Gap junction (GJ) channels mediate direct cell-cell diffusion of cytosolic ions and molecules. Each channel is la multimer of connexin (Cx) proteins that forms an intercellular pore by docking two hemichannels. Our goal is to elucidate the mechanisms underlying GJ channel formation and gating that will aid in understanding the role of GJs in normal and disease states. We will examine 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).
Specific Aim 1 focuses on formation and function of junctional plaques (JPs). We propose that clustering of GJ channels into JP is central to their ability to function. We will test 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 will determine: 1) the correlation between the size of a JP and functional coupling, 2) the minimal (critical) size of a JP that is required for GJ channels to function, and 3) the spatial distribution of functional GJ channels within a JP by examining fluorescence induced by interaction of Cx-TC or Cx-GFP with fluorescent dyes moving through GJ channels. We will test the hypothesis that JPs grow by laterally attracting (trapping) hemichannels dispersed in the plasma membrane and will examine whether calmodulin (CAM) interacts and co-localizes with GJ channels to regulate JP formation and coupling, directly or through [Ca2+]I and CaM-kinase.
Specific Aim 2 focuses on gating and permeability properties of GJ channels. We propose that there are two distinct gating mechanisms in GJs, fast and slow, and we will examine their biophysical properties in Cxs expressed in CNS. Closure of the fast gate leaves a residual conductance and we will examine conductance and selectivity of open and residual states to assess the electrical barrier/s and channel pore size when the fast gate is closed. We will examine whether the fast Vi gate can serve as a selectivity filter, which partially reduces electrical cell-cell signaling but restricts metabolic communication and chemical signaling.
In Specific Aim 3, we will examine conditions under which some heterotypic junctions exhibit nearly unidirectional electrical signaling and may function as rectifying electrical synapses. We will test the hypothesis that electrical signaling asymmetry and metabolic communication can be effectively modulated through a relatively small change in the difference between pre- and postsynaptic resting potentials. Genetic disorders that have been linked to Cx dysfunction include X-linked Charcot-Marie-Tooth disease, sensorineural deafness, congenital cataractogenesis, etc. Abnormalities in GJ channels play a key role in generating cardiac arrhythmias, uterine malfunction, epileptic seizures and malignant cell growth.
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