title: Unitary permeability of gap junction channels to second messengers measured by FRET microscopy.
abstract: 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 (InsP(3)) 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 InsP(3). The unitary permeabilities to cAMP (47 x 10(-3) +/- 15 x 10(-3) microm(3)/s) and InsP(3) (60 x 10(-3) +/- 12 x 10(-3) microm(3)/s) were similar, but substantially larger than the unitary permeability to lucifer yellow (LY; 7 +/- 3 x 10(-3) microm(3)/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.
authors.affiliation: Istituto Veneto di Medicina Molecolare, Fondazione per la Ricerca Biomedica Avanzata, 35129 Padova, Italy.
authors.forename: Victor H
authors.initials: VH
authors.lastname: Hernandez
authors.forename: Mario
authors.initials: M
authors.lastname: Bortolozzi
authors.forename: Vanessa
authors.initials: V
authors.lastname: Pertegato
authors.forename: Martina
authors.initials: M
authors.lastname: Beltramello
authors.forename: Michele
authors.initials: M
authors.lastname: Giarin
authors.forename: Manuela
authors.initials: M
authors.lastname: Zaccolo
authors.forename: Sergio
authors.initials: S
authors.lastname: Pantano
authors.forename: Fabio
authors.initials: F
authors.lastname: Mammano
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D000072259:Connexin 26; D017630:Connexins / Q000096:biosynthesis; D000242:Cyclic AMP / Q000502:physiology; D004594:Electrophysiology; D031541:Fluorescence Resonance Energy Transfer; D017629:Gap Junctions / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007295:Inositol Phosphates / Q000502:physiology; D015640:Ion Channel Gating / Q000502:physiology; D007473:Ion Channels / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010539:Permeability; D015290:Second Messenger Systems / Q000502:physiology*; D014162:Transfection
chemical_list: D017630:Connexins; D007295:Inositol Phosphates; D007473:Ion Channels; D000072259:Connexin 26; C092449:inositol 1,2,3-trisphosphate; D000242:Cyclic AMP