Figure 4.

Kinetics of single RYR2 channel Ca²⁺ activation and deactivation. Local cytosolic free Ca²⁺ increases around a channel were generated by flash photolysis of caged Ca²⁺. Single RYR2 channels were repeatedly activated by photolytic Ca²⁺ stimuli (>5-s interval). Cytosolic solution contained Tris-HEPES (120/250 mM; pH 7.4), 100 nM of free Ca²⁺, 4 mM DM-nitrophen, 50 µM Rhod-2, and 2 mM glutathione. (A) Six single RYR2 channel sweeps when the lumen-to-cytosol current (3.58 pA) was carried by Ca²⁺ are shown at top. The cytosolic solution was as described above, and the luminal solution contained Ca-HEPES (50/250 mM; pH 7.4). Recordings were made at 0 mV. Open events are upward, and zero current level is marked at left. The top two recordings (gray) were made when the cytosolic bath solution was not being stirred. The lower four recordings were made during continuous, vigorous stirring of the cytosolic bath. This stirring efficiently exchanged the local photolyzed solution near the channel with unphotolyzed (basal free Ca²⁺) solution from the bath, generating transient Ca²⁺ stimuli (rise time, ∼100 µs; decay time constant, ∼15–19 ms). An ensemble trace generated from >40 single-channel sweeps (while stirring) is shown just below the single-channel recordings. Below the ensemble trace is the corresponding recording of the applied local stimulus (while stirring). (B) Like A, but the lumen-to-cytosol current (7.9 pA) was carried by Cs⁺. This channel was not the same as shown in A. The cytosolic solution was as described above, and the luminal solution contained 5 µM of added Ca²⁺ and 250 mM Cs-HEPES, pH 7.4. (C) Calcium activation kinetics. Four ensemble RYR2 activity traces from different RYR2 channels with either Ca²⁺ (filled circles) or Cs⁺ (open circles) as charge carrier were averaged and then fit (lines) by a single-exponential function. (D) Calcium deactivation kinetics. The decay phase and exponential fits of the same averaged ensemble traces described in C are shown. Only a small subset of the points fit is actually shown. (E) Time constants of Ca²⁺ activation and deactivation in response to the applied transient Ca²⁺ stimulus. Ca-On and Cs-On refer to the activation time constants when Ca²⁺ or Cs⁺ was charge carrier, respectively. Ca-Off and Cs-Off refer to the inhibition time constants. Ca-Stim and Cs-Stim refer to Ca²⁺ stimulus decay time constant when Cs⁺ or Cs⁺ was charge carrier, respectively. The asterisk indicates a significant difference (P < 0.02).