Involvement of lysine residues in the gating of the ryanodine receptor/Ca2+-release channel of skeletal muscle sarcoplasmic reticulum.
In this study, the modification of skeletal muscle ryanodine receptor (RyR)/Ca2+-release channel with 7-chloro-4-nitrobenzo-2-oxa-1,3,-diazole (Nbd-Cl) demonstrates that lysyl residues are involved in the channel gating. Nbd-Cl was found to have a dual effect: stimulation and inhibition of ryanodine binding and single channel activities. Nbd-Cl, in a time-dependent manner, first stimulated and subsequently inhibited ryanodine binding to both membrane-bound and purified RyR. Incubation of sacroplasmic reticulum membranes with Nbd-Cl for 5-20 s resulted in enhanced ryanodine-binding activity by 2-4-fold due, to an increased binding affinity by about tenfold, with no effect on the total binding sites (Bmax). However, under prolonged incubation (5-20 min), Nbd-Cl strongly inhibited ryanodine binding by decreasing the Bmax with no effect on the binding affinity. Similar effects of stimulation and inhibition by Nbd-Cl were obtained with single channel activity of RyR reconstituted into planar lipid bilayer. Nbd-Cl initially (within a few seconds) activated the channel to a highly open state, then (within a few minutes) inactivated it to the completely closed state. Nbd-Cl-modified protein, as assayed by ryanodine binding or single channel activities, was stable against thiolysis by dithiothreitol, suggesting Nbd-Cl modification of lysyl residues. Evidence from absorption and fluorescence excitation and emission spectra also demonstrated that lysyl residues in RyR were modified by Nbd-Cl. Spectrophotometric data were used to estimate a ratio of up to 1 mol Nbd bound/mol RyR (tetramer) and up to 4 mol Nbd bound per mol RyR (tetramer) for Nbd-Cl stimulated and inhibited RyR activities, respectively. The results clearly indicate the involvement of two classes of lysyl residues in RyR activity. Modification by Nbd-Cl of the fast-reacting group led to stimulation of ryanodine binding and single channel activities, while modification of the slow-reacting group resulted in inhibition of these activities. Thus, the involvement of lysine residues in the gating of the RyR channel is proposed.
Feng
W
W
Department of Life Sciences Ben Gurion University of the Negev, Beer Sheva, Israel.
Shoshan-Barmatz
V
V
D009327:4-Chloro-7-nitrobenzofurazan / Q000737:chemistry; D000818:Animals; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D015640:Ion Channel Gating; D008239:Lysine / Q000378:metabolism*; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D015220:Calcium Channels; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D009327:4-Chloro-7-nitrobenzofurazan; D008239:Lysine