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

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Abstract

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 SRPT-EKT- 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.

Original languageEnglish (US)
Pages (from-to)C141-C154
JournalAmerican Journal of Physiology - Cell Physiology
Volume315
Issue number2
DOIs
StatePublished - Aug 1 2018

Fingerprint

Connexins
Gap Junctions
Connexin 43
Coloring Agents
Peptides
Inhibitory Concentration 50
Tail
Epithelial Cells
Cell Membrane
Rabbits
Wounds and Injuries

Keywords

  • Ca-wave propagation
  • Connexin 43
  • Gap junction channel inhibitor
  • Gap27
  • Hemichannel inhibitor
  • Lipidated mimetic peptide inhibitor
  • SRPTEK-Hdc

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

Cite this

@article{b3dea55e0e674ea3b8f452978af6abd2,
title = "Lipidated connexin mimetic peptides potently inhibit gap junction-mediated Ca2+-wave propagation",
abstract = "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 SRPT-EKT- 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.",
keywords = "Ca-wave propagation, Connexin 43, Gap junction channel inhibitor, Gap27, Hemichannel inhibitor, Lipidated mimetic peptide inhibitor, SRPTEK-Hdc",
author = "Cotter, {Maura L.} and Boitano, {Scott A} and Josef Vagner and Burt, {Janis M}",
year = "2018",
month = "8",
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doi = "10.1152/ajpcell.00156.2017",
language = "English (US)",
volume = "315",
pages = "C141--C154",
journal = "American Journal of Physiology",
issn = "0363-6143",
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TY - JOUR

T1 - Lipidated connexin mimetic peptides potently inhibit gap junction-mediated Ca2+-wave propagation

AU - Cotter, Maura L.

AU - Boitano, Scott A

AU - Vagner, Josef

AU - Burt, Janis M

PY - 2018/8/1

Y1 - 2018/8/1

N2 - 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 SRPT-EKT- 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.

AB - 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 SRPT-EKT- 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.

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KW - Connexin 43

KW - Gap junction channel inhibitor

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KW - Hemichannel inhibitor

KW - Lipidated mimetic peptide inhibitor

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DO - 10.1152/ajpcell.00156.2017

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JF - American Journal of Physiology

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