) Intracellular calcium (Ca) is a key cellular messenger that triggers a variety of cellular processes. It can enter the cytosol from the extracellular medium or it can be released from intracellular stores via at least two types of channel: the inositol 1,4,5-trisphosphate receptor (InsP3R) and the ryanodine receptor (RyR). The hypothesis of this proposal is that control of the lnsP3R by Ca and lnsP3 allows Ca release from the receptor to be regulated at the subcellular level. This hypothesis will be addressed using biophysical, biochemical and molecular biological techniques. Specific long-term goals of this project are to understand how the lnsP3-gated channel functions, how the cell regulates the channel to optimize cellular responses, and how regulation is altered in pathophysiological Situations. In the proposed experiments, we will investigate the interaction Of Ca and lnsP3 in the regulation of the type I lnsP3R. The Ca dependence of channel activity is a function of the lnsP3 concentration. At low concentrations of lnsP3 Ca can both activate and inhibit channel activity whereas at high concentrations of lnsP3 the inhibition by Ca is decreased. In this proposal the two models that have been put forward to explain this complex interaction between Ca and lnsP3 will be examined. The first model suggests that channel activity depends upon the kinetics and order of binding of lnsP3and Ca. The second model suggests that lnsP3.binding to a recently identified low affinity site of the type I lnsP3R is necessary for sustained channel activity. First, the models will be tested by characterizing the single channel properties of normal and mutant forms of the lnsP3R. Then, the functional consequences of the single channel properties will be investigated in intact cells. Our working hypothesis suggests that components of both models will be important to explain channel function. Identification of the molecular mechanisms controlling these channels will assist in the understanding of processes as diverse as secretion, smooth muscle contraction, and motor learning and may provide insights into conditions such as hypertension and ataxia, processes that involve abnormalities in the activity of this channel.
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