Modification of the ryanodine receptor (RyR)/Ca2+ release channel with 2,4-
dinitrofluorobenzene (DNFB) indicated that two classes of amino group inter
act with the reagent, as can be distinguished on the basis of their reactiv
ity/accessibility and the effects on ryanodine binding and single channel a
ctivities. One group interacted very rapidly (t1/2 < 30 s) at 25 degrees C
with low concentrations of DNFB [C-50 (concentration of DNFB required for 5
0% inhibition or stimulation of ryanodine binding) = 5 mu M]. and at pH val
ues of 6.2 and higher, This interaction resulted in the marked stimulation
of ryanodine binding and the complete inhibition of a single Ca2+ release c
hannel incorporated into planar lipid bilayer, The second group is accessib
le at higher temperatures (37 degrees C); at pH values higher than 7.4 it r
eacted slowly (t1/2 = 20 min) with high concentrations of DNFB (C-50 = 70 m
u M). This interaction led to the inhibition of ryanodine binding and singl
e channel activity. Modification of RyR with DNFB under the stimulatory con
ditions resulted in 3.6-fold and 6-fold increases in ryanodine-binding and
Ca2+-binding affinities respectively. Modification with DNFB under the inhi
bitory conditions resulted in a decrease in the total ryanodine-binding sit
es. The exposure of the RyR single channel to DNFB under both inhibitory an
d stimulatory conditions led to the complete closure of the channel. Howeve
r, when modified under the stimulatory conditions, but not under the inhibi
tory ones, the DNFB-modified closed channel could be re-activated by submic
romolar concentrations of ryanodine, in the presence of nanomolar concentra
tions of Ca2+, The DNFB-modified ryanodine-activated RyR channel showed fas
t transitions between open, closed and several sub-conductance states, and
was completely closed by Ruthenium Red. ATP re-activated the DNFB-modified
closed channel or, if present during modification, prevented the inhibition
of RyR channel activity by DNFB. Neither the stimulation nor the inhibitio
n of ryanodine binding by modification with DNFB was affected by the presen
ce of ATP, By using the photoreactive ATP analogue 3'-O-(4-benzoyl)benzoyl-
[alpha-P-32]ATP we found that DNFB modification had no effect on the ATP-bi
nding site of RyR, The results are discussed with regard to the involvement
of amino group residues in channel gating, ryanodine association/dissociat
ion and occlusion, and the relationship between the open/closed state of th
e RyR and its capacity to bind ryanodine.