Modification of ryanodine receptor/Ca2+ release channel with dinitrofluorobenzene

Citation
N. Hadad et al., Modification of ryanodine receptor/Ca2+ release channel with dinitrofluorobenzene, BIOCHEM J, 342, 1999, pp. 239-248
Citations number
46
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMICAL JOURNAL
ISSN journal
02646021 → ACNP
Volume
342
Year of publication
1999
Part
1
Pages
239 - 248
Database
ISI
SICI code
0264-6021(19990815)342:<239:MORRRC>2.0.ZU;2-D
Abstract
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.