Cytoplasmic Ca2+ transients trigger key cellular events including excitability, cell growth and transmitter release. The resting free Ca2+ concentration (Ca2+)1 is maintained by several buffer mechanisms that include Ca2+ binding proteins sequestration of Ca2+ into rapidly- and slowly-exchanging membrane-bound storage organelles (Ca2+ stores), membrane channels and Ca2+ pumps. The rapidly-exchanging Ca2+ stores of the chicken cerebellum Purkinje cell have been tentatively identified as a subset of smooth-surfaced endoplasmic reticulum and equipped with several protein constituents such as Ca2+-pump, Ca2+ release channels (sensitive either to inositol 1,4,5-trisphosphate or Ca2+, caffeine and ryanodine) and calsequestrin and intraluminal Ca2+ binding protein. The long-term objective of the proposed research is to define the functional role of intracellular rapidly-exchanging Ca2+ stores in chicken cerebellar Purkinje cells. To this end, we propose a multifaceted approach that shall accomplish three major goals. 1) The biochemical purification and characterization of Ca2+ stores will involve measurements of Ca2+ fluxes, and enzymatic and immunological binding assays. 2) The functional characterization of the Ca2+ release channels associated with the purified organelles will involve measurement of Ca2+ fluxes by isotope and spectrophotometric methods and single channel analysis. Moreover, the structure-function relationship of these Ca release channels will be studied by means of anti-peptide antibodies for specific domains of the channel molecule. 3) The definition of the role that plasma membrane receptors activated by excitatory amino acids and associated with either ion channels or second messenger systems play in redistribution of Ca2+ from intracellular Ca2+ stores. Excitatory amino acids will be applied in a concentration clamp system on enzymatically isolated Purkinje cells maintained under a voltage clamp to determine their capacity to increase (Ca2+)1 by allowing permeation through a ligand-gated channel or by activation of a second messenger system that releases Ca2+ from one of the rapidly exchanging intracellular Ca2+ stores. The results from this research will provide new and extensive information on many of the biochemical and biophysical properties of rapidly exchanging Ca2+ stores. The results of this investigation will have health-related implications since neurons rely on redistribution of Ca2+ from intracellular stores for several key functions.
