More than 100 hormones, peptides, and transmitters initiate change in target cells by binding to a class of guanine nucleotide binding (G) protein-linked receptors. Many of these bound receptors evoke a rise in intracellular calcium, a ubiquitous second messenger with control over cellular events ranging from contraction and enzyme modulation to nuclear transcription. A key question in pharmacology and cell biology is how the specific information, preserved in signal transduction through unique transmitter molecules, receptors, and G protein subtypes, is sustained by the calcium signal.
The aim of this grant is to use new techniques, combined with a number of traditional methods, to understand the generation, control, and information content of the calcium signal. There are four specific aims of this proposal, each related to understanding the spatial and temporal information imparted by signal transduction chains evoking calcium release. A powerful experimental model, the Xenopus toad oocyte, will be studied with a unique confocal microscope capable of manipulating and imaging subcellular domains within this single cell. The oocyte is used as the model because it is a standard expression system, has a large cell volume in which to define spatial calclium distribution, and obeys many of the same rules of signal transduction as mammalian cells. The four aims are; first, to define the distribution of receptors expressed on the cell surface in relation to underlying evoked calcium waves; second, to test the mechanisms by which various G protein subunits initiate unique patterns of calcium release; third, to understand the intracellular mechanisms of regenerative calcium release; and fourth, to correlate spatial and temporal control of calcium to the function and distribution of intracellular organelles. Calcium is the most highly regulated ion in cells; normal intracellular concentrations are a 10 million fold less inside than outside the cell. A league of enzymes and pumps regulate its distribution in space and time, and calcium in turn, regulates an array of cellular functions. The experiments proposed in this application will define the cellular control of intracellular calcium in shaping its information content and messenger function.
