Kr. Eager et Af. Dulhunty, Cardiac ryanodine receptor activity is altered by oxidizing reagents in either the luminal or cytoplasmic solution, J MEMBR BIO, 167(3), 1999, pp. 205-214
The location of reactive cysteine residues on the ryanodine receptor (RyR)
calcium release channel was assessed from the changes in channel activity w
hen 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'-dithiodi
pyridine (4,4'-DTDP, 1 mM), as well as the hydrophilic thimerosal (1 mM), a
ctivated 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 ope
n time distribution from one (similar to 2 msec) to three (similar to 1 mse
c; similar to 7 msec; similar to 15 msec) in channels activated by trans 4,
4'-DTDP or cis or trans thimerosal. A longer component (similar to 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 cl
asses of cysteines are available to 3,4'-DTDP or thimerosal, SHa or SHa* ac
tivating 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.