Removal of clustered positive charge from dihydropyridine receptor II-III loop peptide augments activation of ryanodine receptors.
Peptides based on the skeletal muscle DHPR II-III loop have been shown to regulate ryanodine receptor channel activity. The N-terminal region of this cytoplasmic loop is predicted to adopt an alpha-helical conformation. We have selected a peptide sequence of 26 residues (Ala(667)-Asp(692)) as the minimum sequence to emulate the helical propensity of the corresponding protein sequence. The interaction of this control peptide with skeletal and cardiac RyR channels in planar lipid bilayers was then assessed and was found to lack isoform specificity. At low concentrations peptide A(667)-D(692) increased RyR open probability, whilst at higher concentrations open probability was reduced. By replacing a region of clustered positive charge with a neutral sequence with the same predisposition to helicity, the inhibitory effect was ablated and activation was enhanced. This novel finding demonstrates that activation does not derive from the presence of positively charged residues adjacent in the primary structure and, although it may be mediated by the alignment of basic residues down one face of an amphipathic helix, not all of these residues are essential.
Bannister
Mark L
ML
Department of Cardiac Medicine, National Heart and Lung Institute, Imperial College London, Dovehouse Street, SW3 6LY, London, UK.
Williams
Alan J
AJ
Sitsapesan
Rebecca
R
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D008051:Lipid Bilayers / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D009154:Mutation; D009206:Myocardium / Q000378:metabolism; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000378:metabolism*; D012756:Sheep
D020746:Calcium Channels, L-Type; D008051:Lipid Bilayers; D020033:Protein Isoforms; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium