Fig. 3.

CPVT RyR2 mutations increase the sensitivity of single RyR2 channels to luminal Ca²⁺ activation. Single-channel activities of RyR2(wt) (A), N4104K (B), R4496C (C), and N4895D (D) were recorded in a symmetrical recording solution containing 250 mM KCl and 25 mM Hepes (pH 7.4). EGTA was added to either the cis or trans chamber to determine the orientation of the incorporated channel. The side of the channel to which an addition of EGTA inhibited the activity of the incorporated channel presumably corresponds to the cytoplasmic face. The Ca²⁺ concentration on both the cytoplasmic and the luminal face of the channel was adjusted to ≈45 nM. The luminal Ca²⁺ concentration was then increased to various levels by the addition of aliquots of CaCl₂ solution. The control current traces for RyR2(wt) or mutants are shown in a, whereas single-channel current traces at 300 μM luminal Ca²⁺ are depicted in b. The holding potential was -20 mV. Openings are downward. Po, arithmetic mean open time (To), and arithmetic mean closed time (Tc) are indicated on top. A short line to the right of each current trace indicates the baseline. (E) The relationships between Po and luminal Ca²⁺ concentrations of single RyR2(wt) (open triangle), N4104K (filled diamond), R4496C (filled circle), and N4895D (filled square) are shown. Data points shown are mean ± SEM from 21 RyR2(wt), 7 N4104K, 14 R4496C, and 10 N4895D single channels.