V. Shoshanbarmatz et al., CROSS-LINKING OF THE RYANODINE RECEPTOR CA2-MUSCLE( RELEASE CHANNEL FROM SKELETAL), Biochimica et biophysica acta. Biomembranes, 1237(2), 1995, pp. 151-161
The relationship between the tetrameric organization of the ryanodine
receptor (RyR) and its activity in binding of ryanodine was approached
through cross-linking studies using several bifunctional reagents, di
ffering in their linear dimensions and flexibility, as well as in the
reactivity of the active groups. Cross-linking with: 1,5-difluoro-2,4-
dinitrobenzene (DFDNB); di(fluoro-3-nitrophenyl)sulfone (DFNPS), 1-eth
yl-3-(3-(dimethylamino)propyl)carbo (EDC); dimethyl suberimidate (DMS)
; ethylene glycol bis(succinimidylsuccinate) (EGS); and glutaraldehyde
resulted in the disappearance of the, 470 kDa, RyR monomer protein ba
nd with concomitant appearance of additional bands of molecular masses
higher than the monomer. At the relatively low concentrations of the
reagents and the conditions used, RyR is the only cross-linked protein
of SR membranes. The 'new' protein bands cross-react with antibodies
against the RyR and correspond to dimers and tetramers of the RyR subu
nits while trimers were not detectable. DFDNB and DFNPS produced also
a 560 kDa protein band which probably represents an intramolecular cro
ss-linked monomer. The SDS-electrophoretic patterns of the cross-linke
d purified RyR resemble those of the membrane-bound receptor. Ryanodin
e binding to the high-affinity site was inhibited by modification of S
R membranes with DFDNB and DFNPS, but not with DMS, EDC, EGS and gluta
raldehyde, although RyR was completely cross-linked. The inhibition by
DFDNB and DFNPS is due to modification of a specific lysyl residue wh
ich is also involved in the control of Ca2+ release. On the other hand
, cross linking of the RyR with glutaraldehyde or EGS resulted in inhi
bition of ryanodine binding to the low-affinity, but not to the high-a
ffinity binding sites. Thus, the cross-linking of two or more sites in
each monomer (which lead to Fixation of dimers or tetramers) did not
prevent the conformational changes involved in the binding and occlusi
on of ryanodine at the high-affinity site, but inhibited its binding t
o the low-affinity sites.