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Electrical conductance of mouse connexin45 gap junction channels is modulated by phosphorylation
T A van Veen1, H V van Rijen, H J Jongsma
1University Medical Center Utrecht, Department of Medical Physiology, The Netherlands. A.A.B.vanVeen@med.uu.nl
Abstract:
In this study we report about the modulation of connexin45 (Cx45) gap junction channel properties by phosphorylation of the connexin molecules through different protein kinases. Phosphorylation of Cx45 was studied in HeLa cells transfected with mouse Cx45 (mCx45). Using Western blotting (WB) and immunocytochemistry, these cells were found exclusively positive for Cx45 and the protein was separated as a doublet of bands with a calculated mass of 46 and 48 kD. After dephosphorylation using calf intestine phosphatase (CIP), the 48 kD band disappeared almost completely leaving a single band at 46 kD. This effect can be prevented by including phosphatase inhibitors during CIP treatment. These results indicate that the 48 kD signal represents a phosphorylated form of Cx45. To investigate the effects of (de)phosphorylation of Cx45 on the conductive properties of gap junction channels built of this connexin, cell pairs were subjected to dual voltage clamp experiments and coupling was determined before and after addition of PMA, 4alpha-PDD, cAMP, cGMP, and pervanadate to the superfusate. 100 nM of the PKC activating phorbol ester PMA increased normalized junctional conductance by 50.9+/-28%. 100 nM of the inactive phorbol ester 4alpha-PDD had no significant effect. Activation of PKA with 1 mM 8-Br-cAMP decreased coupling by 20.9+/-5.7% while 1 mM 8-Br-cGMP (PKG-activation) was ineffective. 100 microM pervanadate, a tyrosine phosphatase inhibitor, reduced coupling by 43.7+/-11.1%. Single channel measurements, under identical phosphorylating conditions, were not significantly different from each other and all frequency histograms exhibited two conductance peaks at approximately 20 and 40 pS. WB analysis revealed, as compared to control conditions, a relative increase of the 48 kD signal upon stimulation with pervanadate (142+/-42%) and 8-Br-cAMP (50+/-23%) whereas neither stimulation with PMA nor 8-Br-cGMP had a significant effect. These experiments show that electrical intercellular conductance via Cx45 gap junction channels is differentially regulated by phosphorylation. However, regulation does not act by changing single channel conductance, but most likely by modulation of the open probability of Cx45 gap junction channels.
Insights
Connexin45 (Cx45) gap junction channel properties are modulated by protein kinase phosphorylation, affecting electrical intercellular communication. This regulation occurs by altering channel open probability, not single channel conductance.
Area of Science:
- Cellular biology
- Molecular biology
- Biophysics
Background:
- Connexin45 (Cx45) forms gap junction channels crucial for intercellular communication.
- Phosphorylation is a key post-translational modification regulating protein function.
Purpose of the Study:
- To investigate the role of Cx45 phosphorylation by different protein kinases in modulating gap junction channel properties.
- To determine how phosphorylation affects the electrical conductance of Cx45 gap junctions.
Main Methods:
- Western blotting (WB) and immunocytochemistry to identify phosphorylated Cx45.
- Dual voltage clamp experiments to measure gap junctional conductance.
- Single channel recordings to assess channel properties.
Main Results:
- Phosphorylation of Cx45 was confirmed, with a 48 kD band representing the phosphorylated form.
- PKC activation (PMA) increased conductance by ~51%, while PKA activation (cAMP) decreased it by ~21%.
- Tyrosine phosphatase inhibition (pervanadate) reduced coupling by ~44% and increased the phosphorylated Cx45 signal.
Conclusions:
- Cx45 gap junction channel function is differentially regulated by phosphorylation.
- Regulation mechanisms involve modulation of channel open probability, not single channel conductance.