Fast release of neurotransmitters at CNS synapses is vital for brain function. At typical synapses, action potential depolarization of the presynaptic terminal activates a set of voltage gated Ca2+ channels, the N-, P/Q- and possibly R-type channels (known collectively as CaV2 channels) which generate a brief but large rise in free Ca2+ near the Ca2+ sensor molecule synaptotagmin. This unleashes vesicle fusion and transmitter release. We seek to understand the cell biology that puts the Ca2+ channels within the presynaptic active zone in appropriate abundance, stoichiometry and proximity to the release machinery. Our published experiments suggest that the proper deployment of presynaptic Ca2+ channels depends on the operation of 'slots'that interact with channel proteins to control their participation in neurotransmission. The evidence rests on the overexpression of mutant channels engineered to be Ca2+-impermeable. These channels strikingly reduce the ability of wild-type channels to support transmission, as if channels competed for a limited number of binding sites. One class of slots prefers P/Q-type channels. Another class accepts N-type but not P/Q-type channels. The main goal of our proposal is to clarify the nature of these putative CaV2-binding sites. We will delineate their physiological impact, their molecular and cellular basis, and their effect on the spatial organization of CaV2 channels. To achieve these goals, we will capitalize on recent experimental advances in our lab. First, we devised an efficient optical method to measure synaptic vesicle fusion and to dissect the contributions of each CaV2 channel to presynaptic Ca2+ influx. We engineered new red fluorescent protein reporter of exocytosis to be able to monitor simultaneously vesicle fusion and Ca2+ entry through CaV2 channels, using a green Ca2+ reporter, to see how well each CaV2 is coupled to vesicle fusion. Second, we will combine optical tests of synaptic activity with immunocytochemistry to find out if the apparent cap on the number of CaV2 channels mediating vesicle fusion is due to trafficking restrictions, a limited number of synaptic anchoring sites or both. Third, we devised a new approach to test whether potential synaptic anchor proteins really do anchor Ca2+ channels close to the machinery of vesicle fusion. Finally, we will dissect the organization of synaptic landmarks, CaV2 channels and synaptic organizing proteins with the 80-90 nm resolution of STED microscopy. Each CaV2 has a unique sensitivity to membrane potential and neuromodulating hormones and roles in migraine, neuropathic pain and epilepsy. Thus probing channel-slot interactions will help us to understand the regulation of neurotransmission by synaptic activity and neuromodulators in health and diseases.

Public Health Relevance

Normal development and function of the nervous system requires highly controlled communication between neurons that is dependent on calcium ions entering nerve terminals through three types of specialized, voltage-gated calcium channels. Aberrant contributions of a given calcium channel type alters neuronal communication and is linked to familial migraine, epilepsy and intractable, central pain. We aim to clarify how the contributions of these different channel types are controlled by structural proteins in the nerve terminals in order to further our understanding of neuronal communication and to identify novel targets for the treatment of the above mentioned diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS024067-28
Application #
8269862
Study Section
Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
Program Officer
Stewart, Randall R
Project Start
1988-08-01
Project End
2015-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
28
Fiscal Year
2012
Total Cost
$369,688
Indirect Cost
$150,938
Name
New York University
Department
Physiology
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
Cohen, Samuel M; Ma, Huan; Kuchibhotla, Kishore V et al. (2016) Excitation-Transcription Coupling in Parvalbumin-Positive Interneurons Employs a Novel CaM Kinase-Dependent Pathway Distinct from Excitatory Neurons. Neuron 90:292-307
Mullins, Caitlin; Fishell, Gord; Tsien, Richard W (2016) Unifying Views of Autism Spectrum Disorders: A Consideration of Autoregulatory Feedback Loops. Neuron 89:1131-56
Cohen, Samuel M; Li, Boxing; Tsien, Richard W et al. (2015) Evolutionary and functional perspectives on signaling from neuronal surface to nucleus. Biochem Biophys Res Commun 460:88-99
Cohen, Samuel M; Tsien, Richard W; Goff, Donald C et al. (2015) The impact of NMDA receptor hypofunction on GABAergic neurons in the pathophysiology of schizophrenia. Schizophr Res 167:98-107
Rosenberg, Evan C; Tsien, Richard W; Whalley, Benjamin J et al. (2015) Cannabinoids and Epilepsy. Neurotherapeutics 12:747-68
Ma, Huan; Li, Boxing; Tsien, Richard W (2015) Distinct roles of multiple isoforms of CaMKII in signaling to the nucleus. Biochim Biophys Acta 1853:1953-7
Ma, Huan; Groth, Rachel D; Cohen, Samuel M et al. (2014) γCaMKII shuttles Ca²⁺/CaM to the nucleus to trigger CREB phosphorylation and gene expression. Cell 159:281-94
Ma, Huan; Cohen, Samuel; Li, Boxing et al. (2013) Exploring the dominant role of Cav1 channels in signalling to the nucleus. Biosci Rep 33:97-101
Owen, Scott F; Tuncdemir, Sebnem N; Bader, Patrick L et al. (2013) Oxytocin enhances hippocampal spike transmission by modulating fast-spiking interneurons. Nature 500:458-62
Tadross, Michael R; Tsien, Richard W; Yue, David T (2013) Ca2+ channel nanodomains boost local Ca2+ amplitude. Proc Natl Acad Sci U S A 110:15794-9

Showing the most recent 10 out of 55 publications