Development of Potent, Conformation-Specific Inhibitors of Connexin-Mediated Communication

Connexin (Cx) mimetic peptides derived from extracellular loop II (ECL2) sequences of Cx43 (e.g. Gap 27: SRPTEKTIFII; Peptide5: VDCFLSRPTEKT) have been used as reversible, Cx-specific blockers of hemichannel (HCh) and gap junction channel (GJCh) function. These blockers typically require high concentrations (HCh: ~5 µM, 1 hour) to achieve inhibition. We report here that addition of a hexadecyl (Hdc) lipid moiety to the conserved SRPTEKT peptide sequence, SRPTEKT-Hdc, results in a novel, highly efficacious and potent inhibitor of mechanically-induced Ca2+-wave propagation (IC50: 64.8 pM) and HCh-mediated dye uptake (IC50: 45.0 pM) in Madin-Darby Canine Kidney cells expressing mCx43 (MDCK43). The lack of similar effect on dye coupling suggested channel conformation-specific inhibition, and indeed, SRPTEKT-Hdc inhibition of Ca2+-wave propagation and dye uptake was selective for Cx43 channels in the functional configuration governed by phosphorylation at serine S368 (pS368). As such, SRPTEKT-Hdc potently inhibited Ca2+-wave propagation (IC50: 230.4 pM) and dye uptake in MDCK cells expressing single site mutants of Cx43 that mimicked (MDCK43-S368D) or favored (MDCK43-S365A) phosphorylation at S368. In contrast, Ca2+-wave propagation and dye uptake were largely unaffected (IC50: 12.3 μM) by SRPTEKT-Hdc in MDCK43-S368A and -S365D cells, mutations that mimic or favor dephosphorylation at S368. Finally, we preliminarily report SRPTEKT-Hdc inhibition of Cx37-mediated Ca2+-wave propagation and dye uptake in MDCK cells expressing a doxycycline-inducible form of mCx37 (iMDCK37). Inhibition occurred in a manner similar to that observed for wild-type Cx43 channels, highlighting the likely involvement of the identical SRPTEKT sequence shared between ECL2 domains of these two Cx isotypes. Together, these data indicate that SRPTEKT-Hdc is a potent and specific inhibitor of the pS368 phospho-form of Cx43 with potential for cross-isotype inhibition of other SRPTEKT-containing Cx isotypes (possibly also in a phosphorylation-dependent manner)