Functional implications of modifying RyR-activating peptides for membrane permeability

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. The lipoamino acid conjugation increased the apparent permeability of the peptide across the Caco-2 cell monolayer by up to ∼20-fold. Nuclear magnetic resonance showed that the α-helical structure of critical basic residues, required for optimal activation of RyRs, was retained after conjugation. The conjugated peptides were more effective in enhancing resting Ca ²⁺ release, Ca ²⁺-induced Ca ²⁺ release and caffeine-induced Ca ²⁺ release from isolated sarcoplasmic reticulum (SR) than their unconjugated counterparts, and significantly enhanced caffeine-induced Ca ²⁺ release from mechanically skinned extensor digitorum longus (EDL) fibres. The effect of both conjugated and unconjugated peptides on Ca ²⁺ release from skeletal SR was 30-fold greater than their effect on either cardiac Ca ²⁺ release or on the Ca ²⁺ Mg ²⁺ ATPase. A small and very low affinity effect of the peptide in slowing Ca ²⁺ uptake by the Ca ²⁺, Mg ²⁺ 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.