Modification of ryanodine receptor/Ca2+ release channel with dinitrofluorobenzene.
Modification of the ryanodine receptor (RyR)/Ca(2+) release channel with 2,4-dinitrofluorobenzene (DNFB) indicated that two classes of amino group interact with the reagent, as can be distinguished on the basis of their reactivity/accessibility and the effects on ryanodine binding and single channel activities. One group interacted very rapidly (t(1/2)<30 s) at 25 degrees C with low concentrations of DNFB [C(50) (concentration of DNFB required for 50% inhibition or stimulation of ryanodine binding)=5 microM], and at pH values of 6.2 and higher. This interaction resulted in the marked stimulation of ryanodine binding and the complete inhibition of a single Ca(2+) release channel incorporated into planar lipid bilayer. The second group is accessible at higher temperatures (37 degrees C); at pH values higher than 7.4 it reacted slowly (t(1/2)=20 min) with high concentrations of DNFB (C(50)=70 microM). This interaction led to the inhibition of ryanodine binding and single channel activity. Modification of RyR with DNFB under the stimulatory conditions resulted in 3.6-fold and 6-fold increases in ryanodine-binding and Ca(2+)-binding affinities respectively. Modification with DNFB under the inhibitory conditions resulted in a decrease in the total ryanodine-binding sites. The exposure of the RyR single channel to DNFB under both inhibitory and stimulatory conditions led to the complete closure of the channel. However, when modified under the stimulatory conditions, but not under the inhibitory ones, the DNFB-modified closed channel could be re-activated by sub-micromolar concentrations of ryanodine, in the presence of nanomolar concentrations of Ca(2+). The DNFB-modified ryanodine-activated RyR channel showed fast 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 inhibition of ryanodine binding by modification with DNFB was affected by the presence of ATP. By using the photoreactive ATP analogue 3'-O-(4-benzoyl)benzoyl-[alpha-(32)P]ATP we found that DNFB modification had no effect on the ATP-binding site of RyR. The results are discussed with regard to the involvement of amino group residues in channel gating, ryanodine association/dissociation and occlusion, and the relationship between the open/closed state of the RyR and its capacity to bind ryanodine.
Hadad
N
N
Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Feng
W
W
Shoshan-Barmatz
V
V
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000588:Amines / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D004139:Dinitrofluorobenzene / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000187:drug effects; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D011487:Protein Conformation / Q000187:drug effects; D012430:Ruthenium Red / Q000494:pharmacology; D012433:Ryanodine / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D013329:Structure-Activity Relationship
D000588:Amines; D008051:Lipid Bilayers; D019837:Ryanodine Receptor Calcium Release Channel; D012430:Ruthenium Red; D012433:Ryanodine; D000255:Adenosine Triphosphate; D004139:Dinitrofluorobenzene; D002118:Calcium