Figure 3: CaV2.2/TN–XL fusion construct preserves function of channel.
From: Nanodomain Ca2+ of Ca2+ channels detected by a tethered genetically encoded Ca2+ sensor
(a) Exemplar whole-cell currents of HEK293 cells expressing 2 channels (grey) or 2/TN–XL channels (black), using 10 mM Ca2+ as charge carrier, and 10 mM EGTA intracellular Ca2+ buffering. (b), Whole-cell tail-activation curves of 2 (grey) and 2/TN–XL (black), obtained from records as in (a). Data shown as mean±s.e.m. with number of cells in parentheses. (c), Single-channel analysis of 2/TN–XL. Top, On-cell ramp-voltage waveform delivered every 5–15 s. Bottom, exemplar single-channel sweep shown at high-gain magnification (100–200 per patch). 90 mM Ba2+ as charge carrier; 2 kHz low-pass filtering. Leak-subtracted traces were averaged, yielding red I-V curve. Unitary current relation (convex grey curve) was fitted to the open-channel current level using GHK equation10. (d), Multiple exemplar single-channel sweeps from another patch, shown at lower-gain magnification to impart a sense of the overall reproducibility and excellent resolution of elementary events. Format as in c. (e), Single-channel PO–V relation (grey, with fit in black, average of n=4 patches), determined by dividing I-V curve (c, red trace) by unitary current relation (c, grey, GHK fit). PO–V curve was calibrated in voltage by aligning the single-channel activation curve with the whole-cell curve reproduced from (b) (open circles). From calibrated PO–V curve, the Po at 30 mV is 0.69. (f) Immunoblots of HEK293 cell lysates probed with anti-GFP antibody; (left) 2/TN–XL at ~320 kD; (middle) TN–XL-Ras at ~71 kD; (right) control (untransfected) cells for reference. (g) Lack of lower molecular-weight species in leftmost lane of (f) excludes proteolysis of sensor from channel.
