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