The overall aim is to improve our understanding of cell coupling regulation. Cell coupling enables cells to integrate their response to stimuli by providing free exchange of ions, metabolites and messengers via discrete channels. Channel gating regulates the degree of independece of neighboring cells and can be both a safety mechanism and a factor in the pathogenesis of diseases. While an understanding of channel structure and composition has recently improved substantially and many uncoupling treatments and agents have been identified, still vague is our knowledge on channel regulation, on the meaning of channel protein phosphorylation, on gating structures and gating mechanisms. Recently, we have succeeded in reconstituting gap junction channels in liposomes and bilayers at the tip of patch clamp pipettes, providing stronger evidence for the existence of large channels at gap junctions; by studying channel gating electrophysiologically in a two-cell system and osmotically in proteoliposomes we have further supported the involvement of CaM-like proteins in channel regulation and produced evidence for the participation of the C-terminal chain in channel gating; with freeze-fracture, deep-etching and rotary shadowing E.M. we have described bridges between gap junction particles and with C.D. spectroscopy and spectrophotofluorometry we have shown conformational changes in junction proteins with uncouplers. The studies we are proposing will utilize four main approaches: intracellular recording in a two-cell system, channel reconstitution in liposomes and in bilayers on patch pipettes, immunoprecipitation and 2-D SDS-PAGE autoradiography, freeze- fracture, deep-etching and rotary shadowing. The involvement of CaM-like proteins in coupling regulation will be tested in crayfish axons intracellularly injected with anti-CaM antibodies or internally perfused with Ca-modulated proteins, and in artificial lipid systems. The uncoupling mechanism of anesthetics will be studied in crayfish axons with ion selective microelectrodes and in reconstituted channels. The size limit of channel permeants will be determined in proteoliposomes and the electrical properties of the channels will be defined in patch clamp experiments. The participation of the C-terminal chain of channel protein in gating will be tested in reconstituted systems. The extent of gap junction phosphorylation in coupled/uncoupled state will be determined in normal and RSV-transformed cells. The nature of interparticle bridges will be determined in split junctions.
| Young, K C; Peracchia, C (2004) Opposite Cx32 and Cx26 voltage-gating response to CO2 reflects opposite voltage-gating polarity. J Membr Biol 202:161-70 |
