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Asymmetric configurations and N-terminal rearrangements in connexin26 gap junction channels.

ABSTRACT: Gap junction channels are unique in that they possess multiple mechanisms for channel closure, several of which involve the N terminus as a key component in gating, and possibly assembly. Here, we present electron crystallographic structures of a mutant human connexin26 (Cx26M34A) and an N-terminal deletion of this mutant (Cx26M34Adel2-7) at 6-Å and 10-Å resolutions, respectively. The three-dimensional map of Cx26M34A was improved by data from 60° tilt images and revealed a breakdown of the hexagonal symmetry in a connexin hemichannel, particularly in the cytoplasmic domain regions at the ends of the transmembrane helices. The Cx26M34A structure contained an asymmetric density in the channel vestibule ("plug") that was decreased in the Cx26M34Adel2-7 structure, indicating that the N terminus significantly contributes to form this plug feature. Functional analysis of the Cx26M34A channels revealed that these channels are predominantly closed, with the residual electrical conductance showing normal voltage gating. N-terminal deletion mutants with and without the M34A mutation showed no electrical activity in paired Xenopus oocytes and significantly decreased dye permeability in HeLa cells. Comparing this closed structure with the recently published X-ray structure of wild-type Cx26, which is proposed to be in an open state, revealed a radial outward shift in the transmembrane helices in the closed state, presumably to accommodate the N-terminal plug occluding the pore. Because both Cx26del2-7 and Cx26M34Adel2-7 channels are closed, the N terminus appears to have a prominent role in stabilizing the open configuration.

SUBMITTER: Oshima A

PROVIDER: S-EPMC3026138 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Asymmetric configurations and N-terminal rearrangements in connexin26 gap junction channels.

Oshima Atsunori A Tani Kazutoshi K Toloue Masoud M MM Hiroaki Yoko Y Smock Amy A Inukai Sayaka S Cone Angela A Nicholson Bruce J BJ Sosinsky Gina E GE Fujiyoshi Yoshinori Y

Journal of molecular biology 20101120 3

Gap junction channels are unique in that they possess multiple mechanisms for channel closure, several of which involve the N terminus as a key component in gating, and possibly assembly. Here, we present electron crystallographic structures of a mutant human connexin26 (Cx26M34A) and an N-terminal deletion of this mutant (Cx26M34Adel2-7) at 6-Å and 10-Å resolutions, respectively. The three-dimensional map of Cx26M34A was improved by data from 60° tilt images and revealed a breakdown of the hexa ...[more]

PMID: 21094651

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Project description:Electrical synapses between neurons in the mammalian CNS are predominantly formed of the connexin36 (Cx36) gap junction (GJ) channel protein. Unique among GJs formed of a number of other members of the Cx gene family, Cx36 GJs possess a high sensitivity to intracellular Mg2+ that can robustly act to modulate the strength of electrical synaptic transmission. Although a putative Mg2+ binding site was previously identified to reside in the aqueous pore in the first extracellular (E1) loop domain, the involvement of the N-terminal (NT) domain in the atypical response of Cx36 GJs to pH was shown to depend on intracellular levels of Mg2+. In this study, we examined the impact of amino acid substitutions in the NT domain on Mg2+ modulation of Cx36 GJs, focusing on positions predicted to line the pore funnel, which constitutes the cytoplasmic entrance of the channel pore. We find that charge substitutions at the 8th, 13th, and 18th positions had pronounced effects on Mg2+ sensitivity, particularly at position 13 at which an A13K substitution completely abolished sensitivity to Mg2+. To assess potential mechanisms of Mg2+ action, we constructed and tested a series of mathematical models that took into account gating of the component hemichannels in a Cx36 GJ channel as well as Mg2+ binding to each hemichannel in open and/or closed states. Simultaneous model fitting of measurements of junctional conductance, gj, and transjunctional Mg2+ fluxes using a fluorescent Mg2+ indicator suggested that the most viable mechanism for Cx36 regulation by Mg2+ entails the binding of Mg2+ to and subsequent stabilization of the closed state in each hemichannel. Reduced permeability to Mg2+ was also evident, particularly for the A13K substitution, but homology modeling of all charge-substituted NT variants showed only a moderate correlation between a reduction in the negative electrostatic potential and a reduction in the permeability to Mg2+ ions. Given the reported role of the E1 domain in Mg2+ binding together with the impact of NT substitutions on gating and the apparent state-dependence of Mg2+ binding, this study suggests that the NT domain can be an integral part of Mg2+ modulation of Cx36 GJs likely through the coupling of conformational changes between NT and E1 domains.

Dataset Information

Asymmetric configurations and N-terminal rearrangements in connexin26 gap junction channels.

Publications

Asymmetric configurations and N-terminal rearrangements in connexin26 gap junction channels.

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