Nature Methods
- 4, 353 - 358 (2007)
Published online: 11 March 2007; | doi:10.1038/nmeth1031
Unitary permeability of gap junction channels to second messengers measured by FRET microscopyVictor H Hernandez1, 5, Mario Bortolozzi1, 5, Vanessa Pertegato1, 2, Martina Beltramello1, Michele Giarin3, Manuela Zaccolo1, 2, Sergio Pantano1, 4 & Fabio Mammano1, 3, 41
Istituto Veneto di Medicina Molecolare, Fondazione per la Ricerca Biomedica Avanzata, 35129 Padova, Italy. 2
Istituto Dulbecco Telethon, Fondazione per la Ricerca Biomedica Avanzata, 35129 Padova, Italy. 3
Dipartimento di Fisica 'G. Galilei' dell'Università di Padova, 35131 Padova, Italy. 4
Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia, 35131 Padova, Italy. 5
These authors contributed equally to this work.
Correspondence should be addressed to Fabio Mammano fabio.mammano@unipd.it Gap junction channels assembled from connexin protein subunits mediate intercellular transfer of ions and metabolites. Impaired channel function is implicated in several hereditary human diseases. In particular, defective permeation of cAMP or inositol-1,4,5-trisphosphate (InsP3) through connexin channels is associated with peripheral neuropathies and deafness, respectively. Here we present a method to estimate the permeability of single gap junction channels to second messengers. Using HeLa cells that overexpressed wild-type human connexin 26 (HCx26wt) as a model system, we combined measurements of junctional conductance and fluorescence resonance energy transfer (FRET) emission ratio of biosensors selective for cAMP and InsP3. The unitary permeabilities to cAMP (47  10-3  15 10-3  m3/s) and InsP3 (60 10-3 12 10-3 m3/s) were similar, but substantially larger than the unitary permeability to lucifer yellow (LY; 7 3 10-3 m3/s), an exogenous tracer. This method permits quantification of defects of metabolic coupling and can be used to investigate interdependence of intercellular diffusion and cross-talk between diverse signaling pathways.
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