Destabilization of Ca2+ homeostasis plays a critical role in the ischemic neuronal death that takes place during a stroke. To identify pharmacological targets to improve the outcome of stroke, one needs to measure the intracellular Ca2+ concentration ([Ca2+]i). The most common currently used method to measure [Ca2+]i is to monitor the signal of small molecule Ca2+ indicators such as fura-2 or fura-2FF. Unfortunately, this method suffers from a lack of selectivity. Namely, these indicators bind not only Ca2+ but also Zn2+ and both [Ca2+]i and [Zn2+]i increase in ischemic neurons. Consequently, this method yields ambivalent results. A more selective method to monitor [Ca2+]i is needed. Genetically engineered Ca2+ indicators, cameleons, constitute a novel promising research tool to monitor [Ca2+]i. However, it is not yet known how specific for [Ca2+]i versus [Zn2+]i this tool is. The goal of the proposed research is to clarify this issue.
We aim to characterize the sensitivity of the D3cpv cameleon to zinc and determine whether this tool can be used to monitor the [Ca2+]i when [Zn2+]i elevations take place concurrently. To this end, the D3cpv fluorescent signal will be monitored in primary mouse hippocampal neurons transfected with D3cpv while the neurons will be exposed to various Zn2+ concentrations in the presence of an agent that transports Zn2+ across the plasma membrane. The reliability of D3cpv as a tool to monitor [Ca2+]i will be tested in the experiments in which D3cpv and fura-2FF signals will be monitored simultaneously in glutamate-treated neurons. These experiments will determine whether using D3cpv, one can resolve fura-2FF signal ambiguities.

Public Health Relevance

The proposed research will examine whether a novel genetically engineered Ca2+ indicator, the D3cpv cameleon, reliably reports intracellular Ca2+ concentrations when intracellular Zn2+ elevations take place concurrently. The research is relevant to public health because intracellular Ca2+ and Zn2+ increases play a role in the mechanisms of neuronal death during stroke.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Small Research Grants (R03)
Project #
1R03NS077095-01
Application #
8226533
Study Section
Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
Program Officer
Stewart, Randall R
Project Start
2011-08-15
Project End
2013-07-31
Budget Start
2011-08-15
Budget End
2012-07-31
Support Year
1
Fiscal Year
2011
Total Cost
$79,521
Indirect Cost
Name
University of Illinois at Chicago
Department
Psychiatry
Type
Schools of Medicine
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Kiedrowski, Lech (2014) Proton-dependent zinc release from intracellular ligands. J Neurochem 130:87-96
Kiedrowski, Lech (2012) Cytosolic acidification and intracellular zinc release in hippocampal neurons. J Neurochem 121:438-50