pH Regulation of Connexin 43: Intermediary Steps

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DESCRIPTION (provided by applicant): Connexins are integral membrane proteins that oligomerize to form intercellular channels called gap junctions. Recently, we have shown direct, pH-dependent intramolecular association between the carboxyl terminal domain and the second half of the cytoplasmic loop of Cx43. The structural bases, kinetics and functional consequences of this association are the central topics of this grant. Specific Aim 1. To generate a structural model of the interaction between the carboxyl terminal and the cytoplasmic loop domains of Cx43. Our experiments show identifiable regions of organized secondary structure in the cytoplasmic loop and in the CT domains of Cx43. The structures are likely to change once the domains are bound. We will correlate structural predictions with site-directed mutagenesis and kinetic binding assays using surface plasmon resonance (SPR) to identify the crucial binding sites. Specific Aim 2. To characterize the role of the CT-CL interaction in the formation of functional gap junction plaques. Selected mutations in the CL region cause the loss of identifiable gap junction plaques in transfected cells. However, the same mutant does form gap junction plaques if the CT domain is also deleted from the sequence. We hypothesize that a specific sequence in Cx43CT prevents gap junction formation unless masked by a corresponding sequence in the CL domain. Specific Aim 3. To establish whether the CT-CL interaction is involved in determining the single channel properties and pH gating behavior of Cx43. We propose that binding of the cytoplasmic loop to the CT domain mediates the single channel and "pH gating" behavior of Connexin 43. Results will be correlated with structural predictions and binding profiles (Aim 1). These data will yield a global picture of the functional consequences resulting from the interaction of the CT with the cytoplasmic loop.