The long term goal of the proposed studies remains the definition of water transport mechanisms across biological membranes at the physiological, biochemical and molecular levels. In this revised proposal, efforts will be focused on the identification and structure-function analysis of biologically important water transporting proteins.
Specific Aim 1 : To identify and characterize novel mammalian water transporters. Based on the tissue distribution of water channels identified thus far, additional physiologically important water channels must exist. A PcR/homology cloning strategy has already yielded 3 new full-length and 3 partial-length cDNAs. We propose: a. to obtain full length cDNAs corresponding to the putative new water channels, b. characterize protein function by expression in Xenopus oocytes, and c. determine tissue distribution by Northern blot, in situ hybridization and immunostaining.
Specific Aim 2 : To quantify the biophysical transporting properties of water channels. The key functional parameters that characterize water channel proteins will be measured, including: single channel water permeability, solute selectivity, osmotic-to-diffusional water permeability ratio, and Arrhenius activation energy. In addition, putative inhibitory and activating compounds will be tested. Experiments will be carried out on cloned proteins CHIP28, WCH-CD, MIWC, GLIP, and the new water channels identified in aim 1. Novel aspects of this aim include measurement of osmotic and diffusional water permeability by new fluorescence methods, and liposome reconstitution of proteins purified from a baculovirus/Sf9 expression system.
Specific Aim 3 : To study selected features of water channel structure. Initial experiments indicated that CHIP28 is assembled in membranes as tetramers in which individual monomers function independently and contain multiple membrane-spanning helical domains. There are three subaims: a. To determine whether WCH-CD and MIWC form oligomers in membranes by freeze fracture electron microscopy and electron crystallography. b. To determine the transmembrane topology of MIWC by translation of selected mutated, chimeric and flagged cDNAs. We will also test the provocative hypothesis that functional maturation of CHIP28 occurs between the endoplasmic reticulum and plasma membrane and is associated with a change in topology. c. Fluorescence energy transfer and time-resolved fluorescence will be applied to measure selected distances between labeled residues in CHIP28 and to test whether HgCl2 inhibition influences protein segmental dynamics.
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