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Structure/Function Analysis of Cx43 Gap-Junctional Channels

        Connexins are the proteins that form the gap-junctional channels that mediate cell-to-cell permeation of ions and hydrophilic molecules of molecular weight of up to 1,000 Daltons. Gap-junctional channels mediate electrical and chemical coupling between neighboring cells. Connexins are essential for embryonic development and normal function of cells and tissues, and they also participate in pathological processes, both genetic and acquired. Cx43 is a connexin isoform expressed in a variety of organs, including heart, brain, liver, kidney and uterus, as well as in capillary endothelial cells. Cx43 gap-junctional channels conduct the impulse in the cardiomyocytes. Cx43 is also essential for the normal development of the heart since Cx43 deletion in mice results in cardiac malformations and is lethal. Recent observations in mammalian cardiomyocytes, astrocytes and kidney proximal tubule cells, suggest that ATP depletion activates Cx43 hemichannels. Hemichannel activation may be involved in the cell injury and death under ischemia, in pathological conditions such as myocardial infarction, stroke and ischemic renal tubule necrosis.
        Gap-junctional channels are formed by end-to-end docking of hemichannels (also known as connexons), one from each of two adjacent cells. Hemichannels are connexin hexamers associated in a symmetric arrangement. The pore of a gap-junctional channel consists of three regions in series: the transmembrane region of one cell, the extracellular space between the two cells (gap), and the transmembrane region of the other cell. Although elucidation of the structure and properties of the entire pore length is essential in order to understand the function of gap-junctional channels and hemichannels, currently we focus on the transmembrane pore region.

Schematic connexin representation. Hemichannels (connexin hexamers or connexons) and gap-junctional channels. M1-M4 represent the four transmembrane helices. The intracellular sequences (N-terminal region, intracellular (IC) loop and the C-terminal domain) are involved in channel gating and regulation, and the two extracellular loops (EC1 and EC2) are essential for docking with connexons of the neighboring cell.

Structural model of the hemichannel pore. Schematic representation of the transmembrane α helix hemichannel model viewed from the cytoplasmic side. The helices from one monomer are labeled, and they are customarily named from A to D because they have not been assigned to a primary sequence.

        Electron cryomicroscopy and image analysis of two-dimensional crystals of a recombinant Cx43 with truncation of the C-terminal domain produced a three-dimensional density map that showed that the transmembrane pore of each hemichannel is formed by 24 closely packed α helices. Based on site-directed mutagenesis studies, the extracellular loops have been suggested to be β sheets, but the structure may be complex, perhaps a combination of β sheets with α helices that extend from the membrane-pore helices. Because of the insufficient structure resolution and the fact that the intra- and extracellular loops are not defined, the transmembrane helices have not being assigned. Therefore, the molecular bases for permeation through the gap-junctional channel and hemichannel pores cannot be inferred from the available structure. Our research aims to address this gap in knowledge using improved and novel approaches. Our main approach is the use a Cx43 expression/purification/ reconstitution system, where we calculate inter-helix distances from luminescence resonance energy transfer (LRET) data. LRET allows for accurate estimations of intra- and inter-molecular distances. We generate hemichannels of controlled composition formed by single-Cys Cx43 mutants, and use LRET in hemichannels containing a single donor and known number of acceptors at pre-determined locations (i.e., donor and acceptor bound to the same position residue in different subunits).

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