Abstract
The ryanodine receptor type 1 (RyR1) and type 2 (RyR2), but not type 3 (RyR3), are efficiently activated by 4-chloro-m-cresol (4-CmC). We previously showed that a 173-amino acid segment of RyR1 (residues 4007-4180) is required for channel activation by 4-CmC (Fessenden, J. D., Perez, C. F., Goth, S., Pessah, I. N., and Allen, P. D. (2003) J. Biol. Chem. 278, 28727-28735). In the present study, we used site-directed mutagenesis to identify individual amino acid(s) within this region that mediate 4-CmC activation. In RyR1, substitution of 11 amino acids conserved between RyR1 and RyR2, but divergent in RyR3, with their RyR3 counterparts reduced 4-CmC sensitivity to the same degree as substitution of the entire 173-amino acid segment. Further analysis of various RyR1 mutants containing successively smaller numbers of these mutations identified 2 amino acid residues (Gln(4020) and Lys(4021)) that, when mutated to their RyR3 counterparts (Leu(3873) and Gln(3874)), abolished 4-CmC activation of RyR1. Mutation of either of these residues alone did not abolish 4-CmC sensitivity, although Q4020L partially reduced 4-CmC-induced Ca(2+) transients. In addition, mutation of the corresponding residues in RyR3 to their RyR1 counterparts (L3873Q/Q3874K) imparted 4-CmC sensitivity to RyR3. Recordings of single RyR1 channels indicated that 4-CmC applied to either the luminal or cytoplasmic side activated the channel with equal potency. Secondary structure modeling in the vicinity of the Gln(4020)-Lys(4021) dipeptide suggests that the region contains a surface-exposed region adjacent to a hydrophobic segment, indicating that both hydrophilic and hydrophobic regions of RyR1 are necessary for 4-CmC binding to the channel and/or to translate allosteric 4-CmC binding into channel activation.
Highlights
Striated muscle contraction is triggered by Ca2ϩ release from intracellular stores through the ryanodine receptor (RyR),2 a large homotetrameric channel protein embedded in the membrane of the sarcoplasmic reticulum (SR)
Our results demonstrate that the differential sensitivity to 4-CmC between ryanodine receptor type 1 (RyR1) and RyR3 can be attributed to 2 amino acid residues in RyR1, Gln4020 and Lys4021 and their RyR3 counterparts Leu3873 and Gln3874
To determine whether 4-CmC acts on the analogous region of RyR2, this segment was substituted into RyR3 to create chimera 1 rev RyR2 (Fig. 1A)
Summary
Construction of Mutated RyR1 and RyR3 cDNAs—cDNAs encoding the rabbit isoforms of RyR1, RyR2, and RyR3 have previously been cloned into pHSVprPUC enabling the generation of HSV amplicons required for transduction of 1B5 myotubes [11]. Quantification of Ca2ϩ Transients—The relative amount of Ca2ϩ released in response to each RyR agonist was estimated by calculating the average change in Fluo-4 fluorescence during application of the agonist This mean value was normalized to the resting fluorescence of the cell obtained 10 s prior to the addition of the agonist. The cis chamber was virtually encoding the RyRs. After 2 h at 37 °C in antibiotic-free medium, grounded, whereas the trans chamber was connected to the virus was removed and the myotubes were incubated for 2 days head stage input of an amplifier Calcium Imaging—RyR-expressing myotubes were chal- obtained by introducing rabbit skeletal muscle junctional SR lenged with successive additions of 80 mM KCl, 40 mM caffeine, vesicles to the cis chamber to induce fusion with the bilayer and 0.5 mM 4-CmC dissolved in imaging solution Secondary Structure Prediction—The secondary structure predictions were performed using the Chou-Fasman method for ␣-helical predictions [14], the Kyte-Doolittle method for hydropathy predictions [15], and the method of Emini et al [16] for surface probability prediction
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
