Fig. 7.

Effects of Mg²⁺/ATP treatment at subactivating cytosolic [Ca²⁺]. a A representative experiment showing a single RyR2 channel gating in the presence of 50 μM cytosolic free Ca²⁺. Addition of 1 mM cytosolic EGTA to lower cytosolic [Ca²⁺] to <1 nM completely closes the channel. Subsequent addition of 5 mM Mg²⁺ and 1 mM ATP has no effect on the RyR2 channel. Cytosolic washout back to control solutions (50 μM free Ca²⁺) causes the channel to reopen, showing the reversible nature of the Mg²⁺/ATP treatment. Readdition of 1 mM cytosolic EGTA is able to close the channel again, showing no long-term modification of the channel by Mg²⁺/ATP. Arrows and dotted lines indicate closed and open channel levels, respectively. b Western blot analysis showing the phosphorylation of RyR2 at serine-2809 that occurs at nanomolar cytosolic [Ca²⁺] (<1 nM) for the indicated conditions. EGTA (10 mM) was present throughout all incubations. For both gels, cardiac HSR was incubated with 5 mM Mg²⁺ (lane 1), 5 mM Mg²⁺ + 1 mM ATP (lane 2) and 5 mM Mg²⁺ + 1 mM ATP + 1 U of PKA per microgram of protein (lane 3). All lanes were loaded at 30 μg total protein. Western blots were probed with antibodies specific for RyR2 dephosphorylated at serine-2809 (RyR2-2809deP, left gel) or for RyR2 phosphorylated at serine-2809 (RyR2-PS2809, right gel). Size markers are indicated in kilodaltons. The histogram shows the average percentage change in phosphorylation levels with respect to the control (5 mM Mg²⁺ only) by probing the blot with either RyR2-2809deP or RyR2-PS2809. Data are expressed as mean ± SD (n = 3)