Small Ankyrin 1 Organizes the Ca2+ Stores of the Sarcoplasmic Reticulum in Skeletal Muscle

Abstract

Excitation-contraction (EC) coupling in striated muscle requires the coordinated functions of the transverse (t-) tubules, sarcoplasmic reticulum (SR) and the sarcomeric contractile proteins. The transient rise in [Ca2+]i that activates contraction depends on Ca2+ release from the SR. Maintaining the structural and functional integrity of the SR is crucial to maintaining the efficiency of EC coupling in skeletal muscle. Small ankyrin 1 (sAnk1, Ank1.5), a small alternatively spliced product of the ANK1 gene, is an integral membrane protein of the network compartment of the SR (nSR). sAnk1 binds obscurin, a giant cytoskeletal protein that surrounds the sarcomere at the level of the M-band and Z-disc. Our earlier results show that reducing the expression of sAnk1 with siRNA decreases the protein level, but not the mRNA, of the SR Ca2+ pump, SERCA, and may lead to a decrease in SR [Ca2+] load. We used Fluo-5N, a low affinity Ca2+ indicator, which we specifically loaded into the SR lumen, as a reporter of SR Ca2+ stores in control and siRNA treated myofibers isolated from rat flexor digitorum brevis muscle. The intensity of Fluo-5N fluorescence and its distribution in the SR lumen were altered when sAnk1 expression was reduced, consistent with a loss of nSR. Further, fluorescence recovery after photobleaching (FRAP) experiments showed that even small changes in sAnk1 expression altered the interconnectivity of the SR. Functional measurements of SR Ca2+ dynamics also suggested an important role for sAnk1 in maintaining EC-coupling. Our results are consistent with the hypothesis that sAnk1 is essential for the integrity of the nSR compartment and its Ca2+ stores in skeletal myofibers.