L-type Ca2+ channels regulate various functions spanning from neuronal excitability to gene transcription. The long term interest of this grant is to determine the molecular mechanisms that control activity and functional availability of the L-type channel Cav1.2. Our strong preliminary data indicate that a-actinin binds directly to the central pore-forming Cav1.2 subunit. Calmodulin binds to the same region as a-actinin and displaces it from Cav1.2 in the presence but not absence of Ca2+. Knock-down of a-actinin with siRNA or expression of two different dominant negative a-actinin fragments lead to a -50% reduction in the current density of Cav1.2 and substantially reduce peripheral Cav1.2 localization as detected by immunofluorescence. We hypothesize that a-actinin fosters surface expression of Cav1.2 by inhibiting its endocytosis. We further hypothesize that displacement of a-actinin by Ca2+/calmodulin promotes endocytosis of Cav1.2. We will define residues on Cav1.2 that are critical for a-actinin but not CaM binding and vice versa. We will inhibit the interactions between a-actinin and Cav1.2 by: a) expressing dominant negative a-actinin constructs;b) knocking down a-actinin with siRNA (we already established the siRNAs);c) expressing Cav1.2 with residues mutated that are crucial for a-actinin binding. Whether surface expression is reduced following these manipulations as compared to control conditions will be evaluated by biochemical and cell biological methods. We will investigate the role of calmodulin in dislocating a-actinin from Cav1.2 upon Ca2+ influx, thereby perhaps promoting Cav1.2 internalization, by: a) overexpressing Ca2+ binding-deficient calmodulin mutants that act as dominant negative constructs for a number of ion channels with respect to the regulation of their gating by Ca2+/calmodulin, including Cav1.2;b) expressing mutant Cav1.2 that does not bind calmodulin but still interacts with a-actinin. Ca2+-dependent internalization will be compared between control conditions (e.g., overexpression of wt calmodulin or wt Cav1.2) and test conditions (e.g., dominant negative calmodulin or mutant Cav1.2). Cav1.2 channel activity is strongly increased in aged rats. The L-type channel inhibitor nimodipine impressively improves learning capabilities of aged rodents. Hence, an increase in Cav1.2 channel activity is thought to contribute to the etiology of senile symptoms and Alzheimer's disease. Cav1.2 has also been implicated in depression and anxiety disorders. Our work on the regulation of surface expression of Cav1.2 by the interplay between a-actinin and calmodulin will fill a critical gap in our understanding of how Cav1.2 surface expression and localization is regulated. It will thereby contribute to the development of treatment of these diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
5R01AG017502-10
Application #
8127925
Study Section
Special Emphasis Panel (ZRG1-MDCN-G (02))
Program Officer
Wise, Bradley C
Project Start
1999-12-01
Project End
2013-09-30
Budget Start
2011-07-01
Budget End
2013-09-30
Support Year
10
Fiscal Year
2011
Total Cost
$293,452
Indirect Cost
Name
University of California Davis
Department
Pharmacology
Type
Schools of Medicine
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
Tseng, Pang-Yen; Henderson, Peter B; Hergarden, Anne C et al. (2017) ?-Actinin Promotes Surface Localization and Current Density of the Ca2+ Channel CaV1.2 by Binding to the IQ Region of the ?1 Subunit. Biochemistry 56:3669-3681
Turner, Matthew; Anderson, David E; Rajan, Sahana et al. (2016) Chemical shift assignments of the C-terminal EF-hand domain of ?-actinin-1. Biomol NMR Assign 10:219-22
Zhang, Yonghong; Matt, Lucas; Patriarchi, Tommaso et al. (2014) Capping of the N-terminus of PSD-95 by calmodulin triggers its postsynaptic release. EMBO J 33:1341-53
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Núñez-Santana, Félix Luis; Oh, Myongsoo Matthew; Antion, Marcia Diana et al. (2014) Surface L-type Ca2+ channel expression levels are increased in aged hippocampus. Aging Cell 13:111-20
Hell, Johannes W (2014) CaMKII: claiming center stage in postsynaptic function and organization. Neuron 81:249-65
Chen, Chao-Yin; Matt, Lucas; Hell, Johannes Wilhelm et al. (2014) Perampanel inhibition of AMPA receptor currents in cultured hippocampal neurons. PLoS One 9:e108021
Hall, Duane D; Dai, Shuiping; Tseng, Pang-Yen et al. (2013) Competition between ?-actinin and Ca²?-calmodulin controls surface retention of the L-type Ca²? channel Ca(V)1.2. Neuron 78:483-97
Zhang, Mingxu; Patriarchi, Tommaso; Stein, Ivar S et al. (2013) Adenylyl cyclase anchoring by a kinase anchor protein AKAP5 (AKAP79/150) is important for postsynaptic ?-adrenergic signaling. J Biol Chem 288:17918-31
Hamilton, Andrew M; Oh, Won Chan; Vega-Ramirez, Hugo et al. (2012) Activity-dependent growth of new dendritic spines is regulated by the proteasome. Neuron 74:1023-30

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