Lipidated connexin mimetic peptides potently inhibit gap junction-mediated Ca2+-wave propagation

Connexin (Cx) mimetic peptides (e.g., Gap27: SRPTEKTIFII; Peptide5: VDCFLSRPTEKT) reversibly inhibit hemichannel (HCh) and gap junction channel (GJCh) function in a concentration- and time-dependent manner (HCh: similar to 5 mu M, <1 h; GJCh: similar to 100 mu M, > 1 h). We hypothesized that addition of a hexadecyl tail to SRPTEKT (SRPTEKT-Hdc) would improve its ability to concentrate in the plasma membrane and consequently increase its inhibitory efficacy. We show that SRPTEKT-Hdc inhibited intercellular Ca2+-wave propagation in Cx43-expressing MDCK and rabbit tracheal epithelial cells in a time (61-75 min)- and concentration (IC50: 66 pM)-dependent manner, a concentration efficacy five orders of magnitude lower than observed for the nonlipidated Gap27. HCh-mediated dye uptake was inhibited by SRPTEKT-Hdc with similar efficacy. Following peptide washout. HCh-mediated dye uptake was restored to control levels, whereas Ca2+-wave propagation was only partially restored. Scrambled and reverse sequence lipidated peptides had no detectable inhibitory effect on Ca2+-wave propagation or dye uptake. Cx43 expression was unchanged by SRPTHKT-Hdc incubation; however, Triton-insoluble Cx43 was reduced by SRPTEKT-Hdc exposure and reversed following washout. In summary, our results show that SRPTEKT-Hdc blocked HCh function and intercellular Ca2+ signaling at concentrations that minimally affected dye coupling. Selective inhibition of intercellular Ca2+ signaling, likely indicative of channel conformation-specific SRPTEKT-Hdc binding, could contribute significantly to the protective effects of these mimetic peptides in settings of injury. Our data also demonstrate that lipidation represents a paradigm for development of highly potent. efficacious, and selective mimetic peptide inhibitors of hemichannel and gap junction channel-mediated signaling.