Activation and inhibition of skeletal RyR channels by a part of the skeletal DHPR II-III loop:: Effects of DHPR Ser687 and FKBP12

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, Thr(671)-Leu(690); A2, Thr(671)-Leu(690) with Ser(687) Ala substitution; NB, Gly(689)-Lys(708) 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 similar to 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 mu M Al or 50 nM A2. The greatest A1-induced increase in mean current (sixfold) was seen with ion nM cis Ca2+. Inhibition by A1 was greatest at +40 mV (or when permeant ions flowed from cytoplasm to SR lumen) with 100 mu M 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 Ser(687), 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.