The goal of the proposed research is to understand the signal transduction pathway mediated by receptor-triggered calcium spiking. Calcium spikes or oscillations are repetitive transient increases in cytosolic calcium concentration that are induced by constant extracellular stimuli such as hormones or neurotransmitters. The intensity of the stimulus often determines the spike frequency. We will study how calcium spikes are generated and whether the spike frequency can control the activity of calcium-dependent enzymes. Calcium/calmodulin dependent protein kinase II (CaM kinase) is chosen as a model enzyme because of its ubiquitous distribution and its involvement in important cell functions such as the regulation of ion-channels, carbohydrate metabolism, cytoskeletal organization and neurotransmitter release.
The specific aims of the proposed research are based on our preliminary findings that different mechanisms generate localized and long-ranged calcium spikes and that the activation of CaM kinase by calcium spikes is associated with a calmodulin trapping process. Calmodulin trapping is mediated by autophosphorylation of CaM kinase and leads to a 1000-fold increase in calmodulin binding affinity of CaM kinase. The following two hypothesis will be tested: (i) Localized subcellular calcium spikes are generated by calcium-gated opening of calcium channels and long-ranged spikes are mediated by IP3 diffusion and by coupling of IP3 and calcium concentration. This hypothesis is tested by measuring calcium and IP3 diffusion in PC12 cells using confocal calcium imaging. The gating of IP3- receptor and ryanodine receptor calcium channels by calcium will be investigated by 45Ca flux studies. (ii) The activation of CaM kinase by calcium spikes is enhanced by calmodulin trapping, a process that may be involved in the decoding of the frequency of calcium spiking. This hypothesis is tested by investigating the activation of recombinant and rat brain CaM kinase by fluorescence anisotropy measurements of calmodulin trapping, by photobleaching measurements of calmodulin diffusion and by peptide mapping of autophosphorylated CaM kinase. The calcium and calmodulin dependence of these processes are of importance for the understanding of CaM kinase activation by calcium spikes. The proposed study of calcium-mediated signal transduction may contribute to the understanding of a number of disorders such as hypertension which can be treated with, calcium channel blockers or manic depressive illness which responds to lithium, an inhibitor of the inositolphosphate metabolism.
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