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bioRxiv · 10.1101/311613

Calcium-Calmodulin Gating of Connexin43 Gap Junctions in the Absence of the pH Gating Domain

Veenstra, R. D.
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Abstract

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 Ca2+ 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 (gj) to zero within 15 min of perfusion, while this response was prevented by omitting 1.8 mM CaCl2 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 Ca2+/CaM gating response of Cx43 gap junctions was also examined. Internal addition of a Cx50 cytoplasmic loop CaMBD peptide (200 nM) prevented the Ca2+/ionomycin-induced decrease in Cx43 gj, while 100 M Gap19 peptide had no effect. Lastly, the transjunctional voltage (Vj) 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 gj from inactivating potentials was abolished by CT truncation of Cx43. We conclude that CT pH gating 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. Sequence-specific Cx CaMBD mimetic peptides act by binding Ca2+/CaM non-specifically and the Cx43 mimetic Gap19 peptide has no effect on this chemical gating mechanism.

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Veenstra, R. D.. 2018-04-30. Calcium-Calmodulin Gating of Connexin43 Gap Junctions in the Absence of the pH Gating Domain. https://doi.org/10.1101/311613

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