Feliksas Bukauskas

Gap junction channel de novo formation and gating

Our major goal is to study de novo formation and gating of gap junction (GJ) channels by combining electrophysiological and imaging methods in living cells. GJ channels are assembled from connexin (Cx) protein and mediate direct cell-cell diffusion of cytosolic ions and molecules. We use cells expressing wild type Cxs and Cxs fused with color variants of green fluorescent protein (Cx-GFP) or Cxs containing the tetracysteine motif (Cx-TC). We propose that clustering of GJ channels into junctional plaques (JPs) is central to their ability to function and we are testing the hypothesis that JP formation starts with aggregation of hemichannels into hemichannel plaques (HPs), followed by superposition of HPs from apposing cells, hemichannel docking and channel pore opening. We propose that JPs grow by laterally attracting hemichannels dispersed in the plasma membrane and examine whether calmodulin and other cytosolic proteins interact and co-localize with GJ channels to regulate JP formation and coupling. We focus on gating and permeability properties of GJ channels. We propose that there are two distinct gating mechanisms in GJs, fast and slow. Closure of the fast gate leaves a residual conductance and we examine conductance and selectivity of open and residual states to assess the electrical barrier/s and channel pore size of open and gated channel. We suggest that the fast gate can serve as a selectivity filter, which partially reduces electrical cell-cell signaling but restricts metabolic communication and chemical signaling. We examine conditions under which some heterotypic junctions exhibit nearly unidirectional electrical signaling and may function as rectifying electrical synapses. We study Cxs involvement in apoptosis development and correlation between connexin expression and cell-to-cell signaling involved in apoptosis and necrotic spread.

 

Selected Publications