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Neurons and ?-cells of the pancreas express connexin36, forming gap junction channels that exhibit strong cationic selectivity.

ABSTRACT: We examined the permeability of connexin36 (Cx36) homotypic gap junction (GJ) channels, expressed in neurons and ?-cells of the pancreas, to dyes differing in molecular mass and net charge. Experiments were performed in HeLa cells stably expressing Cx36 tagged with EGFP by combining a dual whole-cell voltage clamp and fluorescence imaging. To assess the permeability of the single GJ channel (P(?)), we used a dual-mode excitation of fluorescent dyes that allowed us to measure cell-to-cell dye transfer at levels not resolvable using whole-field excitation solely. We demonstrate that P(?) of Cx36 for cationic dyes (EAM-1? and EAM-2?) is ~10-fold higher than that for an anionic dye of the same net charge and similar molecular mass, Alexa fluor-350 (AFl-350?). In addition, P(?) for Lucifer yellow (LY²?) is approximately fourfold smaller than that for AFl-350?, which suggests that the higher negativity of LY²? significantly reduces permeability. The P(?) of Cx36 for AFl-350 is approximately 358, 138, 23 and four times smaller than the P(?)s of Cx43, Cx40, Cx45, and Cx57, respectively. In contrast, it is 6.5-fold higher than the P(?) of mCx30.2, which exhibits a smaller single-channel conductance. Thus, Cx36 GJs are highly cation-selective and should exhibit relatively low permeability to numerous vital negatively charged metabolites and high permeability to K?, a major charge carrier in cell-cell communication.

SUBMITTER: Bukauskas FF

PROVIDER: S-EPMC3626077 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Neurons and β-cells of the pancreas express connexin36, forming gap junction channels that exhibit strong cationic selectivity.

Bukauskas Feliksas F FF

The Journal of membrane biology 20120630 5-6

We examined the permeability of connexin36 (Cx36) homotypic gap junction (GJ) channels, expressed in neurons and β-cells of the pancreas, to dyes differing in molecular mass and net charge. Experiments were performed in HeLa cells stably expressing Cx36 tagged with EGFP by combining a dual whole-cell voltage clamp and fluorescence imaging. To assess the permeability of the single GJ channel (P(γ)), we used a dual-mode excitation of fluorescent dyes that allowed us to measure cell-to-cell dye tra ...[more]

PMID: 22752717

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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

Neurons and ?-cells of the pancreas express connexin36, forming gap junction channels that exhibit strong cationic selectivity.

Publications

Neurons and β-cells of the pancreas express connexin36, forming gap junction channels that exhibit strong cationic selectivity.

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