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. 2001 Feb 13;98(5):2865–2870. doi: 10.1073/pnas.041608898

Figure 4.

Figure 4

Ryanodine-pretreated E4032A channels become responsive to RyR agonists. (A) In an E4032A-expressing 1B5 myotube pretreated with 500 μM ryanodine for 30 min, responses to 40 mM KCl, 40 mM caffeine and 0.5 mM 4-chloro-m-cresol are restored. (Bars = 0.05 340/380 units vs. 50 sec.) (B) The degree of restoration of E4032A activity by ryanodine is indicated. A small percentage of the total number of cells examined for changes in calcium respond to RyR agonists in dishes containing untreated E4032A-expresssing 1B5 cells (clear bar). Upon treatment with 500 μM ryanodine for 24 h, the percentage of total cells responding to each of the RyR agonists significantly increased (Black bars: *, P < 0.001). (Inset): E4032A-expressing 1B5 myotubes were incubated with increasing concentrations of ryanodine for 24 h. The percentage of the total number of cells examined that responded to 40 mM caffeine is plotted vs. the ryanodine concentration used in the preincubation. (C) Ryanodine restores skeletal-type excitation–contraction coupling of E4032A. Addition of 40 mM KCl (black bar) to an E4032A-expressing cell pretreated with 500 μM ryanodine for 24 h produced calcium transients in both the presence and absence of extracellular calcium, indicating a functional interaction between RyR and DHPR. (Bar = 0.05 340/380 units vs. 60 sec.) (D) E4032A activity can be restored by ryanodine in single-channel studies. Single-channel measurements of E4032A channels reconstituted in BLM were conducted as described in Materials and Methods. E4032A channels isolated from 1B5 cells pretreated for 24 h with 200 μM ryanodine are active at 7 μM calcium cis (upper trace). Of 15 channels reconstituted, all exhibited substate behavior approximating 3/4, 1/2, and 1/4 transitions whose frequency of occurrence were approximately the same. Of these reconstitutions, 50% of the channels exhibited frequent transitions to full open similar to wt. For the channel shown, the open probability for transitions from closed to 1/4 state (dashed line) and 1/4 state to fully open were 0.83 and 0.028, respectively. In E4032A channels pretreated with ryanodine, channel activity depended on the level of calcium in the cis chamber of the bilayer, since lowering the level of calcium to 3.4 nM (≈1 min before recording) fully inactivated the channel (lower trace).