palmoplantar keratoderma [39]. No other dominant mutations are found along the TM2 helix of Cx26. It has been suggested that the highly non2conservative amino acid substitution of a Trp into the interface between the a2helical TM2 region and the rigid b2sheet forming E1 is likely to alter the secondary structure of the protein [40]. However, functional studies have recently deter- mined that the absence of gap2junctional communication is due to defects in the assembly of mutant hemichannels into full gap2junction channels, whereas the hemichannels per se retain partial channel activity when examined in the unpaired configu- ration [27]. Consistent with these findings, TM2 in our model has Arg75 pointing towards the interior of the connexin four2helix bundle, in good position to form a double salt bridge interaction with Glu41 and Glu186 (Figure S2c). Thus we conclude that neutralization of the positive charge at position 75 generates important rearrangements at the extracellular side of the TM segment, near the maximum constriction zone of the channel [10]. Proceeding along TM2 we meet Trp77. The recessive mutation Cx26W77R causes deafness [41] and prevents the formation of homotypic junctional channels in heterologous expression systems [42]. Accordingly, of the 48 cysteine substitutions made at Cx32 only W77C and two others (W133C, and T134C) resulted in nonfunc- tional channels [13]. We can rationalize these outcomes by noting that this is the only Trp near the extracellular membrane leaflet (Figure S2b) and, in our model, it lies in good position to establish specific interactions with cholesterol molecules, which may play a role in connexins trafficking or plasma membrane localization [43]. Although modification of several residues in TM2 may critically affect the permeability properties of the channel, this is not the case for Leu83 in Cx32 and the corresponding residue, Phe83, in Cx26. Indeed, Cx26F83L is a polymorphism [42]. In our model Leu83 is packed against Ser198 (TM4), which is frequently substituted among different connexin isoforms by other small residues, such as Ala, leaving enough space to accommodate the L83F mutation. Indeed, replacement of the Phe side chain in our model does not generate steric contacts at all, consistent with the lack of phenotypes for this substitution (Figure S2d). In stark contrast, the next residue in the line, Val84, is invariant in all connexins. Cx32V84I is a physicochemically moderate mutation that was found to cause CMTX disease [44] (see also Supplementary Table 2 at http://bioinfo.tau.ac.il/,sarel/GJ. html,) whereas Cx26V84L is a rare mutation associated to nonsindromic deafness [32]. It leaves the unitary ionic conduc- tance of homotypic Cx26 channels unaffected but has a deep impact on the permeability to InsP3 [33], a second messenger that plays numerous key roles in Ca2+ homeostasis. Leu is slightly bulkier than Val, and our model places Val84 facing the helix bundle in close contact with Met34 (TM1). Mutations of Met34 to smaller residues (Ala, Thr) lead to a progressive reduction of unitary conductance [13]. We have recently shown that Cx26M34T is a pathogenic mutation implicated in hereditary deafness [17]. Hence we speculate that, in a sort of domino effect, substitution of Val84 by bulkier residues, such as Leu or Ile, may distort the tertiary structure through clashes with Met34, forcing TM1 to further expose its positively charged Arg32 into the pore lumen (Figure S2e). This would modify the possible interaction of Arg32 with negatively charged InsP3 molecules traversing the pore, ensuing in a reduced unitary permeability for this second messenger. Replacing Val84 with the smaller Ala should not trigger these changes. Indeed, the related mutation Cx26V84A is classified as a polymorphism [45], consistent with this conclusion. Mutations in the next two polar residues, Ser85 and Thr86, have been reported to reduce open channel probability at junctional potential, Vj = 0 (Cx32S85C, [46,47] and Cx32T86A/ All-Atoms Connexon Model V/L/N, [23]). For Thr86, this outcome has been interpreted as due to the loss of hydrogen bonding to the backbone carbonyl of Ile82 [23]. In our model, Ser85 and Thr86 point towards the lipid phase and form H2bonds with the carbonyl oxygen of, respectively, Leu81 and Ile82 one helix turn above. Similar features were detected in the course of our MD simulation (Figure S2a). Interestingly, early compilations of the Cx26 sequence reported a Ser at codon 86 [48], see also: http://www.ncbi.nlm. nih.gov/entrez/viewer.fcgi?val = AAD21314.1), implying that T86S, which maintains intact the H-bonding capabilities, is simply a polymorphism. Next, mutation Cx32P87A is yet another cause of the CMTX disease; nonetheless it forms functional channels with properties that differ mildly from those of wild2type controls when expressed in pairs of Xenopus oocytes [23,49]. The subtle effects of P87A suggest that the phenotype is caused by impaired exchange of solutes larger than simple ions [50]. Furthermore, exchanging Pro with Val rather than Ala at codon 87 prevents expression of junctional currents in oocytes, suggesting that a progressive raise in the side chain bulkiness at position 87 may cause increasing levels of distortion in the tertiary and/or quaternary structure. However, the conformations of Val in a2helical environments are restricted to one rotamer population due to steric hindrances with the backbone of the preceding turn [51]; hence, an alternative possibility is that the conformational restriction of the Val side chain imparts local rigidity to the a2helix. Pathological effects have been reported for other residues of Cx26 along TM2, with a periodicity of nearly 324 residues (see http://ca. expasy.org/uniprot/P29033). Thus, similar to Cx26V84L [33], the deafness2linked Cx26A88S mutation [52] specifically affects the intercellular exchange of larger molecules but leaves the ionic permeability intact [53]. Two other related mutations, Cx30A88V [54] and Cx32A88V [55] are respectively associated with hydrotic ectodermal displasia, a skin disorder, and CMTX. Mutation of the next residue, Leu89, to Ile (as in Cx26L89I) results in simple polymorphisms without known deleterious effects [56], in agreement with its orientation towards the lipid phase (Figure S2f). However, Cx32L89P has been reported to be related with inherited peripheral neuropathies [31]; indeed Leu substitu- tion with Pro is more severe, having the potential to alter the helical structure of TM2. Mutations at Leu90 are associated to CMTX in Cx32 [31], and to hearing loss in Cx26 varying from moderate to profound [57,58]. In expression studies, Cx26L90P proteins were detected by immunofluorescence at contacting HeLa cell plasma mem- branes [59] but failed to induce the formation of homotypic channels [42]. The same fate occurred to Cx32L90H [50]. Cx26L90P mutant channels inserted into the plasma membrane of oocytes at a degree similar to wild type Cx26 but failed to induce any measurable macroscopic current; yet, co2expression of mutant and wild type channels, resembling the situation of heterozygous individuals, did not significantly affect the electro- physiological properties of the wild type Cx26, consistent with a recessive phenotype [60]. In summary, these results indicate that the above mutations at codon 90 do not prevent protein targeting to the plasma membrane, but severely compromise homotypic channel function. In our model Leu90 is packed within the hydrophobic environment of the four helix bundle (Figure S2g). Thus the introduction of a polar residue, as in the L90H mutation, may significantly distort the tertiary structure of individual connexins. As observed above, important changes to the secondary structure of TM2 are also expected in the L90P mutation due to the introduction of Pro that may destabilize the helical conformation near the cytoplasmic mouth of the pore. PLoS ONE | www.plosone.org 7 July 2008 | Volume 3 | Issue 7 | e2614