Bastadins relate ryanodine-sensitive and ryanodine-insensitive ca2+
efflux pathways in skeletal sr and bc3h1 cells.
Pessah, Isaac N., Tadeusz F. Molinski, Trena D. Meloy, Patty Wong,
Edmond D. Buck, Paul D. Allen, and F. C. Mohr and Matthew M. Mack.
Departments of Molecular Biosciences and Chemistry, University of
California Davis, CA 95616, and (Department of Anesthesia, Brigham
and Womenaes Hospital, Boston, MA 02147
APStracts 3:0324C, 1996.
Bastadins potently interact with the FKBP12/ryanodine receptor (Ry1R)
complex in skeletal muscle to enhance a high affinity ryanodine- (RY)
binding conformation (Mack et al. J. Biol. Chem. 269: 23236-23249,
1994). Bastadins are used to examine the relationship between RY
-sensitive and RY-insensitive Ca2+ efflux pathways which coexist in
junctional SR vesicles from rabbit skeletal muscle and differentiated
BC3H1 cells. Complete block of caffeine-sensitive Ca2+ channels with
micromolar RY or ruthenium red (RR) does not alter the steady-state
loading capacity of SR. Inhibition of SERCA pumps with thapsigargin
unmasks a RY- and RR-insensitive Ca2+ efflux pathway. Bastadin 5
alone does not inhibit Ca2+ efflux unmasked by inhibition of SERCA
pumps, but in combination with blocking concentrations of RY or RR
eliminates the RY-insensitive Ca2+ [circumflex]oleak[diaeresis]o and
enhances steady-state loading capacity of SR vesicles 2.5-fold. These
actions of bastadins occur in the same concentration range which
enhances the number of high-affinity binding sites for [3H]ryanodine
(EC50s 2M). Similar effects on SR Ca2+ transport are found with FK506
and RY in combination. Block of Ry1R in intact BC3H1 cells with
ryanodine does not eliminate the prominent Ca2+ leak unmasked by
thapsigargin. A membrane-permeant mixture of bastadins in combination
with ryanodine nearly eliminates the Ca2+ leak unmasked by
thapsigargin, even though the Ca2+ stores are replete. The
requirement of both a known Ry1R blocker and bastadins in combination
provides a pharmacological link between ryanodine-sensitive Ca2+
channels and ryanodine-insensitive leak pathways in isolated
junctional SR and BC3H1 cells. These results taken together strongly
suggest that bastadins, through their modulatory actions on the
FKBP12/Ry1R complex, convert ryanodine-insensitive leak states into
ryanodine-sensitive channels which recognize [3H]ryanodine with high
affinity.
Received 17 June 1996; accepted in final form 16 September 1996.
APS Manuscript Number C355-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 5 November 1996