This proposal will continue to elucidate the regulation of neuronal function by calcium-binding proteins (CBPs) including calmodulin (CaM)-dependent second messenger systems. Calcium (Ca2+), one of the most important cellular signals, can control events with timings as diverse as neurotransmitter release and neuronal degeneration. Yet, the dynamics of its interactions with CBPs are little known, least of all at the time-scale needed to resolve its distinct roles in neuronal signaling The proposed studies will provide detailed insights into the functioning of endogenous neuronal Ca2+ -buffers, their interactions and competition for Ca2+ with Ca2+ effector systems, and their combined roles in determining the specific vulnerability of central neurons. The main hypothesis is as follows: the roles of neuronal CBPs as intracellular Ca2+ buffers or as competitors for specific Ca2+-dependent regulatory events are defined by their unique Ca2+-binding kinetics. In turn, the distinct kinetics and selective interactions of CBPs with Ca2+-effector systems are responsible for their diverse roles in regulating neuronal excitability, synaptic integration, and selective neuronal vulnerability. This hypothesis will be addressed in the following specific aims: 1) to resolve the Ca2+-binding kinetics of three neuronal CBPs including calbindin-D28K (CB28K) Parvalbumin (PV), and calretinin (CR) at or near physiological conditions; 2) to determine the physiological Ca2+-binding kinetics of substrate-free CaM, and of CaM bound to some effectors including the Ca2+/CAM-dependent protein kinase II (CaMK II), the Ca2+/CaM-dependent serine (Ser)/threonine (Thr) phosphatase calcineurin (CN), and the CaM-binding domain of the SK2 CA2+ dependent potassium (K+) channel; 3) to ascertain the role of CB28K in neuronal Ca2+ handling and in short-term plasticity at specific synapses; 4) to determine how CR alters neuronal excitability and short-term plasticity at synapses between mossy cells and granule cells of the hippocampal formation; 5) to uncover the relationship between the vulnerability of murine mossy cells and their CR content. These five aims will be accomplished by measuring Ca2+-binding to CBPs after ultra-fast photolysis of caged Ca2+ followed by computing the binding rate constants through compartmental kinetic modeling. High-resolution electrophysiological recordings combined with measurements of intracellular Ca2+ dynamics at the sub-millisecond time-scale will be done in identified neurons of brain slices prepared from mice with genetically altered levels of CBPs. For the first time, the physiological Ca2+-binding properties of CBPs will be linked to their functions as Ca2+ buffers, regulators of cellular excitability, and determinants of selective neuronal vulnerability. Understanding the precise relationship between intracellular Ca+ -binding and Ca2+ effector mechanisms will uncover the complex involvement of Ca2+ in the triad of neuronal excitability, plasticity, and vulnerability. This triad is critical in severe neurological maladies including ischemia/stroke neurodegenerative disorders, and epilepsy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS027528-11
Application #
6723640
Study Section
Special Emphasis Panel (ZRG1-BDCN-2 (01))
Program Officer
Stewart, Randall
Project Start
1990-08-01
Project End
2007-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
11
Fiscal Year
2004
Total Cost
$325,969
Indirect Cost
Name
University of California Los Angeles
Department
Neurology
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Faas, Guido C; Mody, Istvan (2012) Measuring the kinetics of calcium binding proteins with flash photolysis. Biochim Biophys Acta 1820:1195-204
Faas, Guido C; Raghavachari, Sridhar; Lisman, John E et al. (2011) Calmodulin as a direct detector of Ca2+ signals. Nat Neurosci 14:301-4
Gordey, M; Mekmanee, L; Mody, I (2001) Altered effects of ethanol in NR2A(DeltaC/DeltaC) mice expressing C-terminally truncated NR2A subunit of NMDA receptor. Neuroscience 105:987-97
Otis, T S; De Koninck, Y; Mody, I (1994) Lasting potentiation of inhibition is associated with an increased number of gamma-aminobutyric acid type A receptors activated during miniature inhibitory postsynaptic currents. Proc Natl Acad Sci U S A 91:7698-702
Mody, I; De Koninck, Y; Otis, T S et al. (1994) Bridging the cleft at GABA synapses in the brain. Trends Neurosci 17:517-25
Mody, I; Staley, K J (1994) Cell properties in the epileptic hippocampus. Hippocampus 4:275-80
De Koninck, Y; Mody, I (1994) Noise analysis of miniature IPSCs in adult rat brain slices: properties and modulation of synaptic GABAA receptor channels. J Neurophysiol 71:1318-35
Soltesz, I; Zhou, Z; Smith, G M et al. (1994) Rapid turnover rate of the hippocampal synaptic NMDA-R1 receptor subunits. Neurosci Lett 181:5-8
Kohr, G; Mody, I (1994) Kindling increases N-methyl-D-aspartate potency at single N-methyl-D-aspartate channels in dentate gyrus granule cells. Neuroscience 62:975-81
Mody, I; Soltesz, I (1993) Activity-dependent changes in structure and function of hippocampal neurons. Hippocampus 3 Spec No:99-111

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