Figure 5.

Monitoring global and microdomain changes in SR Ca²⁺ with globally expressed and targeted G-CatchER⁺

(A) Representative plasmid construction and expression of globally expressed G-CatchER⁺ and targeted G-CatchER⁺-JP45 in the skeletal muscle SR. (Left panel) Global expression of G-CatchER⁺. G-CatchER⁺ contains the calreticulin signal peptide at the N terminus and the KDEL ER/SR retention sequence at the C-terminus. Resulting expression of the sensor follows a uniform distribution pattern throughout the SR monitoring global Ca²⁺. (Right panel) RyR1 microdomain targeted G-CatchER⁺ with JP45. G-CatchER⁺ resides at the C-terminus of JP45. Resulting expression of the sensor positions it near the lumenal opening of RyR1 in proximity to CASQ1 polymers that establish a large proximate Ca²⁺ pool in the TC necessary for E-C coupling.

(B) Wild-type FDB fibers were transfected with G-CatchER⁺ and G-CatchER⁺-JP45. Images reveal differential expression patterns for the targeted and un-targeted probe. Intensity changes from G-CatchER⁺ and G-CatchER⁺-JP45 fibers plotted against fiber length. Arrows correspond to arrows on G-CatchER⁺-JP45. A clear distinction is seen in the localization of G-CatchER⁺ and G-CatchER⁺-JP45.

(C) Ca²⁺ transients in FDB fibers were recorded upon stimulation with a 100-Hz electrical pulse for 300 ms. Black trace is fluorescence intensity monitored with G-CatchER⁺ expressed globally, and the red trace is G-CatchER⁺-JP45 located at the lumenal side of the junctional face membrane of the terminal cisternae.

(D) HILO imaging in C2C12 myotubule cells for G-CatchER⁺ (black, n = 6) and G-CatchER⁺-JP45 (red, n = 3) in response to 500-μM 4-cmc with representative cells for imaging. Scale bars, 10 μm in panel (B) and 20 μm in panel (D).

See also Figure S5.