Gap-junctional single-channel permeability for fluorescent tracers in mammalian cell cultures

Area of Science:

• Cell Biology
• Biophysics
• Molecular Biology

Background:

• Gap junctions mediate direct cell-to-cell communication.
• Quantifying gap-junction permeability is crucial for understanding cellular functions.
• Existing methods may not fully capture the complexity of transport through gap junctions.

Purpose of the Study:

• To develop and validate a simple dye transfer method for quantifying gap-junction permeability.
• To investigate the relationship between ionic conductance and dye permeance.
• To determine single-channel permeance values for specific tracer dyes in different cell lines.

Main Methods:

• Utilized a patch-type micropipette for dye perfusion (calcein, Lucifer yellow) into single cultured cells.
• Employed a low-light charge-coupled device camera to monitor dye spreading.
• Applied compartment modeling and curve fitting to estimate permeation rates and single-channel permeance.

Main Results:

• Established a correlation between permeation rates and total ionic conductance.
• Observed a wide scatter in single-channel permeances, deviating from a simple linear pore model.
• Quantified specific ranges of single-channel permeances for calcein and Lucifer yellow in BICR/M1R(k), HeLa-Cx43, and HeLa-Cx46 cells.

Conclusions:

• The developed dye transfer method effectively quantifies gap-junction permeability.
• Gap-junction permeability exhibits complex regulation, with single-channel permeance varying significantly for different molecules.
• An unknown mechanism likely modulates the differential permeability of gap junctions for ions and larger molecules.