Regulation of intestinal calcium transport by 1,25(OH)2D occurs at multiple steps and by multiple mechanisms. This proposal focuses on the events between entry of calcium across the brush border membrane and accumulation of calcium in vesicles forming in the terminal web which transport calcium through the cell to the basolateral membrane. Our hypothesis is that the 105-110kD calmodulin binding protein linking the plasma membrane to the actin core of the microvillus, brush border myosin 1 (myosin 1), is responsible for the transport of calcium out of the microvillus and into these vesicles. We postulate that 1,25(OH)2D regulated the function of myosin 1 by altering its binding to calmodulin (the major calcium binding protein in the microvillus) as well as its binding to the plasma membrane and the actin core. Such regulation occurs in part through the induction by 1,25(OH)2D of a tropomyosin like protein, intestinal leucine zipper protein (ZP), which governs the actin myosin 1 interaction. Furthermore, 1,25(OH)2D induced changes in the calcium concentration of the microvillus alter the mechanical and enzymatic (ATPase) properties of myosin 1, contributing to the regulated transport of the calmodulin bound calcium within the microvillus. We will test this hypothesis first by purifying and determining the function of ZP, focussing on its regulation of Actin/ myosin 1 interactions including myosin 1 motility studies. We will then evaluate the roles of calcium and calmodulin in controlling myosin 1 function including its interactions with actin and ZP. Finally, we will examine the induction of ZP by 1,25(OH)2D, and correlate the cellular location of ZP induction with the 1,25(OH)2D induced changes in calcium transport, calmodulin levels, and myosin 1 activity along the villus.
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