Measurements of Triadic Calcium in Differentiated Muscle Fibers using a Gcamp Probe Targeted to the Junctional Sr Membrane

Abstract

With the aim of gaining further insights into sarcoplasmic reticulum (SR) Ca2+ release function in skeletal muscle, we used a domain consisting of the 306 residues (T306) of the N-terminal end of triadin to target the Ca2+-sensitive probe GCaMP6f to the junctional SR membrane of muscle fibers. Muscles from mouse were electroporated with plasmid constructs encoding GCaMP6f or T306-GCaMP6f. Muscle fibers were isolated 1-2 weeks later and voltage-activated fluorescence changes were studied with line-scan confocal microscopy under voltage-clamp control. GCaMP6f fluorescence was homogenous throughout the transfected fibers and experienced large changes upon membrane depolarization in the absence of exogenous Ca2+ buffer in the intracellular solution (e.g. mean relative change of ∼20 in response to a 0.5 s-long pulse from −80 to −15 mV). When compared with simultaneously measured rhod-2 Ca2+ transients, GCaMP6f transients showed obvious signs of kinetic delays with, for instance, the time constant of decay after the end of short depolarizing pulses being 4-5 times that of the rhod-2 transients. In contrast, T306-GCaMP6f expression was localized to the vicinity of one or of a few nuclei. Within these spots, fluorescence yielded a transverse banded pattern consistent with triadic localization. Upon membrane depolarization, T306-GCaMP6f generated fluorescence transients of faster kinetics and much lower relative amplitude than T306-free GCaMP6f (e.g. mean relative change of 3.2 in response to a 0.5 s-long pulse to −15 mV), suggesting reduced affinity and faster kinetics of the SR membrane-bound probe as compared to the free probe. In several fibers, voltage-induced fluorescence changes at different positions of the scanned line showed time-course discrepancies which may reflect distinct behaviors of ryanodine receptor activation. Supported by CNRS, INSERM, Université Lyon 1 and AFM-Téléthon MyoNeurAlp 2.3.1.3.