Aldolase potentiates DIDS activation of the ryanodine receptor in rabbit skeletal sarcoplasmic reticulum.
DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate), an anion channel blocker, triggers Ca2+ release from skeletal muscle SR (sarcoplasmic reticulum). The present study characterized the effects of DIDS on rabbit skeletal single Ca2+-release channel/RyR1 (ryanodine receptor type 1) incorporated into a planar lipid bilayer. When junctional SR vesicles were used for channel incorporation (native RyR1), DIDS increased the mean P(o) (open probability) of RyR1 without affecting unitary conductance when Cs+ was used as the charge carrier. Lifetime analysis of single RyR1 activities showed that 10 microM DIDS induced reversible long-lived open events (P(o)=0.451+/-0.038) in the presence of 10 microM Ca2+, due mainly to a new third component for both open and closed time constants. However, when purified RyR1 was examined in the same condition, 10 microM DIDS became considerably less potent (P(o)=0.206+/-0.025), although the caffeine response was similar between native and purified RyR1. Hence we postulated that a DIDS-binding protein, essential for the DIDS sensitivity of RyR1, was lost during RyR1 purification. DIDS-affinity column chromatography of solubilized junctional SR, and MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analysis of the affinity-column-associated proteins, identified four major DIDS-binding proteins in the SR fraction. Among them, aldolase was the only protein that greatly potentiated DIDS sensitivity. The association between RyR1 and aldolase was further confirmed by co-immunoprecipitation and aldolase-affinity batch-column chromatography. Taken together, we conclude that aldolase is physically associated with RyR1 and could confer a considerable potentiation of the DIDS effect on RyR1.
Seo
In-Ra
IR
Department of Life Science, Gwangju Institute of Science and Technology, Gwangju, Korea.
Moh
Sang Hyun
SH
Lee
Eun Hui
EH
Meissner
Gerhard
G
Kim
Do Han
DH
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002846:Chromatography, Affinity; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D047468:Immunoprecipitation; D015640:Ion Channel Gating / Q000187:drug effects*; D009124:Muscle Proteins / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000302:isolation & purification / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism; D012680:Sensitivity and Specificity; D014316:Tritium
D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D014316:Tritium; D012433:Ryanodine; D005634:Fructose-Bisphosphate Aldolase; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium