Accessing gap-junction channel structure-function relationships through molecular modeling and simulations.

BACKGROUND: Gap junction channels (GJCs) are massive protein channels connecting the cytoplasm of adjacent cells. These channels allow intercellular transfer of molecules up to ~1 kDa, including water, ions and other metabolites. Unveiling structure-function relationships coded into the molecular architecture of these channels is necessary to gain insight on their vast biological function including electrical synapse, inflammation, development and tissular homeostasis. From early works, computational methods have been critical to analyze and interpret experimental observations. Upon the availability of crystallographic structures, molecular modeling and simulations have become a valuable tool to assess structure-function relationships in GJCs. Modeling different connexin isoforms, simulating the transport process, and exploring molecular variants, have provided new hypotheses and out-of-the-box approaches to the study of these important channels. METHODS: Here, we review foundational structural studies and recent developments on GJCs using molecular modeling and simulation techniques, highlighting the methods and the cross-talk with experimental evidence. RESULTS AND DISCUSSION: By comparing results obtained by molecular modeling and simulations techniques with structural and functional information obtained from both recent literature and structural databases, we provide a critical assesment of structure-function relationships that can be obtained from the junction between theoretical and experimental evidence.

Identification of amino acid residues lining the pore of a gap junction channel.

Gap junctions represent a ubiquitous and integral part of multicellular organisms, providing the only conduit for direct exchange of nutrients, messengers and ions between neighboring cells. However, at the molecular level we have limited knowledge of their endogenous permeants and selectivity features. By probing the accessibility of systematically substituted cysteine residues to thiol blockers (a technique called SCAM), we have identified the pore-lining residues of a gap junction channel composed of Cx32. Analysis of 45 sites in perfused Xenopus oocyte pairs defined M3 as the major pore-lining helix, with M2 (open state) or M1 (closed state) also contributing to the wider cytoplasmic opening of the channel. Additional mapping of a close association between M3 and M4 allowed the helices of the low resolution map (Unger et al., 1999. Science. 283:1176-1180) to be tentatively assigned to the connexin transmembrane domains. Contrary to previous conceptions of the gap junction channel, the residues lining the pore are largely hydrophobic. This indicates that the selective permeabilities of this unique channel class may result from novel mechanisms, including complex van der Waals interactions of permeants with the pore wall, rather than mechanisms involving fixed charges or chelation chemistry as reported for other ion channels.

Molecular and functional diversity of neural connexins in the retina.

Electrical synapses (gap junctions) in neuronal circuits have become a major focus in the study of network properties such as synchronization and oscillation (Galarreta and Hestrin, 1999; Gibson et al., 1999). Despite the recent progress made in unraveling the contribution of gap junctions to network behavior, little is known about the molecular composition of the junctional constituents. By cloning gap junction proteins [connexins (Cxs)] from zebrafish retina and through functional expression, we demonstrate that the retina possesses a high degree of connexin diversity, which may account for differential functional properties of electrical synapses. Three new Cxs, designated as zebrafish Cx27.5 (zfCx27.5), zfCx44.1, and zfCx55.5, and the carp ortholog of mammalian Cx43 were cloned. By in situ hybridization and in situ RT-PCR, we demonstrate that the four fish connexin mRNAs show differential localization in the retina. Transient functional expression in paired Xenopus oocytes and in the neuroblastoma N2A cell line indicate an extreme range of electrophysiological properties of these connexins in terms of voltage dependence and unitary conductance. For instance, the new zfCx44.1 exhibited high sensitivity to voltage-induced closure with currents decaying rapidly for transjunctional potentials >10 mV, whereas zfCx55.5 channels showed an opposite voltage dependence in response to voltage steps of either polarity. Moreover, although zfCx44.1 channels showed unitary conductance as high as any previously reported for junctional channels (nearly 300 pS), zfCx55. 5 and zfCx27.5 exhibited much lower unitary conductances (<60 pS).

Aberrant gating, but a normal expression pattern, underlies the recessive phenotype of the deafness mutant Connexin26M34T.

Mutations in the gene GJB2, encoding the gap junction protein Connexin26 (Cx26), are the most prevalent cause of inherited hearing loss, and Cx26M34T was one of the first mutations linked to deafness (Kelsell et al., 1997; Nature 387, 80-83). We report the first characterization of the gating properties of M34T, which had previously been reported to be nonfunctional. Although homotypic mutant channels did not produce detectable currents, heterotypic pairings with wtCx26 confirmed that M34T formed intercellular channels, although the gating properties were altered. Cx26M34T displayed an inverted response to transjunctional voltage (Vj), mediating currents that activate in a time- and Vj-dependent manner. These characteristics suggest that the channel population is only partially open at rest, consistent with previous reports that dye transfer in M34T-expressing cells is reduced or abolished (e.g., Thonnissen et al., Human Genet. 111, 190-197). To investigate the controversial recessive/dominant behavior of this mutant, we coexpressed M34T with wtCx26 RNA at equimolar levels, mimicking the situation in heterozygotic individuals. Under these conditions, M34T did not significantly reduce Cx26/Cx26 coupling, or alter the electrophysiological properties of the wt channels, consistent with the recessive nature of the allele. Overexpression of the mutant did have some inhibitory effects on conductance, possibly explaining some of the previous reports in exogenous expression systems and some patients. Consistent with its electrophysiological behavior, we also show that M34T localizes to cell junctions in both transfected HeLa cells and patient-derived tissue.

Site-directed mutagenesis reveals putative regions of protein interaction within the transmembrane domains of connexins.

Through cysteine-scanning mutagenesis, the authors have compared sites within the transmembrane domains of two connexins, one from the alpha-class (Cx50) and one from the beta-class (Cx32), where amino acid substitution disrupts the function of gap junction channels. In Cx32, 11 sites resulted in no channel function, or an aberrant voltage gating phenotype referred to as "reverse gating," whereas in Cx50, 7 such sites were identified. In both connexins, the sites lie along specific faces of transmembrane helices, suggesting that these may be sites of transmembrane domain interactions. In Cx32, one broad face of the M1 transmembrane domain and a narrower, polar face of M3 were identified, including one site that was shown to come into close apposition with M4 in the closed state. In Cx50, the same face of M3 was identified, but sensitive sites in M1 differed from Cx32. Many fewer sites in M1 disrupted channel function in Cx50, and those that did were on a different helical face to the sensitive sites in Cx32. A more in depth study of two sites in M1 and M2 of Cx32 showed that side-chain length or branching are important for maintenance of normal channel behavior, consistent with this being a site of transmembrane domain interaction.