The overall aim is to learn more about the mechanism(s) which regulate cell communication via low resistance junctions by focusing on uncoupling. Uncoupling is mainly a safety device which enables cells to survive the death of their neighbors by the occlusion of intercellular channels, but is also believed to be involved in the pathogenesis of various pathological processes. Although a good deal is known about uncoupling agents and treatments which cause uncoupling, fundamental questions on uncoupling mechanism and channel structure are still unanswered. We do not know whether uncoupling agents such as Ca++ and H+ act directly on channel proteins or on associated molecules; although channel occlusion is believed to result from conformational changes in channel protein, there is no experimental evidence to support it; while clever approaches have been developed in intact cells, the independent role of Ca++ and H+ in uncoupling is still questioned; finally the very nature of gap junction architecture is still unclear. We are proposing several new approaches to these problems. The possible involvement of calmodulin in uncoupling, recently supported by preliminary data (29, 30, 31), will be tested by attempting to localize calmodulin (immunocytochemically) in isolated junctions and intact cells and by studying the effects of calmodulin inhibitors and specific anti-calmodulin antibodies on junction structure and channel permeability regulation. In view of the vast present knowledge on calmodulin structure and function, evidence for calmodulin participation in uncoupling would lead to a full understanding of cell coupling regulation. Various ions and protein reagents will be tested in their capacity to induce or inhibit changes in junction particle array previously shown to parallel uncoupling. The effects of uncoupling agents on the junction protein conformation and the interaction between this protein and calmodulin will be determined by spectrofluorometry and CD measurements. Attempts to study channel conductance and regulation in isolated junctions will be made by incorporating junctional vesicles into bilayers and by patch-clamping isolated junctions. The effect of hypertonic solutions on junction particle density will be studied as the first step in testing an hypothesis on the mechanism for particle aggregation in gap 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 |
