Theoretical and experimental investigation of gap junctional conductance and permeability

Abstract

Gap junctions are intercellular pores providing the primary means for direct exchange between cells, of ions and metabolites up to molecular weights of about 1000. They are essential for normal physiological function and development. The pivotal cellular role of gap junctions has fueled concentrated investigation of their transport properties. Transport processes through gap junctions fall into two categories, conduction and permeation . This thesis addresses both these modes of transport. In the first part, we have developed a hindered convection-diffusion (microscopic conductance) model of ion transport through gap junctions. This model addresses important phenomena of differential geometrical exclusion of ions in the pore cross section, not fully considered to date. It is used to explore the influence of charged pore walls and varying geometries on the transport characteristics of these channels (Chapters 2 and 3 ). The formulation of the model and the method of solution of the governing transport equations are detailed in Chapter 2. Chapter 3 describes the application of this model to predict the conductance and selectivity characteristics of gap junctions. The predicted conductance of a neutral channel using a geometry based on a recently published structure (Fleishman et al, 2004) comes out to be higher than the experimentally observed conductance value, which is also seen using the classical theoretical formalism (Goldman-Hodgkin-Katz equation). This apparent discrepancy between the predicted and the experimentally observed conductance is explored in this thesis. Gap junctions show marked permselectivity towards fluorescent dyes and natural metabolites, which is not only dependent on the size and charge, but also on the structure of permeant molecules. To assess the influence of dye structure on permeation through Cx32 and Cx43 homotypic channels, we studied the permeability of these channels, expressed in paired Xenopus (frog) oocytes, with two structurally different fluorescent dyes (Alexa488 and Lucifer Yellow) as probes (Chapter 4 ). Following characterization of cytoplasmic transport limitations, our dye transfer experiments reveal that the single-channel permeability of Lucifer Yellow is 5-6 times lower than Alexa488 for both channels. This suggests that the structural differences between the dyes significantly affect unitary channel permeability. Further, a macroscopic model was developed to calculate the junctional and non-junctional permeabilities in the process of dye transfer through gap junctions and dye leak through hemichannels (the precursors to gap junctions) in paired oocytes and tested against the previously acquired data from K. Spaeth in the Nicholson Laboratory (Chapter 5).