title: Functional implications of modifying RyR-activating peptides for membrane permeability.
abstract: 1. Our aim was to determine whether lipoamino acid conjugation of peptides that are high-affinity activators of ryanodine receptor (RyR) channels would (a) render the peptides membrane permeable, (b) alter their structure or (a) reduce their activity. The peptides correspond to the A region of the II-III loop of the skeletal dihydropyridine receptor. 2. The lipoamino acid conjugation increased the apparent permeability of the peptide across the Caco-2 cell monolayer by up to approximately 20-fold. 3. Nuclear magnetic resonance showed that the alpha-helical structure of critical basic residues, required for optimal activation of RyRs, was retained after conjugation. 4. The conjugated peptides were more effective in enhancing resting Ca2+ release, Ca2+-induced Ca2+ release and caffeine-induced Ca2+ release from isolated sarcoplasmic reticulum (SR) than their unconjugated counterparts, and significantly enhanced caffeine-induced Ca2+ release from mechanically skinned extensor digitorum longus (EDL) fibres. 5. The effect of both conjugated and unconjugated peptides on Ca2+ release from skeletal SR was 30-fold greater than their effect on either cardiac Ca2+ release or on the Ca2+ Mg2+ ATPase. 6. A small and very low affinity effect of the peptide in slowing Ca2+ uptake by the Ca2+, Mg2+ ATPase was exacerbated by lipoamino acid conjugation in both isolated SR and in skinned EDL fibres. 7. The results show that lipoamino acid conjugation of A region peptides increases their membrane permeability without impairing their structure or efficacy in activating skeletal and cardiac RyRs.
authors.affiliation: Division of Molecular Bioscience, John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra, ACT 2601, Australia. angela.dulhunty@anu.edu.au
authors.forename: Angela F
authors.initials: AF
authors.lastname: Dulhunty
authors.forename: Louise
authors.initials: L
authors.lastname: Cengia
authors.forename: Jacqui
authors.initials: J
authors.lastname: Young
authors.forename: Suzy M
authors.initials: SM
authors.lastname: Pace
authors.forename: Peta J
authors.initials: PJ
authors.lastname: Harvey
authors.forename: Graham D
authors.initials: GD
authors.lastname: Lamb
authors.forename: Yiu-Ngok
authors.initials: YN
authors.lastname: Chan
authors.forename: Norbert
authors.initials: N
authors.lastname: Wimmer
authors.forename: Istvan
authors.initials: I
authors.lastname: Toth
authors.forename: Marco G
authors.initials: MG
authors.lastname: Casarotto
mesh_terms: D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D018938:Caco-2 Cells; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D018929:Cell Culture Techniques; D002463:Cell Membrane Permeability / Q000187:drug effects* / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D006801:Humans; D008051:Lipid Bilayers; D008969:Molecular Sequence Data; D015394:Molecular Structure; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects* / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
chemical_list: D008051:Lipid Bilayers; D010446:Peptide Fragments; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D000409:Alanine; D002118:Calcium