SUBCONDUCTANCE STATES IN SINGLE-CHANNEL ACTIVITY OF SKELETAL-MUSCLE RYANODINE RECEPTORS AFTER REMOVAL OF FKBP12

FKBP12 was removed from ryanodine receptors (RyRs) by incubation of ra bbit skeletal muscle terminal cisternae membranes with rapamycin. The extent of FKBP12 removal was estimated by immunostaining Western blots of terminal cisternae proteins. Single FKBP12-depleted RyR channels, incorporated into planar lipid bilayers, were modulated by Ca2+, ATP, ryanodine, and ruthenium red in the cis chamber and opened frequently to the normal maximum conductance of similar to 230 pS and to substate levels of similar to 0.25, similar to 0.5, and similar to 0.75 of the maximum conductance. Substate activity was rarely seen in native RyRs . Ryanodine did not alter the number of conductance levels in FKBP12-d epleted channels but, at a membrane potential of +40 mV, reduced both the maximum and the substate conductances by similar to 50%. FKBP12-st ripped channels were activated by a 10-fold-lower [Ca2+] and inhibited by a 10-fold-higher [Ca2+], than RyRs from control-incubated and nati ve terminal cisternae vesicles. The open probability (P-o) of these FK BP12-deficient channels was greater than that of control channels at 0 .1 mu M and 1 mM cis Ca2+ but no different at 10 mu M cis Ca2+, where channels showed maximal Ca2+ activation. The similar to 0.25 substate was less sensitive than the maximum conductance to inhibition by Ca2and was the dominant level in channels inhibited by 1 mM cis Ca2+. The results show that FKBP12 coordinates the gating of channel activity i n control and ryanodine-modified RyRs.