I would also like to thank the members of my committee for their guidance on my projects and on my career development throughout my graduate school. I would also like to thank the Vanderbilt International Scholar Program for supporting my graduate studies at Vanderbilt.
Introduction overview
An overview of voltage-gated Ca 2+ channels
Ligand-gated Ca 2+ channels and voltage-gated Ca 2+ channel
Among them, L-type Ca2+ channels are activated at high membrane potential and show slow inactivation when using Ba2+. Therefore, T-type Ca2+ channels are also referred to as low-voltage-activated (LVA) channels and the rest as high-voltage-activated (HVA) channels (Catterall, 2011).
Molecular components of voltage-gated Ca 2+ channels
Based on their physiological and pharmacological properties, voltage-gated Ca2+ channels can be classified into five different types: L-, P/Q-, N-, R- and T-type (Fig. Voltage dependence and inactivation kinetics of P /Q-, N- and R-type Ca2+ channels lie between L- and T-type Ca2+ channels and are more similar to L-type than T-type (Nowycky et al., 1985; Randall and Tsien, 1995).
Functions of neuronal L-type Ca 2+ channels
Role of LTCCs in synaptic plasticity
Pharmacological studies have shown that both short- and long-term eCB-dependent inhibition of transmission requires postsynaptic activation of L-type Ca2+ channels (some also require metabotropic glutamate receptors), followed by presynaptic activation of eCB receptors (Mathur and Lovinger, 2012) . . Although the details are not yet fully known, it is believed that ECB synthesis and/or mobilization requires Ca2+ influx through L-type Ca2+ channels.
Role of LTCCs in excitation-transcription coupling
Activated CaMKIV then phosphorylates transcription factor CREB at the Serine133 site to increase transcription of downstream immediate early genes such as c-fos, Arc and Homer1a (Bito et al., 1996; Deisseroth et al., 1996; Hardingham et al., 2001 ). Among the three pathways mentioned above, L-type Ca 2+ channels are preferentially associated with those involving active transport of NFATc4 or Ca 2+ /calmodulin ( Deisseroth et al., 1996 ; Graef et al., 1999 ; Wheeler et al., 2012 ).
Other specialized functions of LTCC in specific brain regions
In SNc dopaminergic neurons, Ca2+ influx through CaV1.3 L-type Ca2+ channels is coupled to SK channels, and together they are required for autonomous firing (Guzman et al., 2009; Putzier et al., 2009). Interestingly, this appears to correlate with increased Ca2+-dependent proteolysis of L-type Ca2+ channels in aged mice (Michailidis et al., 2014).
Regulation of neuronal L-type Ca 2+ channels
- L-type Ca 2+ channel localization
- Interaction with scaffold proteins
- L-type Ca 2+ Channel clustering
- Regulation of L-type Ca 2+ channels by Ca 2+ -binding proteins
- Regulation of L-type Ca 2+ channels by phosphorylation
- Regulation of L-type Ca 2+ channels by G proteins
- Alternative splicing, RNA-editing, and Proteolysis
Catterall's group first showed that AKAP15 binds to the leucine zipper site on CaV1.2 channels (Hulme et al., 2003). A second site of proteolysis within the CaV1.2 II-II linker was recently proposed (Michailidis et al., 2014).
CaMKII as an important Ca 2+ effector
- CaMKII structure and the Ca 2+ regulation
- Functions of CaMKII as a protein kinase
- Functions of CaMKII as a scaffold protein
- Other CaMKII autophosphorylation sites
Phosphorylation of GluN2B Ser1303 by CaMKII promotes the dissociation of preformed complexes of CaMKII and GluN2B (Strack et al., 2000a). More recently, CaMKII has been reported to phosphorylate diacylglycerol lipase- (DAGL), a key enzyme that produces the endocannabinoid 2-arachidonoylglycerol (2-AG) (Shonesy et al., 2013).
LTCC-mediated nuclear CREB signaling
Transcriptional factor CREB and memory formation
They found that disruption of CREB binding to its DNA targets selectively blocked serotonin-induced long-term facilitation, but not short-term facilitation. The importance of CREB in memory retention was further demonstrated in mammals by Silva's laboratory, where they found that CREB mutant mice showed normal short-term memory, but were deficient in long-term memory under cued or contextual conditioning and Morris water maze text (Bourtchuladze et al. al. al., 1994)).
Mechanism of L-type Ca 2+ channel-mediated CREB phosphorylation
The importance of CREB in memory retention was further demonstrated in mammals by Silva's laboratory, where they found that CREB mutant mice showed normal short-term memory but were deficient in long-term memory in cued or contextual conditioning and Morris water maze text (Bourtchuladze et al. al. , 1994). shuttle function through several steps: 1) Ca2+ influx through L-type Ca2+ channels recruits CaMKII/ and CaMKII to the vicinity of the channel; 2) phosphorylation at Thr287 of CaMKII (presumably by CaMKII/) traps calmodulin; 3) dephosphorylation of Ser334 exposes the unique nuclear localization signal of CaMKII through a Ca2+- dependent phosphatase calcineurin; 4) CaMKII then shuttles to the nucleus and delivers calmodulin which is required for CaMKK, CaMKIV and CREB activation.
L-type Ca 2+ channel-mediated E-T coupling in disease and behavior
Ca2+ influx through L-type Ca2+ channels recruits CaMKII and CaMKII to the vicinity of the channel. Recently, it was found that the voltage-induced conformational change of the channel is also required for E-T coupling and that NMDA receptors are somehow functionally linked to L-type Ca2+ channels.
An overview of work reported in this dissertation
The N-terminal domain of the CaV1.3 L-type Ca2+ channel is predicted to be a DNA-binding protein, and overexpression of the N-terminal domain in cultured neurons altered both the transcription and the cell morphology of neurons. In this chapter I show that activated Ca2+/calmodulin-dependent protein kinase II (CaMKII) strongly interacts with a novel binding motif in the N-terminal domain of CaV1 LTCC 1 subunits that is not conserved in CaV2 or CaV3 voltage-gated Ca2+ . .
Experimental procedures
A plasmid encoding CaV1.3 with an N-terminal HA tag (pCGNH-CaV1.3, for co-immunoprecipitation) was prepared by inserting rat CaV1.3 cDNA into pCGN vector (a gift from Dr. Winship Herr , Université de Lausanne, Switzerland, Addgene plasmid ID. 53308). Mouse CaMKII pcDNA was co-transfected with pcDNA empty vector (control) or pCGNH-CaV1.3, 3 and 2- subunits.
Results
A proteomics approach to identify L-type Ca 2+ channel complexes
These data demonstrate that CaMKII isoforms in the brain can directly or indirectly associate with the N-terminal domain of CaV1.3 and CaV1.2. Purified, GST-labeled intracellular domains of CaV1.2 and CaV1.3 were individually incubated with mouse forebrain lysates.
Glutathione-agarose co-sedimentation assays show that there is no reliably detectable interaction of inactive (non-autophosphorylated) conformations of CaMKII with any of the CaV1.3 intracellular domains, but the activated (pre-autophosphorylated, pT286) CaMKII. Activation of CaMKII by binding of Ca2+/calmodulin and Mg-ADP is sufficient for interaction with the CaV1.3 NTD.
CaMKII/NTD interaction is L-type channel specific
Ca2+ channel NTDs are more conserved in membrane-proximal regions, but become more divergent in the distal regions. The CaV1.3 NTD shows the strongest binding to CaMKII, followed by CaV1.2, while interactions with CaV2.2 and CaV3.2 are barely detected.
Molecular determinants for CaV1.3 NTD interaction with CaMKII
We identified two additional CaMKII mutations (V102E and E109K) that also significantly interfered with binding to the CaV1.3 NTD (Fig. 2.5B), while another mutation (Y210E) had no significant impact. A V102E mutation selectively disrupts CaMKII binding to the CaV1.3 NTD: V102/3 are highlighted in red in Panel A.
The Ca V 1.3 NTD is important for CaMKII association with LTCC complexes 63
C2 plots levels of immunoprecipitated HA-CaV1.3 proteins (black) and CaMKII (purple) in the presence of Ca 2+ /calmodulin/Mg 2+ /ATP normalized to levels isolated in the presence of EDTA in each experiment. Similar analysis of co-immunoprecipitation of WT or V102E-CaMKII with WT, 69-93 or RKR-AAA HA-CaV1.3 in the presence of EDTA or Ca2+/calmodulin/Mg2+/ATP.
Ca V 1.3 NTD is required for LTCC-and CaMKII-mediated nuclear signaling
Expression of a nimodipine-resistant CaV1.3-T1033Y mutant rescues nimodipine blockade of pCREB induction by 40 mM KCl. However, deletion of the CaMKII binding domain (69-93) prevents rescue of pCREB signaling by CaV1.3- T1033Y.
Lysates of HEK293T cells co-expressing FRB-CaMKII and CaMKII (1:5 ratio) were mixed with lysates co-expressing HA-FKBP-CaMKII-K43R and HA-CaMKII-K43Rio (1:K43Rio) or absence of 5 nM rapamycin on ice for 5 min. Note that the addition of rapamycin causes a significant reduction in the mobility of both FKBP-tagged and untagged CaMKII.
Discussion
Comparison of NTD with previously identified CaMKII binding domains
The CaMKII-binding domains in the GluN2B subunit of the NMDA receptor and the 1/2 subunits of the VGCC share a similar sequence to the autoregulatory domain of CaMKII, including the presence of an (auto)phosphorylation site (Grueter et al., 2008). In addition, none of these classes of CaMKII-binding domains have appreciable sequence similarity to the CaMKII-binding domains in the CaV1.3 and CaV1.2 NTDs identified here.
Roles of the NTD and other CaMKAPs in LTCC complexes
Nevertheless, we hypothesize that neurons contain multiple subpopulations of CaV1.3 LTCC complexes associated with different -subunit variants. Indeed, the key role for densin in targeting CaMKII to promote Ca2+-dependent facilitation of CaV1.3 LTCCs was noted above (Jenkins et al., 2010).
Role of CaMKII binding to the Ca V 1.3 NTD in E-T coupling
In this study, we focused on the E-T coupling paradigm, which has been shown to depend on signaling within the LTCC nanodomain (Deisseroth et al., 1996; Wheeler et al., 2008). Thus, selective members of the CaV1.3 channel complex, such as Shank3, may play an important role in E-T coupling (Zhang et al., 2005a).
Future directions
Purified GST-tagged CaV1.3 domains of equal mass (2 g) were incubated with purified mouse CaMKII at 30 oC for 10 minutes in the presence of [-32P]ATP. Protein stains (top panels) and autoradiographs (bottom panels) showing mutation of the CaMKII binding site blocks CaMKII phosphorylation of the CaV1.3 NTD.
ROLE OF CAMKII/Ca V 1.3 RKR MOTIF INTERACTION IN CHANNEL CLUSTERING
Experimental procedures
To generate Flag-CaV1.3, the DNA sequence encoding HA in the pCGN vector was first deleted to generate pCGN0 by mutagenesis. The CaV1.3 cDNA sequence was then inserted into pCGNF between the XbaI and BamHI sites using SLIC cloning.
Results
- CaMKII clusters Ca V 1.3 L-type Ca 2+ channels in a Ca 2+ -dependent way
- The CaMKII/RKR motif interaction is required for channel clustering
- Ser1486 of Ca V 1.3 CTD is not significantly phosphorylated by CaMKII
- CaMKII phosphorylates Ca V 1.3 at multiple intracellular domains
- Identification of CaMKII phosphorylation sites within Ca V 1.3
- Ca V 1.3 NTD phosphorylation by CaMKII depends on CaMKII/RKR motif
Deletion of the CaMKII association domain (AD) prevents the Ca2+-dependent increase of HA-CaV1.3 in HA immunoprecipitates. Both wild-type and AD CaMKII co-immunoprecipitate with HA-CaV1.3 in a Ca2+-dependent manner.
Discussion and future directions
- Potential mechanisms for L-type Ca 2+ channel clustering
- Clustering and coupling between Ca 2+ channels and other channels
- Effects of CaMKII phosphorylation sites on Ca V 1.3 channels
In addition to homomeric clustering of L-type Ca2+ channels, it is also possible that L-type Ca2+ channels can be clustered and coupled to other channels to facilitate crosstalk. Collectively, these data identify a novel mechanism for L-type Ca2+ channel regulation by the 2a subunit.
Experimental procedures
16101, for GST-tagged proteins) or Qiagen Ni-NTA agarose (cat. #30210, for His-tagged proteins) following the manufacturer's instructions. nM of GST- and His-tagged proteins were incubated with 20 l of pre-washed glutathione agarose beads in GST pulldown buffers for 1 hour before washing and elution.
Results
- A novel interaction between Ca V 1.2/Ca V 1.3 NTD and subunits
- Characterization of the molecular determinants for NTD/ interaction
- The NTD/ interaction is pH-dependent
- Effects of disrupting NTD/ interaction on voltage-dependent activation of
- NTD/2a interaction modulates Ca 2+ -dependent inactivation of Ca V 1.2 Ca 2+
This suggests that the NTD B1 regions of CaV1.2 and CaV1.3 are sufficient to bind subunits. This suggests that disruption of the NTD/β2a interaction increases the Ca 2+ -dependent inactivation of CaV1.2 channels.
Discussion and future directions
- Comparison of NTD/ interaction with previously identified interactions
- Potential mechanism of NTD/2 regulation
- Potential roles of CaMKII in modulating NTD/2 regulation
Examination of the sequence of the CaV1.3 N-terminal domain revealed that the RKR motif resembles a nuclear localization signal (NLS). We also tested the subcellular localization of the mCherry-tagged CaV1.3 NTD in cultured hippocampal neurons.
Experimental procedures
A DNA fragment encoding the rat CaV1.3 NTD was then inserted into FSVPG upstream of the V5 sequence to generate FSVPG-CaV1.3 NTD. For packaging of lentiviruses expressing CaV1.3 NTD or control GFP, HEK293T cells were divided into two triplicate bottles (Thermo Fisher Scientific, cat. #132913) the day before transfection.
Results
- Ca V 1.3 L-type Ca 2+ channel N-terminal domain may undergo Ca 2+ -dependent
- Ca V 1.3 L-type Ca 2+ channel N-terminal is a nucleus-located protein
- Overexpression of Ca V 1.3 NTD changes the neuronal transcriptome
- Overexpression of Ca V 1.3 NTD changes neuronal morphology
However, mCherry-tagged CaV1.3 NTD is mainly seen overlapping with the DAPI nuclear counterstain. The genes downregulated by overexpressing CaV1.3 NTD were enriched in histones (C) and motor proteins (D).
Discussion and future directions
- Calpain as a potential candidate that mediates Ca V 1.3 NTD proteolysis
- Ca V 1.3 NTD as a nuclear protein
- Potential links between Ca V 1.3 NTD proteolysis and Parkinson Disease
Calmodulin kinase II is involved in voltage-dependent facilitation of the L-type Cav1.2 calcium channel: Identification of the phosphorylation sites. Regulation of Postsynaptic Stability by the L-Type Calcium Channel CaV1.3 and Its Interaction with PDZ Proteins.
CONCLUSIONS AND FUTURE DIRECTIONS
The Ca V 1.3 NTD as a multifunctional regulatory domain
Data described in this thesis highlight the importance of the CaV1.3 N-terminal domain in regulating channel function and signaling in several ways (Fig. In particular, the following immediate directions may help to provide a better understanding of NTD regulation of the channel.
Understanding events within the Ca 2+ channel nanodomain
Visualizing channel clustering
Potential interplays among the RKR motif-binding proteins
Functional regulation of L-type calcium channels via protein kinase A-mediated phosphorylation of the beta(2) subunit. Mechanism and regulation of calcium/calmodulin-dependent protein kinase II targeting the NR2B subunit of the N-methyl-D-aspartate receptor.
