Oscillations in intracellular Ca+ concentration are observed in many, if not all eukaryotic cells. They have been suggested to govern a variety of fundamental biological processes, including secretion, proliferation and development. Oscillations are typically triggered by an extracellular stimulus that provokes Ca+ release from intracellular stores. Cytoplasmic Ca+ concentrations rise as a result. Store depletion is then signaled to a Ca+-specific conductance in the membrane, eliciting a Ca+ flux that replenishes the stores in preparation for subsequent waves of release. Despite the prevalence and importance of Ca+ oscillations, there remain many unanswered questions regarding their origins and function, including the nature of the store depletion signal and means by which they are modulated. This proposal investigates pathways responsible for Ca+ oscillations, using Paramecium as a model system. This unicell uses Ca+ oscillations to effect behavioral responses to extracellular GTP. Paramecium is particularly suited to these studies because evidence suggests that the Ca+-specific membrane conductance that responds to store depletion has gained a voltage sensitivity that makes it particularly easy to elicit and study under voltage clamp. Further, mutants that are unable to respond to GTP are available for use in a genetic dissection of the oscillations and their regulatory pathways. The proposal has three specific aims. First, voltage-clamp techniques are used to investigate two currents that are observed upon GTP application to determine their properties and dependence on the conductance that is believed to be responsible for replenishing the Ca+ stores.
The second aim i nvestigates the pathways that terminate the oscillations during persistent GTP exposure. Evidence suggests that this involves adenylate cyclase and protein kinase A. The target of this pathway is believed to be the store-replenishment current, inactivating it through a shift in voltage sensitivity.
The final aim i dentifies new mutants that are defective in sensing and desensitization to GTP. These mutants can help dissect the physiology of the oscillations and their regulatory pathways and ultimately can provide the means to identify the genes involved in this Ca+ signaling phenomenon.
| Politi, Huguette C; Preston, Robin R (2003) Is it time to rethink the role of Mg2+ in membrane excitability? Neuroreport 14:659-68 |
| Haynes, W John; Kung, Ching; Saimi, Yoshiro et al. (2002) An exchanger-like protein underlies the large Mg2+ current in Paramecium. Proc Natl Acad Sci U S A 99:15717-22 |
