intro.html

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    <P>Meanwhile, during the past decade large-scale molecular
dynamic (MD) simulations have been used as a complementary
tool to observe, with atomistic detail, the structure and
dynamics of water inside these protein cavities (24–28).
Connexins (Cxs) are a family of vertebrate proteins that
constitute gap junction channels (GJCs). GJCs connect the
cytoplasm of adjacent cells by the end-to-end docking of
two connexin hemichannels producing a hydrophilic path
between cells (29,30). The intercellular transfer through
GJCs occurs by passive diffusion allowing the exchange
of water, ions (e.g., Naþ, Kþ, and Ca2þ), small molecules
such as peptides, metabolites, and signaling molecules
(e.g., cAMP and inositol 1,4,5-trisphosphate) (29,31).
Each hemichannel is formed by the oligomerization of six
Cx subunits. Each Cx exhibits a characteristic topology of
four transmembrane (TM) helices designated TM1–TM4,
two extracellular loops termed E1 and E2, and one intracellular
or cytoplasmic loop termed CL. Malfunction of GJCs
or hemichannels is associated to pathologic conditions and
genetic disorders that produce skin diseases, neurodegenerative
and developmental diseases, cataracts, and most cases
of hereditary deafness (see comprehensive reviews in previous
studies (32,33)). In particular, mutations in the gene
encoding the human Cx26 (hCx26) account for a large proportion
of genetic deafness, leading either to nonsyndromic
or syndromic disease. In syndromic deafness the hearing
loss is associated with abnormal epidermal keratinisation
(reviewed in (33)).
<br/><br/>
Despite the broad interest to further understand, at the
molecular level, functional changes produced by mutations
on Cx-based channels, there are still many unanswered
questions regarding structure-function relationships, permselectivity,
and gating mechanisms (34–37). In particular,
the ordering, structure, and dynamics of water inside Cx
GJCs and hemichannels remain largely unknown. Recently,
the x-ray crystallographic structure of the hCx26 channel
(38), solved at 3.5A˚ -resolution, has provided a structural
basis for many other Cx channels also serving as a starting
point to study structure-function relationships of these
channels (39–43). One of many approaches to do so is by
conducting systematic studies aimed to determine the presence
and role played by structural water within proteins
(8,13). However, the hCx26 crystallographic structure provided
neither evidence on the presence of water molecules
inside the main pore nor the existence of other relevant
pockets that could be hydrated. Moreover, to our knowledge,
neither theoretical nor experimental attempts to identify
the presence and role of water inside Cx structures have
been conducted so far.
<br/><br/>
To gain insights on the structure-function relationships
coded into the molecular architecture of the hCx26 hemichannel,
we conducted extensive MD simulations aimed to
characterize the structure and dynamics of water molecules
within this protein. By doing so, we have identified and characterized,
to our knowledge, a novel water pocket, termed the
IC pocket, which lays at the intracellular side of all hCx26
located in-between the four transmembrane helices of each
monomer. Relying on these data and complemented with
multiple sequence alignments, we elaborated on the functional
role of the IC pocket: the position of Arg143 may regulate
the IC pocket volume, modulating the dynamics of the
N-terminal helix (NTH). To evaluate our functional hypothesis,
a hemichannel permeability assessment was conducted
through time-lapse imaging of ethidium (Eth) uptake,
comparing hCx26Wt and mutants R143A, R143E, R143Q,
and R143K. As a whole, our data suggest that the IC pocket
and, furthermore, Arg-143 plays an essential role on the
modulation of the hCx26 hemichannel permeability.
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