The lipidated connexin mimetic peptide SRPTEKT-Hdc is a potent inhibitor of Cx43 channels with specificity for the pS368 phospho-isoform.

Connexin (Cx) mimetic peptides derived from extracellular loop II sequences (e.g., Gap27: 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 (~5 µM, <1 h for HCh; ~100 µM, >1 h for GJCh) to achieve inhibition. We have shown that addition of a hexadecyl (Hdc) lipid tail 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 rat Cx43 (MDCK43). The lack of similar effect on dye coupling (NBD-MTMA) suggested channel conformation-specific inhibition. Here we report that SRPTEKT-Hdc inhibition of Ca2+-wave propagation, dye coupling, and dye uptake depended on the functional configuration of Cx43 as determined by phosphorylation at serine 368 (S368). Ca2+-wave propagation was enhanced in MDCK cells expressing single-site mutants of Cx43 that mimicked (MDCK43-S368D) or favored (MDCK43-S365A) phosphorylation at S368. Furthermore, SRPTEKT-Hdc potently inhibited GJCh-mediated Ca2+-wave propagation (IC50 230.4 pM), dye coupling, and HCh-mediated dye uptake in MDCK43-S368D and -S365A cells. In contrast, Ca2+-wave propagation, dye coupling, 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. Together, these data indicate that SRPTEKT-Hdc is a potent inhibitor of physiological Ca2+-wave signaling mediated specifically by the pS368 phosphorylated form of Cx43.

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: ~5 µM, <1 h; GJCh: ~100 µM, > 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 SRPTEKT- 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.

The membrane scaffold CD82 regulates cell adhesion by altering α4 integrin stability and molecular density.

Hematopoietic stem/progenitor cell (HSPC) interactions with the bone marrow microenvironment are important for maintaining HSPC self-renewal and differentiation. In recent work, we identified the tetraspanin protein, CD82, as a regulator of HPSC adhesion and homing to the bone marrow, although the mechanism by which CD82 mediated adhesion was unclear. In the present study, we determine that CD82 expression alters cell-matrix adhesion, as well as integrin surface expression. By combining the superresolution microscopy imaging technique, direct stochastic optical reconstruction microscopy, with protein clustering algorithms, we identify a critical role for CD82 in regulating the membrane organization of α4 integrin subunits. Our data demonstrate that CD82 overexpression increases the molecular density of α4 within membrane clusters, thereby increasing cellular adhesion. Furthermore, we find that the tight packing of α4 into membrane clusters depend on CD82 palmitoylation and the presence of α4 integrin ligands. In combination, these results provide unique quantifiable evidence of CD82's contribution to the spatial arrangement of integrins within the plasma membrane and suggest that regulation of integrin density by tetraspanins is a critical component of cell adhesion.