Three-dimensional structure of a human connexin26 gap junction channel reveals a plug in the vestibule

Abstract

Connexin molecules form intercellular membrane channels facilitating electronic coupling and the passage of small molecules between adjoining cells. Connexin26 (Cx26) is the second smallest member of the gap junction protein family, and mutations in Cx26 cause certain hereditary human diseases such as skin disorders and hearing loss. Here, we report the electron crystallographic structure of a human Cx26 mutant (M34A). Although crystallization trials used hemichannel preparations, the density map revealed that two hemichannels redocked at their extracellular surfaces into full intercellular channels. These orthorhombic crystals contained two sets of symmetry-related intercellular channels within three lipid bilayers. The 3D map shows a prominent density in the pore of each hemichannel. This density contacts the innermost helices of the surrounding connexin subunits at the bottom of the vestibule. The density map suggests that physical blocking may play an important role that underlies gap junction channel regulation. Our structure allows us to suggest that the two docked hemichannels can be independent and may regulate their activity autonomously with a plug in the vestibule.connexin channels ͉ electron crystallography ͉ intercellular communication ͉ membrane protein structure ͉ two-dimensional crystals

Site-Directed Mutagenesis Reveals Putative Regions of Protein Interaction within the Transmembrane Domains of Connexins

“…Helices are arranged as proposed earlier by Skerrett et al (2002), including the close apposition documented for E146 and C201 in the closed state. This arrangement of helices is in general agreement with the recently published crystal structure of Cx26 (Oshima et al 2007). Protein function was unaffected by tryptophan insertion at positions highlighted in green, suggesting that these residues face a lipid or aqueous environment.…”

“…The relative location of mutationsensitive sites within helices is shown in Figure 3, providing a set of data that can be used in future models of gap junction structure and function. The model describes the arrangement of helices around the gap junction pore, consistent with data described above and also with pore-lining studies of Cx32 (Skerrett et al 2002) and the crystal structure of Cx26 (Oshima et al 2007). By using helix wheel representations, the tilts of the helices with respect to the membrane identified in the Unger (1999) and Oshima (2007) models are not reflected, which can affect the alignment of residues.…”

“…The model describes the arrangement of helices around the gap junction pore, consistent with data described above and also with pore-lining studies of Cx32 (Skerrett et al 2002) and the crystal structure of Cx26 (Oshima et al 2007). By using helix wheel representations, the tilts of the helices with respect to the membrane identified in the Unger (1999) and Oshima (2007) models are not reflected, which can affect the alignment of residues. Nonetheless, this representation serves to demonstrate that the mutation sensitive sites can be clustered at regions of helix-helix contact, predominantly within a subunit, but also between subunits.…”

“…These two models agree on the assignment of M3 (the main pore-lining helix) and M4, but reverse the assignments of M1 and M2. The recently published crystal structure of Cx26 provides support for the assignments proposed in Skerrett et al (2002), based on the location of an electron dense cytoplasmic structure that is likely to be the cytoplasmic loop connecting M2 and M3 (Oshima et al 2007).…”

Functional consequences of novel connexin 26 mutations associated with hereditary hearing loss

“…13 -15 The threedimensional structure of M34A, a human Cx26 mutation, reveals a prominent electron density in the pore of each hemichannel, suggesting that physical blocking may be important in gap junction channel regulation. 16 Although W24X is known to be a common Cx26 mutation in India, 17,18 the full spectrum of mutations of this gene occurring in India is not known. We present here the results of a study of Cx26 in 530 individuals exhibiting non-syndromic, sensorineural hearing loss, in which we identified four novel Cx26 mutations and 14 mutations that have been described earlier.…”