INTRACELLULAR MESSENGER FUNCTIONS OF INOSITOL PHOSPHATES

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Inositol phosphates are used as intracellular messengers within a signalling pathway which serves to control many diverse cellular functions, such as neurotransmitter and hormone responses, secretion, muscle contraction and phototransduction. Disorders of this signalling pathway have been implicated in diseases including tumorigenesis and manic depressive illness, and the relevance of this system to clinical studies will almost certainly grow as we come to understand it more. Great progress has recently been made in the biochemical elucidation of the pathways leading to the formation of several different inositol phosphate compounds. However, the physiological study of the actions of these compounds is less well advanced, and although it is thought that they act primarily by raising intracellular free calcium levels, little is known about the specific actions of many of them, or about the mechanisms by which they regulate calcium fluxes across intracellular and cell surface membranes. The main object of this proposal is to study the messenger functions of inositol phosphates, by using electrophysiological and optical techniques to measure membrane currents and intracellular calcium. Xenopus oocytes will be used as a convenient model cell system, as these cells have a well characterized phosphoinositide signalling system, and their large size facilitates many procedures including voltage-clamp recording and micro-injection. The initial aim is to characterize the abilities of different inositol phosphates to liberate intracellular calcium, to activate influx of calcium across the plasma membrane, and to activate membrane conductances independent of calcium. Subsequently, the properties of inositol phosphateactivated calcium channels will be examined in detail by voltage- and patch-clamp recording of currents across the plasma membrane, and by reconstituting channels from internal membranes into lipid bilayers. This will give information about the kinetics and conductances of single channels, their ionic specificity, modulation by inositol phosphates and other second messengers, and blocking by calcium antagonists and other pharmacological agents. Similar studies will also be made of the calcium-activated chloride channels which mediate the final electrical response to phosphoinositide activation in the oocyte, and of any calcium-independent currents which are found to be modulated by inositol phosphates. Recordings of intracellular calcium will be made to determine whether the oscillatory membrane current response to inositol phosphates arises from an oscillatory liberation of calcium, and the feedback mechanism responsible for this process will be studied. The oocyte will also be used as a translation system for exogenous mRNA, to see whether calcium channels, or other components of the phosphoinositide signalling system, can be functionally expressed by mRNA from brain or salivary gland.