A biophysical approach to the study of structure and function of connexin channel nanopores
Abstract
Objective: Connexins are transmembrane proteins that form intercellular junctional channels in vertebrates and are known or suspected to be involved in a wide variety of biological processes including cardiac development and function, hearing, haematopoesis, regeneration, lens transparency, fertility, immune system function and protection from oxidative stress. Connexin mutations can cause developmental and physiological defects, and link to various 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. Study design : 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 . Results: The unitary permeabilities to cAMP (47 ± 15 × 10 –3 μm 3 /s) and InsP 3 (60 ± 12 × 10 –3 μm 3 /s) were similar, but substantially larger than the unitary permeability to Lucifer Yellow (LY; 7 ± 3 × 10 –3 μm 3 /s), an exogenous tracer. Conclusion: This method permits quantification of defects of metabolic coupling and can be used to investigate interdependence of intercellular diffusion and cross-talk between diverse signalling pathways.