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, 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 ~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 μM 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 μM 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.