Calcium-calmodulin gating of a pH-insensitive isoform of connexin43 gap junctions

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 (g_j) 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 g_j 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 g_j, while 100 μM Gap19 peptide had minimal effect. The investigation of the transjunctional voltage (V_j) gating properties of NMVM Cx43-M257 gap junctions confirmed the loss of the fast inactivation of Cx43-M257 g_j, but also noted the abolishment of the previously reported facilitated recovery of g_j from inactivating potentials. We conclude that the distal CT domain of Cx43 contributes to the V_j-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.