Ca(2+) sparks operated by membrane depolarization require isoform 3 ryanodine receptor channels in skeletal muscle

Abstract

Stimuli are translated to intracellular calcium signals via opening of inositol trisphosphate receptor and ryanodine receptor (RyR) channels of the sarcoplasmic reticulum or endoplasmic reticulum. In cardiac and skeletal muscle of amphibians the stimulus is depolarization of the transverse tubular membrane, transduced by voltage sensors at tubular-sarcoplasmic reticulum junctions, and the unit signal is the Ca(2+) spark, caused by concerted opening of multiple RyR channels. Mammalian muscles instead lose postnatally the ability to produce sparks, and they also lose RyR3, an isoform abundant in spark-producing skeletal muscles. What does it take for cells to respond to membrane depolarization with Ca(2+) sparks? To answer this question we made skeletal muscles of adult mice expressing exogenous RyR3, demonstrated as immunoreactivity at triad junctions. These muscles showed abundant sparks upon depolarization. Sparks produced thusly were found to amplify the response to depolarization in a manner characteristic of Ca(2+)-induced Ca(2+) release processes. The amplification was particularly effective in responses to brief depolarizations, as in action potentials. We also induced expression of exogenous RyR1 or yellow fluorescent protein-tagged RyR1 in muscles of adult mice. In these, tag fluorescence was present at triad junctions. RyR1-transfected muscle lacked voltage-operated sparks. Therefore, the voltage-operated sparks phenotype is specific to the RyR3 isoform. Because RyR3 does not contact voltage sensors, their opening was probably activated by Ca(2+), secondarily to Ca(2+) release through junctional RyR1. Physiologically voltage-controlled Ca(2+) sparks thus require a voltage sensor, a master junctional RyR1 channel that provides trigger Ca(2+), and a slave parajunctional RyR3 cohort.

Fig. 1.

Markers of expression in adult muscle. (a and b) Images of fluorescence from a mouse transfected with endoplasmic reticulum–GFP cDNA, showing expression in FDB and interosseus muscles (a) at efficiency close to 100% (b). (c) Overlay of confocal images of fluorescence of YFP (red) and blue fluorescence of a FDB fiber transfected with YFP-tagged RyR1 and stained with nuclear-selective Hoechst 33342. Arrows mark paranuclear areas of high YFP content. (d) YFP fluorescence at high magnification showing distribution in dual rows. (e) FDB transfected with RyR3 immunostained with anti-RyR3 Ab. (f) Immunostained control FDB. (g and h) FDB transfected and stained and at higher magnification to show doublets of stained rows. These rows correspond to triad junctions, as clarified by the diagram. (i) Location of triads and channels. In the mouse and other mammals there are two transverse tubules per sarcomere, located at the ends of the I band, so that two triads from neighboring sarcomeres (marked by the bracket) are close to each other; in amphibians, birds, and fish, a single triad is located at the center of the I band. DHPR (red) face RyR1 (green) in the T–SR junction. In nonmammals, RyR3 (blue) are located in the parajunctional SR membrane. The presence of doublets in the stained image (d and h, bracket) indicates that in transfected cells the foreign RyRs migrate to triads.

Fig. 2.

Spontaneous events in fibers from transfected muscle. (a) Line scans in an intact fluo-4-loaded RyR3-transfected fiber featuring small sparks. (b) Intact fiber from another mouse showing large and more frequent sparks, sometimes propagating in space. (c) Variety of events in a RyR3-transfected cell voltage-clamped at −80 mV. Note large events near surface. (d) Line scans of fluo-4 fluorescence in two cells transfected with RyR1 and voltage-clamped. Note narrow Ca²⁺“embers.” Line scanning was perpendicular to the fiber axis.

Fig. 3.

Response to voltage clamp of WT and RyR1-transfected fibers. (a1–c1) Line scan images of F(x,t)/F₀(x) in a WT fiber held at −80 mV and subjected to pulses represented at top. (a2–c2) Corresponding images in a YFP-RyR1-transfected cell. Black corresponds to F/F₀ = 0, and yellow corresponds to 2.5 (a and b) or 4 (c). (a3–c3) F/F₀ for WT (black) or RyR1 (red). (a4–c4) Ca²⁺ release flux derived from the image-averaged fluorescence using removal parameters fitted to the WT records.

Fig. 4.

Sparks activated by action potentials in RyR3-transfected muscle. (a) Line scan of a fiber from transfected FDB. The response to 0.5-ms field pulses (b) was composed of sparks. (c–f) Profiles of F/F₀ at arrows. Large sparks occurring spontaneously near the surface (lower edge of image and profile f) were unaffected by V_m.

Fig. 5.

Response to voltage clamp of RyR3-transfected fibers. (a and b) F(x,t)/F₀(x) in scans along line AA of fiber in Inset showing responses to V_m. In the segment marked by the white bracket the response consisted largely of sparks. In the rest of the image (cyan bracket in Inset and a) the response was eventless, like that of WT muscle (Fig. 3a1). White and cyan traces plot F/F₀ averaged over x in the segments marked by color-matching brackets. (c and d) Responses to higher V_m in a second transfected cell. (e and f) Ca²⁺ release flux derived from the color-matched F/F₀ traces in a and b. (g) Release flux derived from traces in c (solid) or d (dashed; note scale and horizontal shift for visibility in records at 10 mV). Unlike the WT (Fig. 3 b1 and c1), these responses show an early peak of fluorescence. Correspondingly, the peak in flux is more marked. Spatial scale is the same everywhere. Green in the color palette corresponds to F/F₀ = 1, and yellow corresponds to 2.5 (a and b) or 3 (c and d).

Fig. 6.

Mechanisms of activation. (a) Ratio of peak over steady release flux vs. pulse voltage (V_m) in WT mouse (black, averages ± SEM, n = 4 fibers), RyR3-transfected fibers (red, n = 11 fibers and 8 mice), and YFP-RyR1 or RyR1 (pink, n = 6 fibers and 6 mice) compared with data from ref. for rat EDL (blue) and frog semitendinosus (green). Note the similarity of frog and RyR3 mouse data. (b) A mechanism for V_m-operated sparks: T membrane depolarization is sensed by DHPRs (red), which activate underlying RyR1 by DICR. Ca²⁺ released through RyR1 then activates nearby RyR3, a CICR mechanism that may engulf a small cluster, to produce sparks. (c and d) The V_m dependence of amplification by CICR. Circles, junctional RyR1 operated by V_m; squares, RyR3 in parajunctional positions, assumed to open if local [Ca²⁺] rises above “threshold.” At low V_m (c) only one RyR1 is activated (red). Curves are profiles of cytosolic [Ca²⁺]; a cluster of RyR3 is activated by CICR (red). At higher V_m (d) two RyR1 channels are activated, but the number of open RyR3 channels (activated by the “sum” [Ca²⁺] profile) increases >2-fold. The diagram was modified from ref. .