Activation and Inhibition of Skeletal RyR Channels by a Part of the Skeletal DHPR II-III Loop: Effects of DHPR Ser 687 and FKBP12

Abstract

Peptides, corresponding to sequences in the N-terminal region of the skeletal muscle dihydropyridine receptor (DHPR) II-III loop, have been tested on sarcoplasmic reticulum (SR) Ca2+ release and ryanodine receptor (RyR) activity. The peptides were: A1, Thr671-Leu690; A2, Thr671-Leu690 with Ser687 Ala substitution; NB, Gly689-Lys708 and A1S, scrambled A1 sequence. The relative rates of peptide-induced Ca2+ release from normal (FKBP12+) SR were A2 > A1 > A1S > NB. Removal of FKBP12 reduced the rate of A1-induced Ca2+ release by approximately 30%. A1 and A2 (but not NB or A1S), in the cytoplasmic (cis) solution, either activated or inhibited single FKBP12+ RyRs. Maximum activation was seen at -40 mV, with 10 microM A1 or 50 nM A2. The greatest A1-induced increase in mean current (sixfold) was seen with 100 nM cis Ca2+. Inhibition by A1 was greatest at +40 mV (or when permeant ions flowed from cytoplasm to SR lumen) with 100 microM cis Ca2+, where channel activity was almost fully inhibited. A1 did not activate FKBP12-stripped RyRs, although peptide-induced inhibition remained. The results show that peptide A activation of RyRs does not require DHPR Ser687, but required FKBP12 binding to RyRs. Peptide A must interact with different sites to activate or inhibit RyRs, because current direction-, voltage-, cis [Ca2+]-, and FKBP12-dependence of activation and inhibition differ.

Functional implications of modifying RyR‐activating peptides for membrane permeability

“…Stock peptide solutions (B2 mM) were prepared in H 2 O and frozen in 20 ml aliquots (Dulhunty et al, 1999).…”

“…Extravesicular Ca 2 þ was monitored at 710 nm with the Ca 2 þ indicator, antipyrylazo III, using a Cary 3 spectrophotometer (Dulhunty et al, 1999). Identical release experiments were performed at 790 nm, to detect Ca 2 þ -independent changes in optical density (OD), which would alter the Ca 2 þ release measurement.…”

“…A 20 amino-acid peptide (peptide A), corresponding to a part of the loop between the second and third membrane-spanning segment of the skeletal dihydropyridine receptor (DHPR), is a part of a naturally occurring muscle protein and is a high-affinity activator of skeletal and cardiac RyR channels (El-Hayek et al, 1995;Dulhunty et al, 1999;Gurrola et al, 1999;Lamb et al, 2000;Stange et al, 2001;Dulhunty et al, 2002). Both the DHPR and the RyR are essential for skeletal muscle contraction (Tanabe et al, 1988).…”

The recombinant dihydropyridine receptor II–III loop and partly structured ‘C’ region peptides modify cardiac ryanodine receptor activity

“…The recombinant SDCL and the acidic C region [18] are high-affinity activators of RyR1 [12,[18][19][20]. The strongly α-helical basic 'A' region peptide [21] also activates RyR1 with high affinity [10][11][12]20,22]. These functional interactions possibly reflect binding reactions that contribute to the protein-protein interactions in skeletal muscle [18,20].…”

“…The following peptides were synthesized and purified (by the John Curtin School of Medical Research Biomolecular Resource Facility) as described previously [21,22,25] …”