Background: Antiarrhythmic peptide (AAP) analogues prevents uncoupling of Connexin 43 (Cx43) gap junctional channels and prevent reentry arrhythmias during ischemia. The exact mode of action of AAPs is still unknown, but either phosphorylation or prevention of dephosphorylation is likely involved. The present study tested the involvement of serine 306 (S306) in Cx43, which undergoes dephosphorylation during ischemia. Methods: Electrical intercellular coupling was measured at pHi=7.2 and during cellular stress (pHi=6.2 and increased [Ca²⁺]i) using the double whole-cell patch clamp method in HeLa cells transfected with WT-Cx43 or Cx43 with mutations that mimic dephosphorylation (S to A) or phosphorylation (S to D) on S306. Results: Electrical coupling was 40% lower in S306A expressing cells and uncoupling was significantly faster and greater during cellular stress compared to WT. In contrast, electrical coupling of S306D expressing cells was not different from WT and S306D channels do not close during cellular stress. The AAP analogue AAP10 (10 nM) prevented uncoupling during cellular stress in both WT and S306A expressing cells, whereas coupling was unaffected in S306D cells. No single channel activity could be recorded during cellular stress, but in cell pairs treated with AAP10, channels with reduced unitary conductance were observed (60 pS versus 100 pS in unstressed WT channels). AAP10 also reduced unitary conductance at pHi=7.2, and thus AAP10 itself rather than cellular stress causes the reduction. Conclusions: AAP10 prevents stress- induced uncoupling and this effect is independent of phosphorylation at S306. Furthermore, AAP10 decreases unitary conductance during both cell stress and pHi=7.2. Since AAP10 reduces unitary conductance but increases macroscopic electrical coupling, we conclude, that AAP10 increases the open probability of the Cx43 gap junction channels.