Intracellular protons and calcium ions are two major chemical factors that regulate connexin43 (Cx43) gap junction communication and the synergism or antagonism between pH and Ca2+ has been questioned for decades. To assess the ability of Ca2+ ions to modulate Cx43 junctional conductance (gj) in the absence of pH-sensitivity, patch clamp experiments were performed on Neuroblastoma-2a (N2a) cells or neonatal mouse ventricular myocytes (NMVMs) expressing either full-length Cx43 or the Cx43-M257 (Cx43K258stop) mutant protein, a carboxyl-terminus (CT) truncated version of Cx43 lacking pH-sensitivity. The addition of 1 μM ionomycin to normal calcium saline reduced Cx43 or Cx43-M257 gj to zero within 15 min of perfusion. This response was prevented by Ca2+-free saline or addition of 100 nM calmodulin (CaM) inhibitory peptide to the internal pipette solution. Internal addition of a connexin50 cytoplasmic loop calmodulin-binding domain (CaMBD) mimetic peptide (200 nM) prevented the Ca2+/ionomycin-induced decrease in Cx43 gj, while 100 μM Gap19 peptide had minimal effect. The investigation of the transjunctional voltage (Vj) gating properties of NMVM Cx43-M257 gap junctions confirmed the loss of the fast inactivation of Cx43-M257 gj, but also noted the abolishment of the previously reported facilitated recovery of gj from inactivating potentials. We conclude that the distal CT domain of Cx43 contributes to the Vj-dependent fast inactivation and facilitated recovery of Cx43 gap junctions, but the Ca2+/CaM-dependent gating mechanism remains intact in its absence. Sequence-specific connexin CaMBD mimetic peptides act by binding Ca2+/CaM non-specifically and the Cx43 mimetic Gap19 peptide has negligible effect on this chemical gating mechanism.

Intracellular protons and calcium ions are two major chemical factors that regulate connexin43 (Cx43) gap junction channels and the synergism or antagonism between pH and Ca 2+ has been questioned for decades. In this study, we assessed whether the calcium gating mechanism occurs independently of the pH gating mechanism by utilizing the Cx43-M257 (Cx43K258stop) mutant, a carboxyl-terminal (CT) truncated version of Cx43 lacking the pH gating domain. Dual whole cell patch clamp experiments were performed on Neuroblastoma-2a (N2a) cells or neonatal mouse ventricular myocytes (NMVMs) expressing either full length Cx43 or Cx43-M257 proteins. Addition of 1 M ionomycin to normal calcium saline reduced Cx43 or Cx43-M257 macroscopic gap junction conductance (g j ) to zero within 15 min of perfusion, while this response was prevented by omitting 1.8 mM CaCl 2 from the external solution or adding 100 nM calmodulin (CaM) inhibitory peptide to the internal pipette solution. The ability of connexin calmodulin binding domain (Cx CaMBD) mimetic peptides and the Gap19 peptide to inhibit the Ca 2+ /CaM gating response of Cx43 gap junctions was also examined. Internal addition of a Cx50 cytoplasmic loop CaMBD peptide (200 nM) prevented the Ca 2+ /ionomycin-induced decrease inCx43 g j , while 100 M Gap19 peptide had no effect. Lastly, the transjunctional voltage (V j ) gating properties of NMVM Cx43-M257 gap junctions were investigated. We confirmed that the fast kinetic inactivation component was absent in Cx43-M257 gap junctions, but also observed that the previously reported facilitated recovery of g j from inactivating potentials was abolished by CT truncation of Cx43. We conclude that CT pH gating domain of Cx43 contributes to the V j -dependent fast inactivation and facilitated recovery of Cx43 gap junctions, but the Ca 2+ /CaMdependent gating mechanism remains intact. Sequence-specific Cx CaMBD mimetic peptides act by binding Ca 2+ /CaM non-specifically and the Cx43 mimetic Gap19 peptide has no effect on this chemical gating mechanism.