanswered.

GPCRs inhibit CaV2.2 channels by at least four distinct pathways. Three use diffusible second messengers activated by Gi/o, Gs, or Gq proteins. Inhibition by these pathways occurs independent of the membrane potential (voltage-independent, VI) and is cell-specific (Surmeier et al., 1995; Ikeda and Dunlap, 1999; Jeong and Ikeda, 2000a; Kammermeier et al., 2000; Suh and 40 Figure-1 Lipscombe

∆18a 37b 18a 37b 18a 37a a Ca V 2.2[∆18a, e37b] b CaV 2.2[e18a, e37b] c CaV 2.2[e18a, e37a] 80 mV 80 mV 80 mV 0 mV 0 mV 0 mV -100 mV -100 mV -100 mV

Con -pp Con -pp Con + pp Con -pp Con + pp Con + pp test potential (mV) test potential (mV) test potential (mV) -60 -40 -20 20 40 60 -60 -40 -20 20 40 60 -60 -40 -20 20 40 60

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-100 -200 (pA/pF) (pA/pF) (pA/pF) 2+ 2+ 2+ Ca Ca I Ca I -150 Con -pp -200 I -300 Con +pp

d CaV 2.2[∆18a, e37b] e CaV 2.2[e18a, e37b] f CaV 2.2[e18a, e37a] Con GTPγS Con GTPγS Con GTPγS -pp -pp +pp -pp -pp +pp -pp -pp +pp -5 -5 -5 -5 -5 -5 -5 10 10 10 25 25 25 25 -5 -5 10 25 10 25 25 10 25 10 10 25 10 test potential (mV) test potential (mV) test potential (mV) -60 -40 -20 20 40 60 -60 -40 -20 20 40 60 -60 -40 -20 20 40 60

-100

-100 -150 (pA/pF) (pA/pF) (pA/pF) 2+ 2+ Con -pp 2+ Ca Ca Ca I I -150 GTPγS -pp I -200 GTPγS +pp -250

Figure 14. Inclusion of e18a renders CaV2.2 susceptible to G protein-mediated voltage-independent inhibition.(A-F) Calcium currents from cells expressing CaV2.2[Δ18a,37b] (A,D), CaV2.2[18a,37b] (B,E), or

CaV2.2[18a,37a] (C,F) along with β3 and α2δ1. Cells were recorded from with internal solution without (Con) or with (GTPγS) 0.4mM GTPγS. Currents were evoked by 50 ms test pulses to voltages shown on X-axis without (-pp) or with (+pp) a 25 ms prepulse to 80 mV. (A,B,C) In the absence of GTPγS channels did not undergo prepulse facilitation. Example currents are shown above IVs. Schematics above currents indicate splice variant expressed with approximate location of alternative exons on channel structure. (D) In the presence of GTPγS, currents from cells without e18a (Δ18a) showed only voltage-dependent inhibition: they recovered fully to the Con levels after prepulse (+pp). Channels expressing e18a (E) showed both voltage-dependent and voltage-independent inhibition: they recovered partially after prepulse (+pp). Channels expressing both e18a and e37a (F) showed even less recovery from GTPγS-mediated inhibition, hence even more voltage-independent inhibition. Example current traces from steps to -5, 10, and 25 mV are shown above IVs. N values: (A,D) Con –pp = 14, Con +pp = 14, GTPγS = 12 , GTPγS +pp = 10 ; (B,E) Con –pp = 19, Con +pp = 19, GTPγS = 15, GTPγS +pp = 11; (C,F) Con –pp = 15, Con +pp = 15, GTPγS = 10, GTPγS +pp = 8 . Scale bars: (A-C) 5 ms, 500 pA;(D-F) 20 ms, 500 pA.

41 Hille, 2002). The fourth pathway, mediated by direct binding of Gβγ to CaV2.2, requires close co-localization of GPCR and CaV2.2 channels (membrane-delimited), and is relieved during strong membrane depolarization (voltage-dependent, VD) (Herlitze et al., 1996; Ikeda, 1996; Jeong and Ikeda, 2000a).

We were struck by the parallels between the cell-specificity of VI inhibition of CaV2.2 channels by GPCRs and the cell-specific expression of CaV2.2 splice isoforms. Indeed we recently showed that an alternatively spliced exon—e37a—of the CaV2.2-encoding gene, Cacna1b, controls Gi/o protein-dependent VI inhibition of CaV2.2 channels (Raingo et al., 2007; Andrade et al., 2010).

Enrichment of e37a in nociceptors suggests why CaV2.2 channels in nociceptors are so sensitive to inhibition byi/o G -coupled GPCRs. We know of only one other alternatively spliced exon in Cacna1b, e18a, that is expressed in a tissue-specific pattern and has the potential to interact with cytoplasmic second messenger cascades (Coppola et al., 1994; Ghasemzadeh et al., 1999; Gray et al., 2007).

A major limitation associated with testing the cellular consequences of alternative splicing is the lack of tools to selectively target specific splice isoforms. Endogenous regulators of alternative splicing exert cell-specific control over splice isoform expression levels, but only one splicing factor, Nova-2, is known to bind and control alternative splicing of CaV2.2 pre-mRNAs (Allen et al., 2010). We aligned Cacna1b gene sequences of several species and confirmed a conserved putative binding site for the Fox family of splicing factors immediately upstream of e18a (Minovitsky et al., 2005). We used this information to test the role of Fox-2 in e18a expression and subsequently used siRNA to Fox-2 to shift the pattern of e18a splicing in neurons.

Here we show that the alternatively spliced exon e18a, which encodes a unique 21 amino acid sequence in the II-III loop of CaV2.2, controls VI inhibition by Gs proteins in neurons. Our new findings reveal an elegant cellular solution for maintaining and independently controlling separate G protein inhibitory pathways to a single class of CaV2.2 channel.

42 Figure-2 Lipscombe

a CaV 2.2[e18a], PTX b CaV 2.2[e18a], RGS2 Con GTPγS Con GTPγS -pp -pp +pp -pp -pp +pp -5 -5 -5 -5 -5 25 10 25 25 -5 10 10 25 25 25 10 10 10

test potential (mV) test potential (mV) -60 -40 -20 20 40 60 -60 -40 -20 20 40 60

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Ca γ Ca I GTP S -pp I -300 GTPγS +pp -200

c CaV 2.2[e18a], ChTX d CaV 2.2[e18a], GRK2 Con GTPγS Con GTPγS -pp -pp +pp -pp -pp +pp -5 -5 -5 10 -5 -5 -5 25 25 25 25 25 25 10 10 10 10 10

test potential (mV) test potential (mV) -60 -40 -20 20 40 60 -60 -40 -20 20 40 60

-50

(pA/pF)

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Figure 15. e18a-mediated voltage-independent inhibition is mediated by Gs and Gβγ.

(A,B) Calcium currents from cells expressing CaV2.2[18a,37b], β3 and α2δ1. Cells were recorded with internal solution without (Con) or with (GTPγS) 0.4mM GTPγS. Currents were evoked by 50 ms test pulses to voltages shown on X-axis without (-pp) or with (+pp) a 25 ms prepulse to 80 mV. Example current traces from steps to -5, 10, and 25 mV are shown above IVs. (A) Currents from cells pretreated with Gi/o inhibitor pertussis toxin (PTX; 500ng/ml, 16hs). All G protein-mediated inhibition was voltage-independent: currents showed no prepulse facilitation in the presence of GTPγS (GTPγS -pp vs. GTPγS +pp). (B) Currents from cells co-transfected with Gq and Gs inhibitor RGS2 showed mostly VD inhibition. (C) Currents from cells pretreated with Gs inhibitor cholera toxin (ChTX; 500ng/ml, 16hs). All G protein-mediated inhibition was voltage-dependent: currents completely recovered to control current density levels (Con) after prepulse (GTPγS+pp). (D) Currents from cells co-transfected with Gβγ scavenger MAS GRK2ct. Cells did not show VD or VI inhibition when Gβγ was scavenged. N values: (A) Con = 17, GTPγS –pp = 21, GTPγS +pp = 18; (B) Con = 17, GTPγS –pp = 21, GTPγS +pp = 18; (C) Con = 12, GTPγS -pp = 9, GTPγS +pp = 10; (D) Con = 17, GTPγS –pp = 17, GTPγS +pp = 15. Scale bars: (A) 1 nA, 5 ms; (B) 500 pA, 5 ms; (C) 100 pA, 10 ms; (D) 300 pA, 5 ms. 43 II. RESULTS

Gs- and Gq-coupled receptor inhibition of CaV2.2 channels is observed in several types of neurons but it is particularly prominent in sympathetic neurons (Zhu and Ikeda, 1994; Surmeier et al.,

1995; Kammermeier et al., 2000). Notably, e18a-containing CaV2.2 mRNAs dominate in adult sympathetic neurons (Coppola et al., 1994; Ghasemzadeh et al., 1999; Pan and Lipscombe, 2000;

Gray et al., 2007). We hypothesized that e18a might control VI inhibition of CaV2.2 channels by

Gs and/or Gq.

E18a is required for VI inhibition of cloned channels independent of Gi/o We first tested the role of e18a in a mammalian expression system using global G protein activation to inhibit CaV2.2 channels lacking (CaV2.2[∆18a]) and containing e18a (CaV2.2[e18a]). GTPgS globally activates all G proteins, inducing maximal G protein inhibition, thus bypassing GPCRs.

We could isolate membrane delimited inhibition by G protein activation, mediated by Gβγ binding to the I-II intracellular CaV2.2 linker, by its voltage-dependence (VD). The presence of VD inhibition also served as a useful control for the integrity of G protein inhibition of CaV2.2 channels.

The cell-specific inhibitory pathways that use Gi/o, Gs, and Gq to activate their distinct signaling cascades (Surmeier et al., 1995; Ikeda and Dunlap, 1999; Jeong and Ikeda, 2000b; Kammermeier et al., 2000; Suh and Hille, 2002) all inhibit CaV2.2 channels by a VI mechanism that persists during strong depolarization. We used depolarizing prepulses to +80 mV to separate VD from VI inhibition of CaV2.2 channels.

In tsA201 cells, in the absence of GTPgS, prepulses do not facilitate CaV2.2 currents (Fig. 14A-

14C). GTPgS inhibits CaV2.2[D18a] channels but exclusively via a VD mechanism; in the presence of GTPgS, strong depolarizing prepulses (+pp) normalize both the voltage-dependence of activation and current amplitudes to control levels (Fig. 14D; Raingo et al., 2007). By contrast, in cells expressing CaV2.2[e18a] channels internal GTPgS activates prominent VI inhibition in addition to VD inhibition (Fig. 14E). Currents are strongly reduced compared to control recordings over a range of test potentials, and significant inhibition remains following prepulses to +80 mV

(Con –pp vs. GTPgS +pp at 5 mV, p = 0.034). Additionally, current densities of cells expressing 44 Figure-3 Lipscombe a b Fox binding site Exon 18a ControlFox-2 siRNA Homo sapiens (human)* TTTTTGCATGTGCAGTTTTGTAAAGCA Pan troglodytes (chimp) TTTTTGCATGTGCAGTTTTGTAAAGCA Macaca mulatta (macaque) TTTTTGCATGTGCAGTTTTGTAAAGCA 50 Canis canis (dog)* TTTTTGCATGTGCAGTTTTGTAAAGCA Felis catus (cat) CTTTTGCATGTGCAGTTTTGTAAAGCA Bos taurus (cow) TTTTTGCATGTGCAGTTTTGTAAAGCA Equus caballus (horse) TTTTTGCATGTGCAGTTTTGTAAAGCA 37 anti-Fox Mus musculus (mouse)* TTCTTGCATGTGCAGTTTTGTAAAGCA Rattus rattus (rat)* TTCTTGCATGTGCAGTTTTGTAAAGCA Ornithorhynchus anatinus (platypus) TTTTTGCATGTGCAGTTTTGTAAAGCA Monodelphis domestica (opossum) TTTTTGCATGTGCAGTTTTGTAAAGCA Gallus gallus (chicken)* TTTT -GCATGTGCAGTTTTGTAAAGCA 25 37 anti-GAPDH c d +18a +18a ∆18a ∆18a 60 60

40 40 % +18a % +18a 20 20

0 1 10 100 none cycB [Fox-2 siRNA] (nM) con 3 siRNA Figure 16. The splicing factor Fox-2 represses e18a inclusion. (A) Genomic alignment: a conserved Fox-2 binding site is located upstream of e18a. Star denotes species used in alignment by Minovitskey and colleagues 21. Putative Fox-1/2 consensus sequence, (U)GCAUG, is 100% conserved in the upstream intron. In most cases, Fox-1/2 proteins act to repress the inclusion of an alternative exon when they bind upstream of the exon. (B) Western blot showing knockdown of Fox protein in cells transfected with siRNA against Fox-2. GAPDH expression is shown below as a loading control, and the experiment was run in triplicate. (C) RT-PCR analysis of +18a and ∆18a channel mRNAs from F11 cells transfected with Fox-2 siRNA. Three independent transfections were analyzed per condition. There was a significant increase in percent of channels containing e18a: 22.5% ± 2.3 of mRNAs from untransfected cells vs 58.4% ± 2.5 in cells transfected with 100nM Fox-2 (star indicates 0 vs. 100 nM, Student’s t-test, p = 0.000129). (D) RT-PCR analysis of +18a and ∆18a channel mRNAs extracted from F11 that were untransfected (none), transfected with siRNA against cyclophillin B (cycB) or transfected with control siRNA (con 3). Above: representative gel; below: average of 3 exps. There is no significant difference in the percentage of channels containing +18a: 20.3 ± 2.3% of mRNAs from cycB cells and 26 ± 1.7% of mRNAs from con 3 cells and 22.5% ± 2.3 from untransfected cells (one-way ANOVA, F = 1.789, p = 0.246).

45 CaV2.2[e18a] channels are significantly larger than those of cells expressing CaV2.2[D18a] channels (at 0 mV, p = 0.048).

E18a-dependent VI inhibition of CaV2.2 channels is independent of PTX-sensitive i/oG proteins (Fig. 15A), conditions that would have completely eliminated e37a mediated VI inhibition of

CaV2.2 channels (Raingo et al., 2007)—PTX occludes VD inhibition of CaV2.2[e18a] channels by internal GTPgS while sparing VI inhibition. GTPgS-mediated VI inhibition of CaV2.2 channels containing both e18a and e37a (Fig. 14F) is greater than the level of VI inhibition exhibited by channels containing e18a (Fig. 14E) or e37a alone (Raingo et al., 2007). We conclude that a PTX- insensitive G protein uses e18a to mediate VI inhibition of CaV2.2 channels. By contrast, VD inhibition of all splice isoforms tested (D18a, e18a, e37b, e37a) uses Gβγ generated from Gi/o protein activation when activated by gGTP S (Fig. 15A; (Raingo et al., 2007)).

E18a mediates VI inhibition by Gs and Gβγ

Gs and Gq mediate the majority of inhibitory actions of PTX-insensitive GPCRs on CaV2.2 channels in neurons (Boehm and Kubista, 2002; Tedford and Zamponi, 2006). The regulator of G protein signaling 2 (RGS2) is used frequently to occlude Gq-mediated signaling, but it is also reported to occlude Gs signaling (Heximer et al., 1997; Roy et al., 2006a; Roy et al., 2006b). In tsA201 cells expressing RGS2, internal GTPgS engaged only VD—not VI—inhibition of CaV2.2[e18a] channels

(Fig. 15B), suggesting Gq and/or Gs is responsible for the VI inhibition. To differentiate between Gs and Gq involvement we pretreated cells with cholera toxin (ChTX), a specific inhibitor of Gs (Fig. 15C). In cells pre-treated with ChTX, similar to our results with RGS2, GTPgS triggered VD—not

VI—inhibition of CaV2.2 channels, indicating a role for sG in e18a-dependent VI inhibition.

Gs protein-dependent inhibition of N-type currents in sympathetic neurons also requiresβ G γ

(Jeong and Ikeda, 1999). By contrast, VI inhibition of N-type currents by i/oG occurs independent of Gβγ (Diversé-Pierluissi et al., 1995; Raingo et al., 2007). We used the Gβγ scavenger MAS GRK2ct, which binds to and interferes with Gβγ signaling (Kammermeier and Ikeda, 1999), and

found that MAS GRK2ct occluded both VD and VI inhibition of CaV2.2[e18a] channels by GTPgS 46 Figure-4 Lipscombe

a mouse age: P0 P5 P9 P11 P18 P30 +18a

∆18a

% +18a: 35 43 43 49 62 68

b Fox-2 siRNA-injected overlay

VIP ω-CTX c d test potential (mV) -40 -20 20 40 600

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-40 2+ (pA/pF) 2+ Ca 200 Ca I -60 Control + pp VIP 0 VIP + pp peak I current (-pA) 0 2 4 6 8 10 12 time (min.)

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Figure 17. Calcium currents from SCG neurons injected with Fox-2 siRNA and uninjected cells are inhibited by the Gs agonist VIP. (A) RT-PCR analysis of e18a in mRNA isolated from mouse SCGs of postnatal ages indicated above gel. Primers flanked the splice site and generated bands of size 291bp (+18a) and 228bp (∆18a). PCR-derived cDNA products were separated on a 8% denaturing polyacrylamide gel. Results are similar to previous analysis of e18a inclusion in rat SCG during development (Gray, Raingo, & Lipscombe, 2007). (B)SCG neurons were injected with Fox-2 siRNA and dextran fluorescein (green) then fixed and stained with an anti-Fox-2 antibody (red). siRNA injected cells show little Fox-2 staining, while uninjected cells show strong Fox-2 expression levels, especially in cell nuclei. (C) Diary plot of an example calcium current recording from an uninjected cell. Plot shows reversible inhibition of current by VIP and inhibition of majority of Ca current by N-type inhibitor ω-conotoxin. (D) IV from example cell showing peak current at +20mV and VD + VI inhibition of Ca current. (E) Example currents from an uninjected (E) a Fox-2 siRNA injected (F), and an uninjected cell pretreated with cholera toxin (G, ChTX; 500ng/ml, 16hrs). Cells were challenged with VIP (10 μM) and currents were evoked with a 50 ms step to 20 mV. (H) Percent inhibition of total calcium currents by VIP; Fox-2 and control siRNAs (siCon) do not affect percent inhibition, while ChTX occludes 90% of the VIP-mediated inhibition. 47 (Fig. 15D). By contrast, MAS GRK2ct occluded only VD not VI inhibition of CaV2.2[D18a, 37a] channels by GTPgS (Raingo et al., 2007). Our data suggest that e18a-containing CaV2.2 clones can support Gs inhibition of N-type currents with properties closely aligned to those observed in neurons.

To test if e18a controls VI inhibition of native CaV2.2 channels in neurons, we needed a way to change levels of e18a-containing CaV2.2 channels in single neurons without altering the total pool of CaV2.2 mRNAs.

Fox-2 represses e18a inclusion A putative Fox protein binding site upstream of e18a (Minovitsky et al., 2005) in Cacna1b (Fig. 16A) is consistent with an exon repressor (Underwood et al., 2005). We used a Fox-2 siRNA previously validated by Douglas Black and colleagues (Underwood et al., 2005), to establish if Fox-2 repressed e18a inclusion in the F11 cell line (dorsal root ganglia/neuroblastoma fusion). F11 cells endogenously express CaV2.2 and Fox-2 and they are relatively easy to transfect. We tested the efficacy of the siRNA by examining Fox-2 protein levels using Western blot analysis (Fig. 16B); there was almost complete Fox-2 protein knockdown in cells transfected with the Fox-2 siRNA. The Fox antibody labeled three bands (Fig. 16B). This result is consistent with the finding that Fox proteins are themselves alternatively spliced (Nakahata and Kawamoto, 2005; Baraniak et al., 2006; Damianov and Black, 2009) and that many splicing factor proteins undergo post-trans- lational modification (for review see (Chen and Manley, 2009)).

We then used RT-PCR to test our hypothesis that Fox-2 represses e18a inclusion. Our RT- PCR analyses show concentration-dependent increases in channel mRNAs containing e18a

(CaV2.2[e18a]) from cells transfected with Fox-2 siRNAs (Fig. 16C; untransfected vs. 100 nM, p = 0.0001). Transfecting siRNA designed against a different region of the Fox-2 gene also increased e18a inclusion but with lower efficiency (64% increase, data not shown). In contrast, the relative abundance of CaV2.2[e18a] and CaV2.2[Δ18a] mRNAs was not significantly different in cells transfected with siRNA against cyclophillin B (a housekeeping gene) or a control non- 48 targeting siRNA (#3 from Dharmacon) when compared to untransfected cells (Fig. 16D; one-way ANOVA, p = 0.246).

With a tool to shift the splicing pattern of e18a, we could test the sensitivity of e18a-containing

CaV2.2 channels to Gs-coupled receptor-dependent inhibition in neurons. siRNA knockdown of Fox-2 in neurons

CaV2.2 channels underlie N-type currents, the major calcium current in sympathetic neurons of superior cervical ganglia (SCG). CaV2.2 channels in SCG neurons are inhibited by several GPCRs, particularly those that use Gs and Gq. The dominant CaV2.2 channels expressed in SCG lack e18a at birth, but by adulthood 68% of CaV2.2 mRNAs contain e18a (mouse: Fig. 17A; rat: Gray et al.,

2007). Our analysis of cloned CaV2.2 channels predicts increasing sensitivity of N-type currents in sympathetic neurons to Gs coupled-receptor inhibition with development. However, a large number of molecular and morphological changes accompany neuronal development, severely complicating our ability to attribute any observed changes in G protein inhibition of N-type to a change in the pattern of e18a splicing. We therefore used siRNA against Fox-2 to increase e18a inclusion in neurons from postnatal day 0-2 mice which express a low level of CaV2.2[e18a] mRNAs (Fig. 17A). Immunofluorescence analyses show strong Fox-2 signal in nuclei of control neurons, consistent with the nuclear localization of splicing factors (Fig. 17B). By comparison, Fox-2 signals are reduced substantially in neurons injected with Fox-2 siRNA (co-injected with dextran fluorescein dye; Fig. 17B).

Loss of Fox-2 does not affect overall inhibition of calcium currents bys G

Vasoactive intestinal peptide (VIP) activates Gs-coupled receptors and inhibits calcium currents in sympathetic neurons (Zhu and Ikeda, 1994; Ehrlich and Elmslie, 1995; Jeong and Ikeda, 2000a) (Fig. 17C-17D). We found that 10 mM VIP inhibits whole cell calcium currents equally in uninjected neurons, neurons injected with Fox-2 siRNA, and neurons injected with a control non-targeting siRNA (Fig. 17E, 17F, 17H). Inhibition by VIP is significantly occluded after inacti-

-4 vating sG proteins by cholera toxin treatment (Uninjected vs. ChTX, p = 1.9*10 , Fig. 17G-17H). 49 Figure-5 Lipscombe a b c d e f 100 75 75 2.2 2+ Uninjected Fox-2 siRNA 2+ Ca

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k Gi/o Gs Gi/o Gs Gq

converging pathways parallel pathways

Figure 18. Fox-2 siRNA injection in SCG neurons switches the voltage-dependence of VIP inhibition of the N-type current to mostly voltage independent inhibition. (A) Example currents from an uninjected cell treated with N-type inhibitor ω-conotoxin (10 μM) followed by treatment with VIP (10 μM) and challenged with a voltage step to 10 mV. (B) Example currents as in A from a cell injected with Fox-2 siRNA. (C-E) Knocking down Fox-2 does not affect the % N-type current, % inhibition of N-type channels or the % inhibition of non-N-type channels. (F) Cells injected with Fox-2 siRNA show significantly increased charge density. Example currents from an uninjected cell (G) and a Fox-2 siRNA injected cell (H). Cells were challenged with VIP (10 μM) and currents were evoked with a 50 ms step to 20 mV without (VIP -pp) or with (VIP +pp) a 20 ms prepulse to 80 mV. (I) Injection with control siRNA does not affect % VI inhibition of total Ca current by VIP while Fox-2 siRNA causes a significant increase in %VI inhibition (VII). (J) Fox-2 siRNA significantly increases the VIP-mediated VI inhibition of N-type currents. (K) Models of GPCR inhibition of CaV2.2 channels. The prevailing view has been that multiple GPCR pathways converge on a single CaV2.2 channel. In this model cell-specificity in these interactions is due to differences in GPCR expression or activity. We propose a different model: there are multiple parallel pathways of GPCR inhibition specified by CaV2.2 splice isoform.

50 The residual component of VIP inhibition of calcium currents after ChTX treatment (Fig. 17H) is observed by others (Zhu and Ikeda, 1994) and is thought to reflect the promiscuity of GPCRs (Hermans, 2003).

To isolate N-type currents from the whole cell calcium current we used the specific CaV2.2 blocker w-conotoxin GVIA (w-CTX). N-type currents comprise ~75% of the total calcium current in newborn mouse SCG (Fig. 18A-18C). The fraction of N-type current (Fig. 18C; p = 0.19), the percentage of N-type current inhibited by VIP (Fig. 18D; p = 0.17), and the percentage of non-N- type current inhibited by VIP (Fig. 18E; p = 0.28) were not significantly affected by the loss of Fox- 2. Our data suggest that the basic inhibitory mechanisms that couple VIP receptors to inhibition of N-type and non-N-type calcium channels are independent of Fox-2 and e18a. However, N-type current density in injected cells was significantly larger than in control cells (p = 0.017, Fig. 18F), consistent with the significantly larger N-type current densities in cells expressing CaV2.2[e18a] compared to those expressing CaV2.2[D18a] channels (Figs. 14A-E).

Loss of Fox-2 increases VI inhibition of N-type currents by sG Finally, we measured the amount of VIP-mediated VI and VD inhibition of total calcium currents and of N-type currents and observed significant differences between neurons containing high and low levels of Fox-2 (Fig. 18G-18J). The percentage of VI inhibition of total calcium current by VIP was not different in uninjected and control siRNA injected cells (Fig. 18I; p = 0.43). By comparison, in neurons injected with Fox-2 siRNA, a significantly larger fraction of VIP inhibition of whole cell calcium current was VI (Fig. 18I; Uninjected vs. siFox; p = 0.001). Furthermore, when we isolated the effects of VIP on N-type currents, we found an even greater shift toward VI inhibition in neurons injected with Fox-2 siRNA: from 35% to 57% VI inhibition (Fig. 18J; p = 0.006). Our analyses in neurons correlate with and strongly support our data from studies of cloned

CaV2.2[D18a] and CaV2.2[e18a] channels in mammalian cell lines. We conclude that cell-specific inclusion of e18a during alternative splicing of CaV2.2 pre-mRNA is negatively controlled by Fox-2 and augments Gs protein inhibition of calcium channels.

51 III. DISCUSSION Alternative splicing is extensive in mammalian nervous systems (Fagnani et al., 2007). Multi-exon ion channel pre-mRNAs are targets of neuronal splicing factors, which are proposed to individu- alize exon composition of ion channel mRNAs according to cell needs. While the global importance of alternative pre-mRNA splicing has been amply demonstrated, our study establishes that a single alternatively spliced exon, controlled by Fox-2, can have a highly specific impact on G protein signaling to a neuronal calcium channel. Collectively, we reveal an elegant cellular mechanism to engage or disengage VI inhibition of CaV2.2 channels by Gi/o and Gs proteins by independent control of e37a and e18a inclusion during pre-mRNA splicing (Raingo et al., 2007; Andrade et al., 2010). We suggest that the classic view of multiple GPCRs converging on a single type of CaV2.2 channel needs to be revised to incorporate CaV2.2 channel isoforms and at least two parallel non-converging inhibitory pathways for Gi/o- and Gs-coupled receptors (Fig. 18K).

Voltage-independent inhibition, not voltage-dependent inhibition, depends on inclusion of alternative exons

A unique characteristic of CaV2.2 channels—by comparison to closely related CaV2.1 channels—is their susceptibility to strong inhibition by numerous GPCRs (Bourinet et al., 1996; Currie and

Fox, 1997; Elmslie, 2003). VD inhibition is observed in all CaV2.2 channel isoforms studied so far, provided that G protein and CaV2.2 channel are in close proximity, and is exhibited independent of alternative splicing. However, we show that CaV2.2 channel susceptibility to VI inhibition by

G proteins and GPCRs depends crucially on exon composition; VI inhibition bys G requires e18a, and previously we demonstrated that VI inhibition by i/oG requires e37a (Raingo et al., 2007; Andrade et al., 2010). Interestingly, VD and VI inhibition differ in the spatial restrictions on their ability to inhibit calcium channels. VD inhibition is membrane-delimited and relies on Gβγ directly binding to CaV2.2. It is highly sensitive to the spatial proximity of ion channel and G protein: the influence of Gβγ falls off sharply with distance (Bernheim et al., 1991). On the other hand, VI inhibition employs diffusible second messengers that can influence distant signaling partners

(Bernheim et al., 1991). Alternative splicing of calcium channels therefore might be important for restricting the influence of GPCRs that mediate VI inhibition through Gi/o or Gs proteins. In this 52 way, distant GPCRs would influence only CaV2.2 channels that contain e18a and/or e37a.

GPCRs also inhibit CaV2.2 channels via Gq by depleting PIP2 (Suh et al., 2010). Although GTPgS did not activate qG inhibition of cloned CaV2.2 channels (Fig. 15A,15C), we did observe Gq-mediated

VI inhibition of CaV2.2 channels, independent of e18a, when we co-expressed muscarinic M1 or angiotensin AT1 receptors that couple to Gq (unpublished data CGP & DL). Thus Gq-coupled receptors appear to inhibit CaV2.2 channels independent of known alternative splice sites in

CaV2.2.

Voltage-independent inhibition via e18a and e37a is additive

Even though their functional effects are indistinguishable, Gs and Gi/o work through different intracellular domains of CaV2.2 and use different second messengers: VI inhibition byi/o G is Gβγ-independent, src tyrosine kinase-dependent, RGS2-insensitive, and PTX-sensitive (Diverse-

Pierluissi et al., 1997; Raingo et al., 2007), whereas VI inhibition by sG is Gβγ-dependent, RGS2- sensitive, PTX-insensitive, and ChTX-sensitive. Consequently, VI inhibition mediated by e18a and e37a is additive (Fig. 14F). CaV2.2 channels containing both e18a and e37a are expressed in a subset of nociceptors (Bell et al., 2004), and they are likely to be strongly inhibited when both

Gs and Gi/o coupled receptors are activated. By contrast, VD inhibition is not additive, consistent with the idea that Gα proteins share common Gβγs (Graf et al., 1992; Jeong and Ikeda, 1999). The modularity and functional autonomy of alternatively spliced exons 18a and 37a enable independent control over the VI inhibitory influence of either G protein on CaV2.2 channels without affecting VD inhibition.

Fox-2 may work in concert with other splicing factors Our Western analyses of cells expressing 100 nM Fox-2 siRNA show almost complete loss of Fox protein, but our RT-PCR analyses reveal the presence of CaV2.2[Δ18a] mRNAs (Fig. 16B,16C). The e18a splice junction may be sub-optimal, explaining the presence of e18a-lacking mRNAs in the absence of a splicing repressor. Alternatively, additional splicing factor(s) may act in concert with Fox-2 to repress inclusion of e18a. This type of combinatorial splicing regulation is well docu- 53 mented (Hertel, 2008; Lipscombe et al., 2008; Chen and Manley, 2009).

Elegant cellular mechanisms have evolved to regulate inclusion of specific exons depending on cell-type and stage of development (Li et al., 2007; Licatalosi and Darnell, 2010). A single splicing factor can coordinate the composition of several genes, but we provide evidence that an individual splicing event can be resolved in the activity of channels in individual neurons. We suggest that alternative splicing is a cellular mechanism for setting the sensitivity of CaV2.2 channels to specific G proteins using parallel independent signaling pathways.

54 Threonine and Lysine Residues in e18a 4 Modulate Expression and G Protein Inhibition of CaV2.2 Channels I. INTRODUCTION In Chapters 2 and 3 I presented data showing that inclusion of alternative e18a affects the properties of CaV2.2. These chapters focused on the susceptibility of e18a-containing channels to Gs-mediated voltage-independent inhibition (VII). There is a second characteristic imparted by e18a inclusion: increased current density. In this Chapter I show that the increase in current density associated with e18a is also β subunit specific. I also present studies on a series of CaV2.2 channel mutants designed to identify the specific amino acids in e18a responsible for G protein- mediated VII and increased current density.

As discussed in Chapter 2, auxiliary β subunits modulate many features of calcium channel activity including modifying susceptibility to G proteins (Feng et al., 2001; Sandoz et al., 2004; Heneghan et al., 2009); voltage-dependent activation and inactivation (Olcese et al., 1994; Pan and Lipscombe, 2000); CaVα subunit trafficking to the membrane (Takahashi and Nagasu, 2005); and proteasomal degradation of CaVα subunits (Waithe et al., 2011). In this thesis I show the results of experiments recording the activity of CaV2.2 channels co-expressed with either β3 or β2a. β subunits bind to the α-interacting domain (AID) in the I-II linker of CaV2.2 (Pragnell et al., 1994). As discussed in Chapters 2 and 3, e18a-containing channels are particularly enriched in SCG neurons. Therefore I have been most interested in assessing CaV2.2 channel activity in cells expressing β subunits most likely to associate with native CaV2.2 channels in sympathetic neurons. All four β mRNAs subunits are expressed in rat SCG tissue (Lin et al., 1996), so these expression studies do not help establish which β subunit is the in vivo binding partner for CaV2.2.

Support for a preferential association of CaV2.2 with β3 comes from the following observations:

β3 binds strongly to CaV2.2, β3 mRNA and protein is widely distributed in brain regions that also express CaV2.2 (Ludwig et al., 1997), and β3 knockout mice show significant decreases in CaV2.2 expression without accompanying changes in CaV1.2, 1.2, 2.1, or 2.3 (Smith et al., 1999; Mu- rakami et al., 2007). However, the functional features of native CaV2.2 currents in SCG neurons bear a much closer resemblance to those of recombinant CaV2.2 channels in cells expressing β2a

(Ikeda, 1991). Therefore, I investigated CaV2.2 channel function with β2a and β3 subunits. In this Chapter I present evidence that β3, and not β2a, facilitates e18a-containing channel currents. 56 Of the 21 amino acids encoded by e18a 7 are serines, 2 are threonines and 1 is a lysine (Fig. 20). As discussed in the Introduction chapter, at least three VII pathways are activated by at least three different Gα proteins, but the precise mechanism of rapid VII of CaV2.2 by any GPCR is not known. VII could occur through a number of mechanisms, including rapid channel internalization, stabilization of an inactive or closed state of the channel and/or uncoupling the voltage- sensor from the pore domain, as has been shown for some potassium channels (Jara-Oseguera et al., 2011). Although there is strong evidence that GqPCR activation leads to PIP2 depletion in the plasma membrane and that this is a crtical step in CaV2.2 channel inhibition (Suh et al.,

2010), the mechanism by which PIP2 depletion inhibits the channel is unknown. Additionally, we have shown that inclusion of alternative e37a in the C-terminus of CaV2.2 leads to VII via Gi/oPCRs dependent on src-tyrosine kinase (Raingo et al., 2007), but the connection between the receptor and the channel has yet to be elucidated.

I was therefore motivated to use site-directed mutagenesis to establish which amino acids in e18a are required for VII of CaV2.2 through Gs, and also which amino acids are necessary for e18a-associated increased current density. Gs activates adenylyl cyclase, which increases cAMP production and PKA activation (Fig 5). Serine/threonine phosphorylation is used to tag proteins and might represent a common signal that promotes CaV2.2 channel internalization or, more generally, inhibition. Additionally, lysine modifications like ubiquitination, SUMOylation and acetylation are closely linked to cellular pathways that modify protein activity and expression.

b3 (37b) Therefore I mutated serine, threonine and/or lysine b2a (37b) β3 (7) 150 (S, T, K) amino acids in e18a and tested the resultant

100 channel’s sensitivity to G protein inhibition and mea- pA/pF sured current densities relative to wild-type CaV2.2 50 β2a (3) control recordings. I first analyzed the e18a amino

peak current density (pA/pF) peak current 0 Δ18adelta +18aplus acid sequence using various posttranslational modifica- Figure 19. β3, but not β2a subunits facilitate tion (PTM) prediction software programs and used this e18a-mediated current density increase. Data from all experiments shown in Chap- first-round analysis to select amino acids within e18a ters 2 and 3 were averaged by isoform and β subunit. to mutate. I present data demonstrating that e18a is a 57 hotspot of trafficking motifs and certain amino acids are critical for G protein modification. Most importantly, when the lysine in e18a was converted to alanine (K3A), CaV2.2[e18a] channels were indistinguishable from CaV2.2[D18a] channels. My data suggest that this lysine is necessary for both e18a-dependent increased current density and Gs-mediated VII.

II. RESULTS β2a does not support an e18a-mediated increase in current density.

In Chapters 2 and 3 I show recordings from CaV2.2[D18a] and CaV2.2[e18a] channels under a variety of conditions – with different GPCRs and with different β subunits. I pooled all of my individual averages from the different experiments to analyze how the presence of e18a and the association of CaV2.2 with different β subunits affects current density. There is an unmistakable trend: CaV2.2 channels expressed with β2a show similar current density regardless of the presence or absence of e18a. By contrast, when expressed with β3, e18a-containing channels show large differences in current density compared to channels that lack e18a (Fig. 19).

PTM Software Predicts Many Modification Motifs in e18a. I analyzed the e18a sequence along with neighboring residues using multiple online PTM prediction software programs. In Fig. 20 I show the results from two such programs. ELM (eukaryotic linear motif; elm.eu.org) prediction of kinase motifs is depicted by lines below the e18a sequence. Above the sequence I show the predicted probability of phosphorylation of specific residues from NetPhos 2.0 software that predicts serine, threonine and tyrosine phosphorylation sites in eukaryotic proteins (http://www.cbs.dtu.dk/services/NetPhos/). Additionally, the PAIL (prediction of acetylation of internal lysines) software predicted aceytlation of the only lysine in e18a (score 1.14; threshold 0.5; http://bdmpail.biocuckoo.org/). Based on these compelling analyses, I made a series of e18a mutants to try to isolate the amino acids necessary for e18a- dependent effects (Fig. 21).

Using the mutants I replicated the original experiments shown in Figures 6 and 14: I coexpressed

CaV2.2 with β3 and α2δ1 in tsA201 cells and measured CaV2.2 currents activated by voltage 58 .795 .837 .871.997.83 .992.732 .702

FVKQTRGTVSRSSSVSSVNSP1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 QQ 14-3-3 SH3 WW GSK-3 CK1

PKA PK Figure 20. Posttranslational modification prediction software predicts phosphorylation of e18a.E18a sequence (black) and 2 neighboring residues (grey) were analyzed using online software. ELM (eukaryotic linear motif; elm.eu.org) prediction of kinase motifs is depicted by lines below the e18a sequence with kinase designation written above (some kinases had multiple predicted motifs, which are grouped by color). Above the sequence is the predicted probability of phosphorylation of specific residues from NetPhos 2.0 software which predicts serine, threonine and tyrosine phosphorylation sites in eukaryotic proteins (http://www.cbs.dtu.dk/services/NetPhos/). steps, with control internal solution or with internal solution containing 0.4 mM GTPγS. I used two different protocols to activate CaV2.2 channels: a single depolarizing step to 0 mV +/- a preceding prepulse (pp) to 80 mV and a full IV from -50 to 60 mV +/-pp. These protocols differentiated VDI from VII. Small changes in series resistance or other unknown cellular mechanisms can cause changes in channel behavior over time. Therefore, the single step protocol is useful because there is only a 6 second delay between +pp and –pp steps, minimizing time dependent changes. However, a single step pulse only samples a single voltage, so I also obtained IVs in the most stable recordings. In Fig. 22 I show IVs and in Fig. 23 I show the data from single test pulses to activate the channels .

I first tested a mutant with all 7 serines mutated to alanines (e18a(SALLA)). Based on the results from the phosphorylation prediction software programs, I was expecting to observe substantial differences in the properties of e18a(SALLA) CaV2.2 channel compared to wild-type. However, surprisingly, currents in cells expressing the e18a(SALLA) mutant were indistinguishable from wild type CaV2.2[e18a] currents (Figs. 22, 23). I next tested two CaV2.2 clones containing additional mutations: one in which both threonines were converted to alanines (e18a(SALLA+TALLA)) and one with the single lysine converted to alanine (e18a(SALLA+K3A)). Both of these clones were different from wild-type channels in interesting ways. E18a(SALLA+TALLA) channel currents were associated with greater current density, decreased VDI, and increased VII compared to wild-type. 59 I II III IV OUT

I II III IV OUTpre-mRNA 18 18a 19 +18a ∆18a IN nal mRNA 18 18a 19 18a18 19

pre-mRNA 18 18a 19 wild-type +18a ∆18a nal mRNAFV18KQTR18aGTV19SRSS18aS18VSSV19NSP S A ALL FVKQTRGTVARAAAVAAVNAP S wAil+d-TtypAe ALL ALL FVKQATRGATVASRASASASVASASVNASP

SALLA SALLA+K3A FVAKQTRGTVARAAAVAAVNAP

SALLA+TALLA T8A FVKQATRGAVASRASSSAAVASSAVNASP +K A SALLA K3A FVAQTRGTVASRASSSAAVASSAVNASP Figure 21. Schematic representing location of e18a-encoded residues and channel mutants. Schematic of CaV2.2 channel in the membraneT8A with approximate location of peptide encoded by e18a in the intracellular II-III linker (green dot). BelowF areV theKQ sequencesTRGA ofV e18aSR clonesSSS created.VSS ArrowsVNS indicateP the mutated residues. K3A FVAQTRGTVSRSSSVSSVNSP

E18a(SALLA+K3A) channels were associated with decreased current density and lacked VII, similar to CaV2.2[D18a] channels. T5 it is not highly conserved and is an alanine in Cacna1b genes of human, pig, and cow so I focused on and generated a T8A mutant. The CaV2.2[e18a(T8A)] clone was similarly associated with increased current density and increased VII, but VDI was not different from wild-type e18a (Fig.s 22, 23).

III. DISCUSSION In this Chapter I show preliminary data to begin to explore the signaling cascade between Gs-coupled receptor and e18a-containing channels. I show that e18a-dependent increases in current density are only observed when β3, but not when β2a is present. I also show that mutation of K3 in e18a is necessary for the e18a-dependent effects on current density and G protein-

60 wild-type

wild-type SALLA

A wild-type B SALLA C SALLA+TALLA 100 100 100 wild-type test pulse (mV) test pulse (mV) + test pulse (mV) SALL0 A SALLA TALLA 0 +0 -40 -20 0 20 40 60 -40 -20 0 20 40 60 S-40ALLA-20 K03A 20 40 60 wild-type SALLA -100 ) -100 ) -100 F F )

+ p p / S A T A F ALL / ALL

+ p A A SALLA K3A / T8A p p A ( (

p ( y y

t -200 -200 -t 200 i i + y t s s SALLA TALLA i

n S A

ALL s n e n e d e

d t

d K A t 3 n S A+ t K A n ALL e 3 T A -300 8 n -300

r -300

Con -pp (11) e

r Con -pp (11) e r Con -pp (7) r u r r c

γ u GTP S -pp (3) GTPγS -pp (9) u

c γ + c GTP S -pp (6) SALLA TALLA GTPγSS ALL+ppA (2+) K3A GTPγS +pp (10) -400 -400 GTPγS +pp (6) K3A -400 T8A

D SALLA+K3A E T8A K3A 100 100

test pulse (mV) K3A test pulse (mV) 0T8A 0 -40 -20 0 20 40 60 -40 -20 0 20 40 60 )

F -100K A -100 )

p 3 / F A p / p ( A

p y ( t

-200 y -200 t s i n s e n d e

d t

t n n e

r -300 -300 e r r

Con -pp (9) r

u Con -pp (7) u c GTPγS -pp (6) c GTPγS -pp (6) -400 GTPγS +pp (4) -400 GTPγS +pp (6)

Figure 22. Voltage-dependence of G protein-mediated inhibition and expression of CaV2.2 channels are modulated by specific e18a residues. Calcium currents from cells expressing various CaV2.2[e18a] clones along with b3 and α2δ1. Cells were recorded from with internal solution without (Con) or with (GTPgS) 0.4mM GTPgS. Currents were evoked by 50 ms test pulses to voltages shown on X-axis without (-pp) or with (+pp) a 25 ms prepulse to 80 mV. N values are shown in parentheses. mediated VII as both are absent when this lysine is replaced by alanine. However, this needs to be confirmed with an e18a mutant that contains only the K3A mutation, and not the 7 mutated serines as well.

β2a is distinguished as a target of palmitoylation. Neuronal β2a is unique among β subunits in that it is capable of being palmitoylated via two N-terminal cysteines (Chien et al., 1996). Palmitoylation is a reversible PTM that is associated with membrane targeting because of the addition of a hydrophobic palmitoyl group. β2a mutants lacking the cysteines that are palmitoylation sites still target to the plasma membrane but showed impaired ability to modulate CaV currents. For example, β2a cysteine mutants fail to 61 facilitate CaV1.2 calcium currents or shift the voltage-dependence of inactivation of CaV2.3; two characteristic features of currents in cells expressing β2a (Chien et al., 1996; Qin et al., 1997; Qin et al., 1998). These publications suggest that palmitoylated β2a subunits interact with and A B 400 100 voltage- 80 dependent 300

pe pe60 -ty -ty 200 A A wild pe wild 40 pe S ALL -ty S ALL -ty A A A A 100 wiplde wiplde ty T ALL S ALL 20 ty T ALL S ALL voltage- - + inhibition % of total - + A A A A A A A A independent wild pe 3 wild pe 3 ALL K ALL ALL K ALL S -ty S ALL + T S -ty S ALL + T A + A + peak current density (pA/pF) peak current A A wild pe A A A A wild pe A A A A ty S ALL ALL T 8 K 3 ty S ALL ALL T 8 K 3 - S ALLS T ALL + - S ALLS T ALL + + A A + A A AA A 3 A AA A 3 A wild ALLAK 8 wild ALLAK 8 ALL S 3 T ALL S 3 T SSALL T ALL K SSALL T ALL K + + + + A AA A A AA A A A K 3 A A K 3 ALL TSALLALL K 3 T 8 ALL TSALLALL K 3 T 8 Figure 23. Current density and voltage-dependenceS + + of GTPγS-mediatedS + + inhibition of e18a mutants. Cells A A A A A A A A A 3 A 3 ALL ALL K 3 8 K ALL ALL K 3 8 K were treated as in Fig. 22, exceptS theyS were+ steppedT from -100 SmV toS only+ 0 mVT with or without a pre- A A A A pulse to 80 mV. In B, black bars represent componentA 3 of inhibition that is VII; grayA 3 bars are VDI. S ALL T 8 K S ALL T 8 K A A K 3 K 3

modulate CaV channels in rather complex ways. It is therefore possible that palmitoylation of β2a subunits obscure the effects of e18a on current density.

β subunits, ubiquitination, and proteasomal degradation.

Two recent studies have shown that ubiquitination of both CaV1.2 and CaV2.2 channels is strongly influenced by auxiliary CaVβ subunits (Altier et al., 2011; Waithe et al., 2011). Both studies converge on a model in which β subunit binding protects channels from proteasomal degradation. CaVβ coexpression prevents CaV1.2 channels from ubiquitination by RFP2 ubiquitin ligase, and from entering the endoplasmic reticulum-associated protein degradation (ERAD) complex, a check-point on the way to proteasomal degradation (Altier et al., 2011). Likewise, a CaV2.2 mutant which cannot bind CaVβ subunits is normally expressed at much lower levels than wild-type

CaV2.2, and proteasomal inhibition led to a much larger increase in ubiquitinated mutant protein (360%) versus wild-type (58%), although not increased surface expression (Waithe et al., 2011).

The nature of the CaVβ subunit might be important in predicting its interaction with the ubiquitin-proteasome system. The SH3 domain of CaVβ2a has been reported to down-regulate CaV1.2 62 channel expression in a dynamin-dependent fashion (Gonzalez-Gutierrez et al., 2007). Further- more, this internalization is dependent on oligomerization of the β subunits (Miranda-Laferte et al., 2011), although others have shown that oligomerization augments expression of CaV1.2 (Tareilus et al., 1997; Lao et al., 2010). These recent papers strongly favor involvement of ubiquitination, β subunit binding, and dynamin-dependent internalization of CaV channels in dynamic control of channel expression. Although it is unclear why β3—not β2a—would promote expression of e18a-containing channels, it is very intriguing given the role of β subunits in trafficking and expression of channels. β subunits bind to the AID region in the I-II linker, which, although far from e18a based on primary amino acid sequence, could be spatially close to the II-III linker in the native folded structure. One possibility is that β subunits directly interact with amino acids in the e18a peptide sequence. Alternatively, β subunits could indirectly couple to e18a via an e18a-specific binding partner that differentially interacts with β3 and β2a. It would be interesting to see how the threonine mutants of CaV2.2 behave in the presence of β2a since they are associated with particularly large current densities over wild-type channels when co-expressed with β3.

Phosphorylation sites in CaV2.2 Phosphorylation prediction software points to many potential sites of S/T phosphorylation in e18a (Fig. 20). Phosphorylation of CaV2.2 at other sites is well documented. CaV2.2 channels are upregulated by many kinases including Ras, ERK, MAPK, CaMKII, and PKC (Ahlijanian et al., 1991;

Martin et al., 2006). The CaVβ subunit can also affect phosphorylation of the associated

CaV channel. For example, when MAPK activity was inhibited in a COS-7 cell line expressing CaV channels, current densities decreased two-fold more in cells expressing β3 as compared to those expressing β2a (Fitzgerald, 2002)d. Either MAPK is less effective at upregulating CaV2.2 channel activity, or phosphorylation by MAPK less effectively couples to increases in current density, in the presence of β2a compared to β3. A similar finding is reported with phosphorylation of

L-type CaV1.3 channels by PKA (Liang and Tavalin, 2007). PKA induces increased current density when either β3 or β2a were co-expressed with CaV1.3, but the time-scale of upregulation was different.The effect of PKA on VCa 1.3 channels was only transient in cells expressing β3, whereas 63 the effect of PKA on CaV1.3 currents was long lasting in cells expressing β2a. The authors of this study suggest that β2a and β3 subunits regulate which amino acids are targets of PKA phosphorylation. At the start of my studies, I hypothesized that e18a was phosphorylated via one or more of its serine and threonine amino acids. However, by converting all serines in e18a to alanines I ruled out their contribution to the e18a effects on current density and G protein modulation. By contrast, threonine 8 did influence current densities but in a manner opposite to my expectations. E18a-containing channels are associated with larger current densities and the presence of VII when compared to D18a channels; thus, I expected that the threonine mutation would either be associated with a decrease in CaV2.2 current density and/or loss of VII. In fact, e18a(SALLA+TALLA) CaV2.2 channels were associated with even larger current densities. Additional experiments are needed to understand the mechanism by which threonine 8 in e18a acts to regulate CaV2.2 current density, but my experiments uncouple changes in current density from changes in VII of CaV2.2 channels.

Lysine modifications of proteins. The most interesting mutant that I generated from a mechanistic perspective was the e18a(SALLA+K3A) mutant (Fig. 22D). Converting the single lysine in e18a to alanine abolished both the greater current density associated with e18a-containing CaV2.2 channels and GTPgS-mediated VII. Lysines are targets of multiple PTMs, including SUMOylation, acetylation, and ubiquitination.

SUMOylation, acetylation, and ubiquitination all involve the covalent attachment of a molecule—SUMO (small ubiquitin-related modifier), acetyl, or ubiquitin—to lysine residues. They are all known to affect a large variety of cellular processes. SUMO proteins can affect protein localization, protein-protein interactions, or activity (Geiss-Friedlander and Melchior, 2007). Although first thought of as a modifier of nuclear proteins, multiple neuronal transmembrane proteins have now been identified as substrates for SUMO including potassium channels and GPCRs

(Wilkinson et al., 2010). For instance, SUMOylation shifts the voltage-dependence of activation of KV2.1 and steady-state inactivation of KV1.5 (Benson et al., 2007; Plant et al., 2011). Acetyla- 64 tion regulates diverse and critical cellular processes(Choudhary et al., 2009). Tau is acetylated, which prevents association of phospho-tau with microtubules, leading to tau aggregation, a hallmark of multiple neurodegenerative diseases including Alzheimer’s Disease (Min et al., 2010; Cohen et al., 2011). Ubiquitin attachment is a frequently used signal to modify synaptic proteins (DiAntonio and Hicke, 2004; Yi and Ehlers, 2007; Tai and Schuman, 2008; Rotin and Staub, 2010). For instance, activity-dependent ubiquitination of postsynaptic AMPA receptors regulates- syn aptic plasticity (Colledge et al., 2003; Patrick et al., 2003; Lin et al., 2011). There is now evidence that CaV1.2 and CaV2.2 channels are likewise regulated by ubiquitination (Altier et al., 2011; Waithe et al., 2011).

Phosphorylation, SUMOylation, acetylation, and ubiquitination often modify the same target and can influence the susceptibility of proteins to other forms of PTMs. For example, transcription factors RUNX3 and smad7 are acetylated, and this acetylation prevents ubiquitin-mediated degradation of the proteins (Gronroos et al., 2002; Jin et al., 2004). Phosphorylation of the huntingtin protein enhances its ubiquitination, SUMOylation, and acetylation, all leading to increased proteasomal degradation (Thompson et al., 2009). Dephosphoryation of a serine residue in transcription factor MEF2A leads to a switch from SUMOylation to acetylation of a nearby lysine residue, which significantly effects postsynaptic differentiation (Shalizi et al., 2006).

We do not yet know which, if any, of these PTMs occur in the amino acids encoded by e18a. However, SUMOylation can probably be ruled out; the majority of SUMO consensus motifs con- form to the pattern: I/V/L K x D/E (Matic et al., 2010). Although e18a has a very close sequence (VKQT), the D/E residue is highly conserved and therefore I hypothesize that SUMO is not the PTM responsible for e18a function.

The data I present in this chapter represent my preliminary investigation into the mechanisms by which e18a is associated with VII via a Gs-dependent pathway. My data do not support a role for serine and threonine dependent phosphorylation but they suggest that the single lysine encoded in e18a is critical for VII mediated by Gs. This is very exciting as it provides a starting point for 65 understanding the mechanism of VII of CaV2.2 channels.

66 5| DISCUSSION In this thesis I show that CaV2.2 channel properties including inhibition by G protein-coupled receptors and levels of channel expression are strongly influenced by the presence of e18a.

G PROTEIN-MEDIATED VII: SIGNALING MOLECULES

Gq-, Gi/o- and GsPCRs can all inhibit CaV2.2 channels by voltage-dependent and voltage-independent mechanisms. VDI seems to always rely on Gβγ binding to the channel but there are unique features depending on GPCR subtype. Likewise, although VII mediated by GqPCRs, Gi/oPCRs and GsPCRs are functionally indistinguishable, each GPCR family uses distinct signaling molecules.

VDI through different Gαs is not additive; are Gβγs shared by different GPCRs?

Gβγ-mediated VDI of CaV2.2 channels by different GPCRs are not additive, suggesting that Gβγs generated from Gα protein activation use the same limited pool of Gβγs and/or they share a common site of action (Graf et al. 1992; Jeong and Ikeda 1999). A number of Gα subunits, including Gq, Gs, and Gt, can all occlude the ability of norepinephrine to inhibit CaV2.2 currents via Gi/o-coupled a2-adrenergic receptors when overexpressed in SCG neurons (Jeong and Ikeda 1999). Thus each Gα can act as a Gβγ sink, preventing the action of Gβγ regardless of which Gα is activated. This suggests little specificity in Gα-Gβγ coupling. Our recordings from neurons lend support to this idea. After showing that e18a-mediated VI inhibition of CaV2.2 requires both Gs and Gβγ in experiments using cloned channels, I went on to test our model in neurons. I used

Fox-2 siRNA in neurons to shift the splicing pattern toward e18a-containing CaV2.2 channels and found GsPCR activation resulted in greater VII compared to uninjected neurons but no change in overall inhibition. Based on these findings, I suggest that VII and VDI share the same pool of Gβγ molecules and that they are a limiting factor. Therefore, as the contribution by e18a-containing channels increases, Gβγ is co-opted by VII pathways giving the apperance of a conversion from VDI to VII. This was shown for Gq signaling in neurons (Kammermeier et al. 2000). An alternative possibility is that VDI and VII may use a different pool of Gβγ molecules but their binding site on

CaV2.2 is the same, thereby setting a ceiling on the level of inhibition.

E18a- and e37a-dependent VII pathways are additive and independent 68 Convergent Inhibition of N-type calcium channel

voltage-independent

GPCR GPCR i/o β voltage- voltage- β s γ γ dependent dependent

Parallel Inhibition of N-type calcium channels

voltage-independent

GPCR GPCR volt.- volt.- i/o β β s γ dep. dep. γ

Figure 24. Schematic of convergent and parallel models of GPCR-mediated inhibition of VCa 2.2. Classi-

cally GPCR-CaV2.2 inhibitory pathways have been thought of as all converging on a single class of calcium channel. However, our data suggest that this idea should be revised to allow for multiple, distinct, independent, and parallel inhibitory pathways that depend both on the identity of the GPCR and the splice

variant of the target CaV2.2 channel.

Even though indistinguishable at the functional level, VII of CaV2.2 initiated by Gs and Gi/o activation involve different structural domains of CaV2.2 and use different second messengers. VII by Gi/o is e37a-dependent, Gβγ-independent, src tyrosine kinase-dependent, RGS2-insensitive, and PTX-sensitive (Diverse-Pierluissi et al. 1997; Raingo et al. 2007); VII by Gs is e18a-dependent, Gβγ-dependent, RGS2-sensitive, PTX-insensitive, and ChTX-sensitive. VI inhibitions by e18a and e37a are additive: CaV2.2 channels containing e18a and e37a exhibit more VI inhibition than channels with either one of the exons alone (Fig. 14F). Hence knowing the precise composition of exons in CaV2.2 channels is critical for predicting how a given channel will respond to GPCR inhibition. Our results also suggest that the classic model of GPCR inhibition of CaV2.2 channels, which has multiple receptors converging on a single channel, should be modified to include -GP

CRs working through parallel pathways with unique actions on different CaV2.2 channel isoforms

(Fig. 24). Our data suggest that different GCPRs use different signaling molecules to inhibit CaV2.2 69 channels through distinct mechanisms that are voltage-independent.

Can VII be explained by co-internalization of CaV2.2 with GPCRs? GPCR agonist exposure activates receptors, but often also initiates cascades leading to receptor desensitization. One mechanism by which GPCRs desensitize is via internalization (Ferguson

2001). CaV2.2 channels are known to internalize along with both ORL-1 receptors and D1Rs in the presence and absence of agonist (Altier et al. 2006; Kisilevsky et al. 2008). Consistent with this, recent proteomic screens and FRET-based studies show tight coupling between certain

GPCRs and CaV2.2 channels (Khanna et al. 2007; Muller et al. 2010; Laviv et al. 2011). This raises the possibility that voltage-independent inhibition by GPCRs might involve co-internalization of

CaV2.2 channels and GPCRs. There are two reasons why I think this is unlikely. First, typical time scales of receptor internalization are relatively long. Estimates of t1/2s for GPCR internalization range from 20 minutes for the delta opioid receptor (Qiu et al. 2007) to 90 minutes for the A1 adenosine receptor (Ferguson et al. 2000). By contrast, GPCR mediated VII of CaV2.2 channels occur on a much faster time scale, from less than 1s to a few seconds (Fig.s 8B, 9B, 10B). Addition- ally, in the studies mentioned above CaV2.2 channels were co-internalized with GCPRs following chronic agonist application of 30 minutes (Altier et al., 2005; Beedle et al., 2004); a time scale at least an order of magnitude too long to account for the GCPR-mediated VII. Second, we see prominent VII of CaV2.2 channels with inclusion of GTPγS inside recording pipettes (eg., Fig. 6B). In these experiments the GPCR is by-passed. Therefore, I conclude that GPCR internalization is not likely to explain rapid VII of CaV2.2[e18a] channels that we report.

Can Gi/o- and Gs-mediated VII be explained by PTM-induced internalization?

There are intriguing similarities between the effects that e18a and e37a have on CaV2.2 activity and GPCR sensitivity. Both exons promote G protein-mediated VII and both lead to increased current density, despite their different sequences, position in the channel, and tissue-distribution. My recent discovery that a lysine in e18a is critical for e18a-dependent VCa 2.2 characteristics (Fig. 22, 23) is potentially interesting in light of recent work from our lab concerning ubiqutination of CaV2.2. Lysines are the sites of ubiquitin attachment. Spiro Marangoudakis has 70 found that e37a-containing channels and e37b-containing channels are differentially ubiquitinated (Marangoudakis et al. in revision). E37b-containing CaV2.2 channels are significantly more ubiquitinated in vivo than e37a-containing channels, which may explain the lower current density associated with e37b channels. The idea is that increased ubiquitination leads to decreased surface CaV2.2 protein expression. The precise lysine(s) that is differentially ubiquitinated in e37b-containing CaV2.2 channels is not known, but ubiquitination is increased in e37a following substitution of a tyrosine to a phenylalanine (the equivalent amino acid in e37b). One possibility is that tyrosine phosphorylation in e37a inhibits ubiqutination of a nearby lysine that is -nor mally ubiquitinated in e37b-containing channels. In e18a, mutating lysine 3 is associated with decreased CaV2.2 current density but VII is occluded. Clearly, experiments are needed to identify the key amino acids that mediate VII and that are responsible for altering current density.

Why doesn’t GTPγS activate Gq-mediated inhibition of CaV2.2 channels in tsA201 cells? In many of our experiments we used GTPγS to globally activate all G proteins in tsA201 cells to study the consequences on cloned CaV2.2 channels. We discovered that e37a-containing

CaV2.2 channels are susceptible to a PTX-sensitive VII pathway and e18a-containing channels are susceptible to a ChTX-sensitive pathway. In these recordings, VDI relied exclusively on Gβγ from Gi/o heterotrimers because VDI activated by GTPγS was completely occluded by PTX (Raingo et al. 2007). By contrast, the VII we observed in channels containing e37a and e18a relied on Gi/o and Gs, and they were completely occluded by PTX and ChTX (Raingo et al. 2007) (Fig. 15A,C). Our data are also interesting because of the absence of engagement of Gq protein-dependent inhibition of CaV2.2 channels by GTPγS. By contrast, CaV2.2 channels were inhibited via GqPCRs in a VD way in cells co-expressing Gq-coupled B2Rs (Fig. 9) and in a VI way with the Gq-coupled M1R and AT1Rs (Fig. 12). Furthermore, the Gs-coupled D1R mediated VDI (Fig 12). One possibility is that concentration of Gi/o and Gs in tsA201 cells is much greater than Gq, or that GTPγS is more effective at activating Gi/o and Gs compared to Gq. Since all G proteins may compete for the same pool of Gβγ or other second messengers and perhaps the same binding sites on CaV2.2, the actions of Gq proteins might be swamped out by the other G proteins. Why was VII by Gs-coupled D1R absent in the presence of e18a? 71 VIP and Gs-coupled D1Rs are known to induce VDI and VII of CaV2.2 channels in neurons. VDI of

CaV2.2 by VIP is independent of cAMP and PKA as shown in adult rat SCG ((Zhu and Ikeda 1994; Ehrlich and Elmslie 1995). Whereas in hamster submandibular ganglia VIP inhibition of calcium currents is mostly VI and is dependent on cAMP and PKA (Kamaishi et al. 2004). Likewise, CaV2.2 currents in midbrain neurons of adult rats are inhibited by D1R in a VI manner dependent on cAMP and PKA (Surmeier et al. 1995). In newborn PFC rat neurons the D1R inhibits N type channel via VD and VI pathways, but only VI inhibition is dependent on PKA (Kisilevsky et al. 2008). It can be difficult to compare different studies but it is possible that GsPCRs differ in their ability to generate cAMP and PKA, and that this, in addition to the splicing of CaV2.2, is an important factor in determining if GsPCRs inhibit CaV2.2 through a VI mechanism. Experiments that I describe below in Future Directions may help shed light on this issue.

DIVERSITY IN THE CALCIUM CHANNEL FAMILY

Why do mammals express 10 different CaV genes?

Mammalian genomes contain 10 genes that encode CaV ion channels (Table 1). All of these genes contain sites of alternative splicing. What are the unique functional attributes of these highly related proteins? Within the CaV1 family, CaV1.2 and CaV1.3 channels have distinct channel properties that coincide with their cell-specific expression. CaV1.3 channels are expressed presynaptically in inner ear hair cells (IHCs) and they activate at potentials approximately 25 mV more hyperpolarized than CaV1.2 (Xu and Lipscombe 2001). This results in channels that open in response to small depolarizations from the resting membrane potential, a feature that makes them ideally suited to generate graded responses in IHCs. CaV1.2 and CaV1.3 channels underlie the majority of L-type currents in excitable cells, they are sensitive to dihydropyridines (DHPs).

However, CaV1.2 channels are mainly postsynaptic, while CaV1.3 channels are found both pre- and postsynaptically. Their biophysical properties are unique and they bind different proteins.

Figure 25 shows the known binding partners for pre- and post-synaptic CaV1.3 channels, postsynaptic CaV1.2 channels, compared to select binding partners of CaV2.2 channels. Amino acid sequence alignments shown in Fig. 3 and the relatively high representation of alternative exons in intracellular domains of CaV channels (Fig. 2) underscore the importance of intracellular domains 72 CaV 2.2 CaV 1.3

ribbon vesicle V-ATPase CSP CaN vesicle MAP1A PP2A SV2 NSF Munc18 VAMP synapsin CaM endophilin SNAP-25 RIM1 dynamin AP-2 vesicle ribeye vesicle CaSK CaBP4 Ub syntaxin Ub dynactin RIM2 CRMP 14-3-3 clathrin whirlin harmonin

laminin

AKAP PP2A AKAP 79/150 erbin 15 CaMKII eIF3 MAP2B MAP2B PKA Ub eIF3 Shank densin

CaV 1.2 CaV 1.3

Figure 25: Synaptic binding partners for CaV channels. Select binding partners for CaV2.2 Select binding partners for CaV2.2, postsynaptic CaV1.2, and pre- and postsynaptic CaV1.3. Proteins shown have been identified by more than one proteomics screen and/or a functional assay. Most G proteins, chaperone, and cytoskeletal proteins are not included. The proteins are generally grouped by function, but location in synapse is schematic. References are as follows: CaV2.2: CSP (Swayne et al., 2006), CRMP (Chi et al., 2009), endophilin (Chen et al., 2003), laminin (Nishimune et al., 2004), MAP1A (Leenders et al., 2008), Ub (Waithe et al., 2011), the rest were identified by (Muller et al., 2010) and either (Khanna et al., 2007a) or (Khanna et al., 2007b). CaV1.2: PP2A (Davare et al., 2000), PKA (Johnson et al., 1994), AKAP79/150 (Oliver- ia et al., 2007), HA (Kochlamazashvili et al., 2010), Ub (Altier et al., 2011), eIF3 (Green et al., 2007), MAP2B

(Marshall et al., 2011). CaV1.3 presynapse: whirlin (Kersten et al., 2010), RIM2 (Gebhart et al., 2010), harmonin (Gregory et al., 2011), Ub (Kawaguchi et al., 2006), ribeye (Sheets et al., 2011), CaBP4 (Yang et al.,

2006). CaV1.3 postsynapse: Shank (Zhang et al., 2005), erbin (Calin-Jageman et al., 2007), densin (Jenkins et al., 2010), CaMKII (Jenkins et al., 2010), eIF3 (Green et al., 2007), AKAP15 (Marshall et al., 2011), MAP2B (Marshall et al., 2011). in defining unique protein-protein interactions

Special considerations related to the large size of CaV channel genes The average number of exons comprising human genes range from 4-8, with only 0.2-6 % of genes containing more than 20 exons (Deutsch and Long 1999; Venter, Adams et al. 2001).

CaV genes are therefore exceptional, containing at least 37 exons, and the human CaV2.2 gene contains ~50. One obvious consequence of the size of these genes is that they encode channels that can interact directly with many more partners than smaller proteins. The binding partners illustrated in Fig. 25 are only a fraction of those found to interact with CaV2.2. Therefore, al- 73 2+ though CaV channels are first and foremost known for their ability to pass Ca in rapid response to membrane depolarization, they are also important scaffolding centers and potentially synaptic organizers.

WHAT IS THE PHYSIOLOGICAL SIGNIFICANCE OF VIP-MEDIATED VII IN SCG NEURONS? It has been known for decades that sympathetic and parasympathetic terminals function in opposition and can influence the activity of one another (Samaan 1935). What could the function of VDI vs. VII inhibition be? VI inhibition is stimulus independent, so even during times of high presynaptic activity the inhibition will remain robust. In contrast, a VD inhibition will be relieved by high activity. E18a is regulated both at the tissue level and also during development. Increas- ing the percentage of channels containing e18a could lead to increased sensitivity to robust inhibition of sympathetic terminals by their opposing parasympathetic terminals, and thus more refined signaling to the target tissue. In Chapter 2 I show data where we challenged SCG neurons with VIP to study the mechanism of G protein inhibition of CaV2.2. What is the physiological relevance of VIP inhibition of SCG channels? SCG neurons supply the main sympathetic innervation of cerebral vessels (Edvinsson and Uddman 2005). Thus, they regulate the sympathetic tone of the vessels that cause the pain associated with headaches. Primary headaches, including migraine and cluster headaches, have a neurovascular origin: headache pain results from vasodilation of cerebral blood vessels that activates the trigeminovascular reflex (Edvinsson and Uddman 2005). VIP is a potent vasodilator specifically implicated in cluster headache: during the headache, patients show overactivity of the parasympathetic nervous system, including facial abnormalities and the release of VIP into cranial venous blood (Jansen-Olesen, Goadsby et al. 1994; Edvinsson and Uddman 2005). The purpose of VIP release from parasympathetic cholinergic neurons is not well understood (Whittaker 2010), and as SCG terminals themselves do not release VIP (Sun, Rao et al. 1992), we speculate that parasympathetic release of VIP onto cerebral vessels could be inhibiting SCG neurons via Gs-coupled heteroreceptors.

There is precedent for stimulus-dependent autonomic nervous system interactions. In the heart, Brack and colleagues have shown that the parasympathetic vagal nerve reduces the activity of 74 the sympathetic system in a frequency dependent manner: the higher the sympathetic activity, the less the parasympathetic terminal can inhibit it (Brack, Coote et al. 2004). Hence one can describe the inhibition as voltage-dependent: strong presynaptic depolarization of the sympathetic terminal prevented the parasympathetic inhibition. Perhaps a similar interaction occurs at the SCG target cerebral vessels, where presynaptic SCG channels containing e18a are more sensitive to inhibition by parasympathetic neuron-released VIP.

FUTURE DIRECTIONS The data presented in Chapter 3 represent the beginning of an investigation into the unique signaling cascade between GsPCRs and e18a-containing CaV2.2 channels. Many questions remain unanswered. Gs activates cAMP, which in turn activates PKA but we lack information about the rest of this signaling pathway. As discussed above, different GPCRs inhibit CaV2.2 channels in unique ways and with different dependencies on these molecules. These could be teased out further with treatments that specifically probed cAMP and PKA. For example, treating CaV2.2-expressing tsA201 cells with the cAMP analog 8-bromo-cAMP could be used to test if constitutively activated cAMP mimics the actions of receptor activation on CaV2.2. Likewise, treatment with the PKA inhibitor PKI could demonstrate if PKA is involved in channel inhibition. My analysis of the K3A mutant of CaV2.2 might implicate posttranslational modification of this lysine in channel function. Our lab currently uses HA-tagged ubiquitin molecules to study ubiquitination of im- munoprecipitated CaV2.2 channels from tsA201 cells transiently expressing these channels. We have yet to look at the difference in ubiquitin levels for CaV2.2[D18a] vs CaV2.2[e18a] channels, but this is obviously an important experiment. Members of the lab are also currently optimizing a mass spectrophotometry approach to look for posttranslational modifications of CaV2.2 channels. If successful, this approach would be highly informative to explore potential differences in phosphorylation and ubiquitination, for example betweenV Ca 2.2 channels with and without e18a. Finally, multiple reagents are available to test for possible involvement of internalization in

VII of CaV2.2 channels. A dominant/negative dynamin mutant and the dynamin inhibitor dynaso- re, could be used to probe for a role for dynamin-dependent internalization of CaV2.2 channels.

75 Our lab is currently creating knock-in mouse lines in which e18a is never or always included. If these mice are viable, they will be invaluable for probing many questions concerning the role of e18a in neuronal function. A similar approach was used to generate e37a-only and e37b-only

CaV2.2 mice that helped the lab show that splicing of CaV2.2 influenced the peripheral analgesic actions of morphine (Andrade, Denome et al. 2010). Our use of Fox-2 siRNAs to shift the balance of splicing from CaV2.2[D18a] to CaV2.2[e18a] channels in SCG neurons allowed us to compare uninjected and injected neurons under the same conditions, but it is labor intensive and Fox-2 has targets other than CaV2.2. The availability of mouse lines expressing CaV2.2 channels that either all contain or all lack e18a will allow us to test the functional importance of this splicing event in other regions of the nervous system where e18a is normally expressed.

76 6| MATERIALS + METHODS Clones and transfection tsA201 cells were grown in DMEM (Sigma) + 10% Fetal Bovine Serum (Gibco) and split when 70% confluent. Cells were transfected when 70% confluent using Lipofectamine 2000 (Invitro- gen) and Opti-MEM (Sigma). Cells were transfected with calcium channel cDNA clones of CaV2.2, β2a or β3, and α2δ1 in a molar ratio of 1:1:1 along with EGFP. GPCRs were cotransfected at the following ratios of CaV2.2:GPCR : AT1R 10:1; B2R 10:1; D1R 1:2; M1R 5:1. The AT1R and B2R were gifts from Mark Shapiro; D1R was a gift from Gerald Zamponi; M1R was a gift from

Stephen Ikeda. The following clones were used and isolated in our lab from rat brain: Cavb3

(M88751), CaVα2δ1(AF286488); CaV2.2[D18a,37b] (AF055477); CaV2.2[18a, 37b] (HQ008360).

The CaV2.2[18a,37a] clone was constructed by cloning the ~4.9 kb AscI -PshAI fragment from

CaV2.2[18a,37b] into the ~7.2 kb AscI - PshAI fragment fromCaV2.2[e37a]. The b2a clone was a gift from David Yue. It was subcloned into pcDNA3.1/(zeo+) using EcoRI.

SCG cell isolation and culture

Superior cervical ganglia were removed from P0-2 mice. We dissociated ganglia in HBSS ( -CaCl2,

-MgCl2 -MgSO4) containing 1 mg/ml Trypsin (TRL3, Worthington) while incubating in a 37°C waterbath for 45-60 min. Following trypsinization, cells were dissociated by 5 min of tituration using a fire-polished pipette. Cells were plated on coverslips coated with laminin (Invitrogen) in a growth media modified from the one used by Mahanthappa and Patterson (Mahanthappa &

Patterson, 1998). Neurons were maintained at 37°C with 5% CO2. 24 hrs after plating, cells were treated with 10µM ara-C (Sigma) to inhibit glia growth.

Electrophysiology tsA201 cells. We performed standard whole cell patch clamp recording (Thaler et al., 2004).

Extracellular solution (in mM): 135 ChCl, 1 CaCl2 (except where noted in text), 4 MgCl2, 10 HEPES, pH 7.2 with CsOH. Internal: 126 CsCl, 10 EGTA, 1 EDTA, 10 HEPES, 4 MgATP, pH 7.2 with CsOH. Borosilicate glass pipettes (Warner Instruments) were pulled with a Sutter puller and fire-polished to a resistance of 2-4 MW. The following drugs were used: Pertussis toxin, cholera toxin, dopamine, GTPgS, and angiotensin II are from Sigma Aldrich. HBSS, DMEM and FBS are from 78 Gibco.

SCG neurons. External solution: 135 TEA-Cl, 4 MgCl2, 10 CaCl2, 10 HEPES, 100 nM TTX, pH 7.2 w/

TEA-OH. Internal: 126 CsCl, 1 EDTA (Cs2), 10 EGTA (Cs4), 10 HEPES, 4 MgATP, pH 7.2 with CsOH. We sylgard-coated and fire-polished electrodes to a resistance of 4–6 MΩ. There is currently no combination of toxins that will completely inhibit all other types of calcium channels while leaving the N-type current untouched. Therefore we isolated the N-type component of inhibition using w-conotoxin (10 mM). We inhibited the N-type component, and then subtracted the non-N-type current from the control current in order to isolate the N-type (as in Andrade et al, 2010). All of the values (Con, VIP, etc.) were calculated by taking the area under the curve during 48 ms of test pulse. All cells did not completely recover from VIP treatment, therefore separate cells were used to calculate the effect of VIP on non-N-type and the effect of VIP on total Ca current. % Inhibition of N-type current = total N-type inhibition / total N-type current = [(Con – VIP) – (ω-CTX – ω-CTX+VIP)] / [Con – ω-CTX]. % VD inhibition of N-type current = VD component of N-type inhibition / total N-type inhibition = [(VIP+pp – VIP) – (ω-CTX+VIP+pp – ω-CTX+VIP)] / [(Con – VIP) – (ω-CTX – ω-CTX+VIP)]. Estimators: ω-CTX = Con * ave inhibition of total Ca current by ω-CTX = 22.6 +/- 3.5% (n=10); ω-CTX+pp = 22.5 +/- 3.7% (n=10). ω-CTX+VIP = Con * ave inhibition of non-N-type by VIP =16.3 +/- 0.8% (n=5); ω-CTX+VIP+pp = 16.3 +/- 0.8% (n=5). P values were obtained using the Student’s t-test unless otherwise indicated.

Data analysis. We used pClamp version 10 software and the Axopatch 200A (Axon Instruments) for data acquisition; data were filtered at 2 kHz (–3 dB) and sampled at 20 kHz. We compensated series resistance by 70–90% with a 7-μs lag and performed online leak correction with a P/–4 protocol. All recordings were obtained at room temperature.

Genomic sequence alignment We used the mouse e18a sequence to BLAT other genomes using the UCSC genome browser.

For each species, we identified the 18a exonic sequence as well as 200 nucleotides of each of the neighboring introns. We then used the alignment tool on Vector NTI Advance 10 (Invitrogen) to 79 align the sequences from all species.

RT-PCR We dissected SCG from wildtype mice (4-10 mice per age group). Ganglia were homogenized in TRIZOL (Invitrogen) using a Polytron homogenizer. We used SuperScript II (Invitrogen) to reverse transcribe 1µg of RNA per group. We used 1µl of this first-strand cDNA in a 25µl PCR reaction containing Taq polymerase (New England Biolabs). E18a RT-PCR primers are: BU2118 5’GGC- CATTGCTGTGGACAACCTT and BD2324 5’CGCAGGTTCTGGAGCCTTAGCT. PCR was performed with a thermocycler (BioRad DNAEngine), using the following program : 1 cycle of 95°C for 2 min; 25 cycles of 95°C for 0.5 min, 60°C for 0.5 min, 72°C for 1 min, a final 6 min extension step at 72°C. The RT-PCR protocol for amplifying cDNA from F11 cells was identical. RT-PCR products were run on 8% denaturing polyacrylamide gels. Gels were stained with SYBR Gold stain (Invitrogen). Unsaturated bands were imaged on a BioRad gel doc station. We used the program Scion Image to analyze unsaturated band densities.

F11 cell culture and siRNA transfection F11 cells are split twice a week and are grown in DMEM culture media plus 10% fetal bovine serum and 1% penicillin/streptomycin. One day before transfection, cells are plated in six-well plates without antibiotics. We used Lipofectamine 2000 (Invitrogen) to transfect cells with siRNAs for 48-60 hrs. siRNAs used (Thermo Scientific Dharmacon): Fox-2 (original sequence provided by Dr. Doug Black (UCLA)): CCUGGCUAUUGCAAUAUUUUU; Fox-2b: UAUUGCAAUAGC- CAGGCCUCUU; siGENOME Cyclophillin β control siRNA: catalog #D-001136-01-05; siGENOME non-targeting siRNA #3: catalog #D-001210-03-05.

Western Blot We used Lipofectamine 2000 (Invitrogen) to transfect 10cm dishes of F11 cells with 0.5µg EGFP (C1, Clontech) alone, EGFP + 6µg Fox-2 plasmid (provided by Douglas Black’s lab; used as a size control), or EGFP + 100nM Fox-2 siRNA for 48 hrs. Media was changed after 6 hrs. We estimate that approximately 25% of cells were transfected with EGFP. RIPA buffer containing protease 80 inhibitors (Roche) was used to lyse the cells. Lysed cells were spun at 13,500 RPM at 4°C for 20 min. We used a BCA kit (Pierce) to determine the protein concentration of the supernatant. We denatured 12 µg of each sample for 10 min at 90°C and then ran samples on a 4% stacking and 15% resolving SDS-PAGE gel. Protein was transferred to a nitrocellulose membrane (Protran, Whatman) and blocked with 5% dry milk in phosphate-buffered saline with 0.1% Tween-20 (PBST) for one hour at room temperature. The blot was then incubated in a rabbit anti-FoxRRM primary antibody that recognizes all Fox proteins (1:2000 in PBST with 1%BSA, gift from Doug Black) for one hour at room temperature. Blot was rinsed and then incubated in a donkey-anti- rabbit horseradish peroxidase secondary (1:7500 in PBST with 1%BSA, Jackson ImmunoResearch) for one hour at room temperature. Proteins were detected with a ECL chemiluminescence kit (Vector). The blot was then stripped with 2-mercaptoethanol and SDS and reprobed with a rabbit anti-GAPDH antibody (1:1000 in PBST with 1%BSA, Cell Signaling).

SCG injections SCG neurons used for electrophysiology were injected 24 hrs after plating; those used for immunocytochemistry were injected 7 days after plating. We used an InjectMan NI2 manipulator and Femtojet microinjector (Eppendorf) for injections. We combined 10µl of 20µM siRNA with 10ul of 8.9mg/ml dextran fluorescein (Invitrogen) and spun down any undissolved dextran by spin- ning the sample at 14,000 RPM for 10 min at 4°C. We then used 2µl of this solution to load the microinjection tip (Eppendorf). Injection parameters: Pi 150 hPa, Ti 0.4 sec., Pc 40 hPa.

Immunocytochemistry SCG neurons were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min. We then blocked the cells for 1 hr at room temperature (RT) in 10% normal goat serum (NGS) in PBS. Cells were incubated in a rabbit anti-Fox-2 antibody (1:500, Bethyl) for 2.5 hrs at room temperature in 3%NGS. Cells were washed in PBS and then incubated in a goat-anti-rabbit rhodamine secondary antibody (1:200, Jackson ImmunoResearch) in 3%NGS for 45 min at RT.

Images were captured with a confocal microscope (LSM 510, Zeiss), and analyzed with AxoVision 4.8.1 (Carl Zeiss Imaging Solutions). 81 Mutagenesis Stratagene Quikchange II kit was used to perform site-directed mutagensis on CaV2.2. The PshAI- KpnI fragment of CaV2.2[e18a] was subcloned into pBluscript II SK+ for mutagenesis PCR. The All S to A clone was made by first deleting a small section of e18a-containing sequence, then insert- ing a mutated piece. The All S + K to A and All S + All T to A clones were then made from the All S to A plasmid. The following forward primers were used for the PCRs:

All S to A_delete:5’ GCAAACTCGAGGTACTGTAACCGCAGCAGAACTCGGCCAAGGCGC All S to A_insert: 5’ CGAGGTACTGTAGCTCGCGCCGCAGCTGTCGCCGCCGTAAACGCACCGCAG- CAGAACTCG All S+K to A: 5’ CCATTGCTGCTTTTGTAGCGCAAACTCGAGGTAC All S+ All T to A: 5’ GCTTTTGTAAAGCAAGCTCGAGGTGCTGTAGCTCGCGCCG S14,20A: 5’ CTCGCAGCTCAGCTGTCTCCAGCGTAAACGCACCGCAGCAG T8A: 5’ GCAAACTCGAGGTGCTGTATCTCGCAGCTCATCTGTCTCC K3A: 5’ CATTGCTGCTTTTGTAGCGCAAACTCGAGGTACTG

The following PCR protocol was used: 1. 95C, 1 min. 2. 95C, 50 sec. 3. 60C, 50 sec. 4. 68C, 16 min., 36 sec. Repeat 2-4 18X 5. 68C, 7 min.

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