V. Serre et al., EFFECTS OF CYSTEINE MODIFICATION ON THE ACTIVITY OF THE CGMP-GATED CHANNEL FROM RETINAL RODS, The Journal of membrane biology, 146(2), 1995, pp. 145-162
The effect of sulfhydryl reagents on the activity of the cGMP-gated ch
annel from bovine retinal rods was studied by measurements of 8-Br-cGM
P-(cGMP)-induced calcium efflux from rod membrane vesicles and records
of 8-Br-cGMP-dependent sodium currents through channels incorporated
into planar lipid bilayers. N-ethylmaleimide and mersalyl (thiol block
ers) as well as diamide (dithiol-disulfide conversion agent) have a du
al effect on the channels activity: at low concentration, they increas
e the apparent affinity for cyclic nucleotide (''activation'') at the
same time inducing a loss of cooperativity for nucleotide binding; at
higher concentration, N-ethylmaleimide and diamide produce a reduction
of the amplitude and initial rate of the calcium release at saturatin
g nucleotide concentration, while mersalyl is shown to reduce the acti
vity of the channels in bilayer experiments (''inhibition''). Nitric o
xide precursors have no effect. The results suggest that blocking at l
east 1 of the 3 cytoplasmic cysteine residues situated close to the cC
MP-binding site in each channel subunit by N-ethylmaleimide, mersalyl,
or diamide (forming a dimer between 2 subunits) increases the affinit
y for the nucleotide. Inhibition is produced by blocking at least one
of the 2 other cytoplasmic sulfhydryl groups (N-ethylmaleimide, mersal
yl, oxidized glutathione) or the 2 others (diamide, intrasubunit bridg
e), and may concern a process of channel inactivation. The 3 cytoplasm
ic sulfhydryl groups are accessible when the channels are in the open
state, but not (or much less) accessible when the channels are in the
closed state.