TY - JOUR
T1 - Cardiac ryanodine receptor activity is altered by oxidizing reagents in either the luminal or cytoplasmic solution
AU - Eager, K. R.
AU - Dulhunty, A. F.
PY - 1999
Y1 - 1999
N2 - The location of reactive cysteine residues on the ryanodine receptor (RyR) calcium release channel was assessed from the changes in channel activity when oxidizing or reducing reagents were added to the luminal or cytoplasmic solution. Single sheep cardiac RyRs were incorporated into lipid bilayers with 10-7 M cytoplasmic Ca2+. The thiol specific-lipophilic- 4,4'-dithiodipyridine (4,4'-DTDP, 1 mM), as well as the hydrophilic thimerosal (1 mM), activated and then inhibited RyRs from either the cis (cytoplasmic) or trans (luminal) solutions. Activation was associated with an increase in the (a) mean channel open time and (b) number of exponential components in the open time distribution from one (~2 msec) to three (-1 msec; -7 msec; ~15 msec) in channels activated by trans 4,4'-DTDP or cis or trans thimerosal. A longer component (~75 msec) appeared with cis 4,4'- DTDP. Activation by either oxidant was reversed by the thiol reducing agent, dithiothreitol. The results suggest that three classes of cysteines are available to 4,4'-DTDP or thimerosal, SHa or SHa* activating the channel and SHi closing the channel. SHa is either distributed over luminal and cytoplasmic RyR domains, or is located within the channel pore. SHi is also located within the transmembrane domain. SHa* is located on the cytoplasmic domain of the protein.
AB - The location of reactive cysteine residues on the ryanodine receptor (RyR) calcium release channel was assessed from the changes in channel activity when oxidizing or reducing reagents were added to the luminal or cytoplasmic solution. Single sheep cardiac RyRs were incorporated into lipid bilayers with 10-7 M cytoplasmic Ca2+. The thiol specific-lipophilic- 4,4'-dithiodipyridine (4,4'-DTDP, 1 mM), as well as the hydrophilic thimerosal (1 mM), activated and then inhibited RyRs from either the cis (cytoplasmic) or trans (luminal) solutions. Activation was associated with an increase in the (a) mean channel open time and (b) number of exponential components in the open time distribution from one (~2 msec) to three (-1 msec; -7 msec; ~15 msec) in channels activated by trans 4,4'-DTDP or cis or trans thimerosal. A longer component (~75 msec) appeared with cis 4,4'- DTDP. Activation by either oxidant was reversed by the thiol reducing agent, dithiothreitol. The results suggest that three classes of cysteines are available to 4,4'-DTDP or thimerosal, SHa or SHa* activating the channel and SHi closing the channel. SHa is either distributed over luminal and cytoplasmic RyR domains, or is located within the channel pore. SHi is also located within the transmembrane domain. SHa* is located on the cytoplasmic domain of the protein.
KW - Luminal oxidation
KW - Reactive disulfides
KW - Ryanodine receptor
KW - Sarcoplasmic reticulum
KW - Sulfhydryl oxidation
UR - http://www.scopus.com/inward/record.url?scp=0033067450&partnerID=8YFLogxK
U2 - 10.1007/s002329900484
DO - 10.1007/s002329900484
M3 - Article
SN - 0022-2631
VL - 167
SP - 205
EP - 214
JO - Journal of Membrane Biology
JF - Journal of Membrane Biology
IS - 3
ER -