Abstract
Calmodulin (CaM) binds to the ryanodine receptor/calcium release channel of skeletal muscle (RyR1), both in the absence and presence of Ca(2+), and regulates the activity of the channel activity by activating and inhibiting it, respectively. Using cryo-electron microscopy and three-dimensional reconstruction, we found that one apoCaM binds per RyR1 subunit along the sides of the cytoplasmic assembly of the receptor. This location is distinct from but close to the location found for Ca(2+)-CaM, providing a structural basis for efficient switching of CaM between these two positions with the oscillating intracellular Ca(2+) concentration that generates muscle relaxation/contraction cycles. The locations of apoCaM and Ca(2+)-CaM at a critical region for RYR1-dihydropyridine receptor interaction are suggestive of a direct role for CaM in the mechanism of excitation-contraction coupling.
Highlights
In skeletal muscle, the calcium release channel/ryanodine receptor (RyR1)1 functions as one of the main regulators of cytoplasmic Ca2ϩ concentration, the signal for muscle contraction, by allowing rapid translocation of Ca2ϩ from the sarcoplasmic reticulum to the cytoplasm [1,2,3]
Using cryo-electron microscopy and three-dimensional reconstruction, we found that one apoCaM binds per RyR1 subunit along the sides of the cytoplasmic assembly of the receptor
Some of the RyR1s are thought to contact directly and to be under the control of dihydropyridine receptors (DHPRs), voltage sensors situated on the cell membrane infoldings (T-tubules) that are juxtaposed to the sarcoplasmic reticulum
Summary
RyR1, skeletal muscle isoform of the ryanodine receptor; CaM, calmodulin; DHPR, dihydropyridine receptor; E-C, excitation-contraction; IpTxa, Imperatoxin A; PIPES, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. At pCa 4, Ca2ϩ-saturated CaM (Ca2ϩ-CaM) binds directly to the RyR1 with a stoichiometry of one mole of apoCaM per RyR1 subunit (i.e. four CaMs per tetrameric RyR1 complex) [11, 12] and partially inhibits channel activity [6, 13], but at pCa 7, Ca2ϩ-depleted CaM (apoCaM) becomes an activator (14 –17). In both cases CaM reduces the direct effect produced by Ca2ϩ alone acting on the RyR1. Part of this study has been published previously in abstract form [33]
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