Inositol 1,4,5-trisphophate (IP3)induced intracellular Ca2+ release is an excitable process which mediates the effects of activated hormone and neurotransmitter pathways. Ca2+ -induced Ca2+ release (CICR) from the IP3-bound IP3 receptor (IP3R) is the fundamental property which generates propagating Ca2+ waves, when coupled to Ca2+ diffusion. These waves annihilate upon collision primarily due to Ca2+ inhibition of Ca2+ release at high Ca2+ concentrations. This proposal focuses on the molecular control of Ca2+ signaling by mechanisms which bind, sequester, and/or pump intracellular Ca2+, namely, Ca2+ binding proteins (CaBPs), Ca2+ ATPases and mitochondria. Overexpression of sacro-endoplasmic reticulum Ca2+ ATPases in Xenopus oocytes increases the frequency and amplitude of IP3-induced Ca2+ waves without affecting wave velocity. It is hypothesized that Ca2+ ATPases affect wave activity by rapidly removing cytosolic Ca2+ and/or increasing Ca2+ store refilling. Injection of oxidizable substrates into oocytes energizes mitochondria, increasing Ca2+ wave amplitude, velocity and interwave period. These effects are blocked by respiratory chain inhibitors and by ruthenium red at the Ca2+ uniporter, indicating that mitochondria modulate Ca2+ signalling by sequestering cystolic Ca2+. Thus, both Ca2+ ATPases and mitochondria play important roles in controlling Ca2+ signalling dynamics.
The Specific Aims are 1) To determine how cytosolic Ca2+ binding proteins, calbindin D28k and parvalbumin, affect Ca2+ wave activity and how these systems act in concert with energized mitochondria to control Ca2+ signals. 2) To study whether low and high affinity Ca2+ buffers alter the progression from Ca2+ sparks/puffs to pulsatile Ca2+ foci, and from foci to Ca2+ waves. 3) To investigate the mechanisms by which mitochondrial Ca2+ cycling (uptake and efflux) affect Ca2+ wave activity. Confocal Ca2+ imaging, electrophysiology, recombinant DNA and pharmacological assays will be used to achieve these goals. The significance of this proposal is threefold: First, intracellular Ca2+ signalling controls fundamental cellular processes ranging from growth, differentiation and secretion to the modulation of gene expression. Second CaBPs and mitochondria appear to exert neuroprotective effects in excitotoxicity and apoptosis. And finally, functional defects in either CaBPs or mitochondria can lead to pathogenic disorders such as neurodegenerative diseases (Alzheimer s; Parkinson's and Huntington's), epilepsy and seizures.
