Development of Potent, Conformation-Specific Inhibitors of Connexin-Mediated Communication
AuthorCotter, Maura Lynn
connexin mimetic peptide
gap junction channel inhibitor
AdvisorBurt, Janis M.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction, presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractConnexin (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; GJCh: ~100 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).
Degree ProgramGraduate College