Engineered Cx40 variants increased docking and function of heterotypic Cx40/Cx43 gap junction channels

Area of Science:

• Cardiovascular Biology
• Cellular Biophysics
• Molecular Cardiology

Background:

• Gap junction (GJ) channels, formed by connexins (Cx), are crucial for rapid electrical signal propagation in the heart.
• Connexin40 (Cx40) and connexin43 (Cx43) are co-expressed in atrial myocytes, suggesting potential heterotypic GJ channel formation.
• Previous studies yielded conflicting results regarding the functional capacity of heterotypic Cx40/Cx43 GJ channels.

Purpose of the Study:

• To investigate methods for enhancing heterotypic GJ channel formation and function between Cx40 and Cx43.
• To identify specific Cx40 variants that improve docking and conductance with Cx43.
• To elucidate the structural and electrostatic factors limiting Cx40-Cx43 heterotypic docking.

Main Methods:

• Utilized designed Cx40 variants (D55N and P193Q) targeting extracellular domains.
• Assessed GJ plaque formation at cell-cell interfaces using model cells co-expressing Cx40 variants and Cx43.
• Quantified coupling conductance (Gj) of heterotypic GJ channels in N2A cells.
• Employed homology modeling to analyze docking interfaces and electrostatic interactions.

Main Results:

• Designed Cx40 variants (D55N and P193Q) significantly increased GJ plaque formation with Cx43.
• The coupling conductance (Gj) of D55N/Cx43 and P193Q/Cx43 heterotypic GJ channels was substantially higher than that of native Cx40/Cx43 channels.
• Homology models revealed that electrostatic interactions and surface complementarity at the docking interface impede Cx40-Cx43 docking.

Conclusions:

• Specific Cx40 variants can overcome natural limitations to heterotypic docking with Cx43.
• Enhancing heterotypic GJ channel function between Cx40 and Cx43 offers a potential strategy to improve atrial myocardium coupling.
• Targeting extracellular domains of connexins provides a viable approach to modulate cardiac electrical synchronization.