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 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.
Dulhunty
A F
AF
Muscle Research Group, John Curtin School of Medical Research, Canberra, ACT 2601, Australia. angela.dulhunty@anu.ed.au
Laver
D R
DR
Gallant
E M
EM
Casarotto
M G
MG
Pace
S M
SM
Curtis
S
S
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry*; D020746:Calcium Channels, L-Type; D002586:Cesium / Q000494:pharmacology; D004553:Electric Conductivity; D020104:Immunophilins / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D022021:Tacrolimus Binding Proteins; D019284:Thapsigargin
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D008051:Lipid Bilayers; D010446:Peptide Fragments; D019837:Ryanodine Receptor Calcium Release Channel; D002586:Cesium; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins; D008274:Magnesium; D002118:Calcium