The long range objective of this work is to use theoretical methods to relate the structure to the function of ion channel proteins. In particular, this proposal outlines some problems in neurophysiology. Electrostatic model calculations and molecular dynamics simulations descriptive of the process of ion permeation through transmembrane channel proteins are discussed. One set of electrostatic model calculations, based upon strictly continuum models, provides ways of determining how the shape of the channel protein, the location and density of charged groups, the composition of the electrolyte and the composition of the lipid affect the electrostatic energy profile governing ion access to and translocation through a channel and the electric field generated by transmembrane potentials. Another approach, by incorporating a simplified molecular treatment of the ion(s), water molecules and protein charges that surround and form the aqueous pore, is designed to rigorously account for molecular aspects of pore electrostatics. While continuing to treat the lipid and bulk water as continua it does not assume that pore water is continuum dielectric; instead, it derives the dielectric properties of pore water and provides an unambiguous way to establish the physical basis for ion channels' low electrostatic energy barriers. The molecular dynamics calculations, using a gramicidinlike channel as a model system, focus on some questions central to channel selectivity: the role of vestibule and binding site structure; the process of sequential resolvation during ion translocation; features promoting multiple occupancy. A proposed calculation modeling proton transport in a gramicidinlike channel may provide insights into the mechanism of proton translocation in protein systems generally. GRANt=R01AR28420 The work proposed in this grant application will extend our knowledge regarding the regulatory aspects of the bone forming cell, the osteoblast. The main thrust of this work is the description of a new role for type 5 tartrate resistant acid phosphatase (TRAP). This enzyme is present in osteoclasts and at sites of resorption and it is our hypothesis that the molecule has growth factor-like effects on bone cells. In preliminary findings we have shown that a molecule which is homologous to osteoclastic TRAP can stimulate isolated progenitor osteoblasts to acquire a more differentiated phenotype. The significance of such an effect is that TRAP, due to its location on the resorbing surface, can act as a site-specific molecule to direct the formation of bone to resorption lacunae. Thus, along with other soluble regulatory factors released during osteoclastic activity, TRAP may participate in local bone remodeling. In the first part if this project we intend 1) to characterize the effects of TRAP on bone cells and other cells, 2) to investigate whether known growth factors interact with it, and 3) to determine if it affects the production of TGF beta and bFGF. The second part of the project involves isolating and identifying the TRAP receptor and using the receptor to further characterize regulatory molecules in bone. (We currently believe that the receptor through which TRAP mediates its effects is the IGF- II/mannose-6- phosphate receptor, but this needs to be proven.) The last part of the project explores second messenger signalling mechanisms for TRAP and the possible role of intracellular calcium as a transducing agent. The importance of this work is that it may, for the first time, shed light on the localized nature of the bone remodelling process. Our central hypothesis is that the large amount of osteoclastic TRAP which remains on the surface of resorption lacunae can act as a site-directing regulatory molecule for the formation of new bone. These findings may also explain the high rates of osteoblastic activity in pathological states with high acid phosphates content, such as prostate metastases to the skeleton.
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