Multiple classes of sulfhydryls modulate the skeletal muscle Ca2+ release channel.
Two sulfhydryl reagents, N-ethylmaleimide (NEM), an alkylating agent, and diamide, an oxidizing agent, were examined for effects on the skeletal muscle Ca2+ release channel. NEM incubated with the channel for increasing periods of time displays three distinct phases in its functional effects on the channel reconstituted into planar lipid bilayers; first it inhibits, then it activates, and finally it again inhibits channel activity. NEM also shows a three-phase effect on the binding of [3H]ryanodine by first decreasing binding (phase 1), followed by a recovery of the binding (phase 2), and then a final phase of inhibition (phase 3). In contrast, diamide 1) activates the channel, 2) enhances [3H]ryanodine binding, 3) cross-links subunits within the Ca2+ release channel tetramer, and 4) protects against phase 1 inhibition by NEM. All diamide effects can be reversed by the reducing agent, dithiothreitol. Diamide induces intersubunit dimer formation of both the full-length 565-kDa subunit of the channel and the 400-kDa generated by endogenous calpain digestion, suggesting that the cross-link does not involve sulfhydryls within the N-terminal 170-kDa fragment of the protein. NEM under phase 1 conditions blocks the formation of the intersubunit cross-links by diamide. In addition, single channels activated by diamide are further activated by the addition of NEM. Diamide either cross-links phase 1 sulfhydryls or causes a conformational change in the Ca2+ release channel which leads to inaccessibility of phase 1 sulfhydryls to NEM alkylation. The data presented here lay the groundwork for mapping the location of one of the sites of subunit-subunit contact in the Ca2+ release channel tetramer and for identifying the functionally important sulfhydryls of this protein.
Aghdasi
B
B
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA.
Zhang
J Z
JZ
Wu
Y
Y
Reid
M B
MB
Hamilton
S L
SL
D000818:Animals; D015220:Calcium Channels / Q000187:drug effects* / Q000378:metabolism; D003958:Diamide / Q000494:pharmacology*; D005033:Ethylmaleimide / Q000494:pharmacology*; D008051:Lipid Bilayers / Q000378:metabolism; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism
D015220:Calcium Channels; D008051:Lipid Bilayers; D003958:Diamide; D012433:Ryanodine; D005033:Ethylmaleimide