Identification of a key determinant of ryanodine receptor type 1 required for activation by 4-chloro-m-cresol.
4-Chloro-m-cresol (4-CmC) is a potent and specific activator of the intracellular Ca2+ release channel, the ryanodine receptor (RyR). We have previously shown that RyR1 expressed in dyspedic 1B5 myotubes is activated by 4-CmC, whereas RyR3 is not (Fessenden, J. D., Wang, Y., Moore, R. A., Chen, S. R. W., Allen, P. D., and Pessah, I. N. (2000) Biophys. J. 79, 2509-2525). To identify region(s) on RyR1 that are responsible for mediating activation by 4-CmC, we expressed RyR1-RyR3 chimeric proteins in dyspedic 1B5 myotubes and then measured 4-CmC-induced increases in intracellular Ca2+. Substitution of the C-terminal third of RyR1 into RyR3 imparted 4-CmC sensitivity to the resulting chimera, thus suggesting that determinants required for activation by 4-CmC are located in this region. We subdivided the C-terminal third of RyR1 into smaller segments and identified two overlapping regions of RyR1 (amino acids 3769-4180 and 4007-4382) that each imparted 4-CmC sensitivity to RyR3. Substitution of the 173 amino acids of RyR1 common to these two chimeras (amino acids 4007-4180) also weakly restored 4-CmC sensitivity in the resulting chimera. To confirm these findings, we created a complementary set of chimeras containing RyR3 substitutions in RyR1. Substitution of the RyR3 C terminus into RyR1 disrupted 4-CmC sensitivity in the resulting chimera. In addition, substitution of the corresponding RyR3 sequence into positions 4007-4180 of RyR1 disrupted 4-CmC sensitivity. Taken together, these results suggest that essential determinants required for activation of RyR1 by 4-CmC reside within a 173-amino acid region between residues 4007 and 4180.
Fessenden
James D
JD
Department of Anesthesia Research, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA. fessenden@zeus.bwh.harvard.edu
Perez
Claudio F
CF
Goth
Sam
S
Pessah
Isaac N
IN
Allen
Paul D
PD
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D003408:Cresols / Q000494:pharmacology*; D015870:Gene Expression; D008970:Molecular Weight; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011189:Potassium Chloride / Q000494:pharmacology; D020033:Protein Isoforms / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D013329:Structure-Activity Relationship; D014162:Transfection; D014316:Tritium
D003408:Cresols; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D014316:Tritium; D012433:Ryanodine; C006984:chlorocresol; D002110:Caffeine; D011189:Potassium Chloride; D002118:Calcium