The osteoclast is in large part responsible for the orderly metabolism and remodeling of the bony skeleton which is required for structural integrity and maintains greater than 99% of the bodies stores of calcium. My laboratory has proposed a mechanism for the vectorial transport of protons into the bone resorption compartment which defines specific macromolecular components as targets for regulation of bone resorption.. Of these ruffled membrane chloride channel which we have purified is a limiting component in the transport of protons for bone resorption. We are proposing to study both the hormonal and genetic regulation of the chloride channel activity in the differentiation of the osteoclast and in the mature bone resorbing cell. Specifically we propose to: 1. Characterize the 62 kDa ruffled membrane chloride channel using biochemical and electrophysiological techniques to: a. obtain amino acid sequence of peptides from the purified channel protein, b. establish method for the immunolocalization and purification of the chloride channel, c. correlate hormonal regulation and in situ phosphorylation with chloride channel activity. 2. Isolate cDNA and genomic clones encoding the 62 kD ruffled membrane chloride channel and use these sequences to: a. characterize expression at RNA and protein levels in cultured osteoclast precursors and in response to hormones known to effect osteoclast function and differentiation, b. identify relevant points of regulation of chloride channel expression, c. Map controlling regions necessary for induction of expression during differentiation and in response to hormonal stimulation, and d. use antisense oligonucleotides derived from the chloride channel gene to selectively suppress expression of the chloride channel in developing osteoclasts in culture and determine the effect of such suppression on bone resorption.
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