Effect of the H29D mutation on cytosolic Ca²⁺ activation of single RyR2 channels.

<p>Single channel activities of RyR2 WT (A) and H29D (B) were recorded in a symmetrical recording solution containing 250 mM KCl and 25 mM Hepes (pH 7.4). EGTA was added to either the <i>cis</i> or <i>trans</i> 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 cytosolic Ca²⁺ concentration was then increased to various levels by an addition of aliquots of CaCl₂ solution. Recording potential was -20mV. Openings are downward, and baselines are indicated (short bars). Open probability (Po), mean open time (To), and mean closed time (Tc) are shown. The relationships between Po and cytosolic Ca²⁺ concentrations (pCa) of single RyR2 WT (filled circles) and H29D mutant (open circles) channels are shown in panel C. Data points shown are mean ± SEM from 7 RyR2 WT and 5 H29D single channels.