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The 3.5 ångström X−ray structure of the human connexin26 gap junction channel is unlikely that of a fully open channel

Francesco Zonta12, Guido Polles3, Maria Federica Sanasi1, Mario Bortolozzi12 and Fabio Mammano124*

Author Affiliations

1 Department of Physics and Astronomy “G. Galilei”, University of Padua, 35131, Padua, Italy

2 Venetian Institute of Molecular Medicine, 35129, Padua, Italy

3 International school for advanced studies (SISSA), 34136, Trieste, Italy

4 CNR Institute of Neurosciences, Padua Section, 35131, Padua, Italy

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Cell Communication and Signaling 2013, 11:15  doi:10.1186/1478-811X-11-15

Published: 27 February 2013


The permeability of gap junction channels to metabolites, and not simply to small inorganic ions, is likely to play an important role in development, physiology as well as in etiology of several diseases. Here, we combined dual patch clamp and fluorescence imaging techniques with molecular dynamics (MD) simulations to investigate the permeation of calcein, a relatively large fluorescent tracer (MW 622 Da) through homomeric gap junction channels formed by wild type human connexin26 (hCx26wt) protomers. Our experimental data indicate that the unitary flux of calcein driven by a 125 μM concentration difference is Jpore = 226 molecule/s per channel. In the light of Eyring transition state theory adapted for the liquid phase, this value corresponds to an energy barrier of ~20 kBT (where kB is the Boltzmann constant and T is absolute temperature). The barrier predicted by our MD simulations, based on the 3.5 Å X–ray structural model of the hCx26wt gap junction channel, is ~45 kBT. The main contributions to the energetics of calcein permeation originated from the interaction between the permeating molecule and the charged aminoacids lining the channel pore. Assigning a fake zero total charge to the calcein molecule yielded a value for the barrier height compatible with the experimental data. These results can be accounted for by two different (although not mutually exclusive) hypotheses: (1) the X–ray model of the hCx26wt gap junction channel is not representative of a fully open state; (2) post translational modifications affecting the hCx26wt protein in our expression system differed from the modifications undergone by the proteins in the conditions used to obtain the crystal structure. Hypothesis (1) is compatible with data indicating that, only 10% or less of the channels forming a gap junction plaque are in the open state, and therefore the averaging procedure intrinsic in the generation of the crystal structure data more closely reflects that of a closed channel. Hypothesis (2) is compatible with recent mass spectrometry data and implies that the charge of several amino acid side chains may have been altered, thus modifying substantially the permeation properties of the channels in living cells.

Connexin26; Calcein; Dual patch clamp; Fluorescence imaging; Umbrella sampling; Potential of mean force; Transition rate; Model