The interaction of fluorescein isothiocyanate with the ryanodine receptor/Ca2+ release channel of sarcoplasmic reticulum.
Fluorescein 5'-isothiocyanate (FITC) markedly inhibited ryanodine binding to rabbit skeletal muscle junctional sarcoplasmic reticulum. Half-maximal inhibition was obtained with about 20 microM and complete inhibition by 80 microM FITC. Inhibition was enhanced in the presence of high salt and at alkaline pH. The pH dependence of the inactivation of ryanodine binding suggested that FITC bound to a very reactive lysine epsilon-amino group with a pK alpha of about 7.5 or above. Kinetic analysis of the time course of inactivation of ryanodine binding by various concentrations of FITC suggested that the inactivation resulted from the modification of 2 or more amino acid residues. The inhibition of ryanodine binding by FITC was partially prevented by ATP, ADP, adenyl imidodiphosphate, and 3-O-(benzoyl)-benzoyl-ATP (Bz-ATP) but not by AMP. FITC modification of sarcoplasmic reticulum membranes inhibited the photoaffinity labeling by [alpha-32P]Bz-ATP of the 450-kDa protein and the ryanodine receptor with half-maximal inhibition at about 100 microM. The results suggest that the inhibition of ryanodine binding is mainly due to FITC modification of sites which are not involved in ATP binding. The FITC moiety is bound to the 160-, 96-, 76-, and 60-kDa ryanodine receptor tryptic fragments, and the FITC site is apparently on the 21.5-, 18-, and 17-kDa fragments which are formed by the V8 protease. Covalent modification by FITC dramatically affected the activity of single Ca2+ release channels incorporated into planar lipid bilayers. FITC caused a marked increased in channel open probability mainly to a noisy approximately 60% subconductance state. FITC-modified channels were no longer affected by ryanodine but were still abolished by Mg2+ and ruthenium red. We suggest that FITC modifies reactive lysine residues involved in channel activation by transmembrane charge movement in the t-tubular system.
Orr
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I
Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
Martin
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C
Ashley
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Shoshan-Barmatz
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D000227:Adenine Nucleotides / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008051:Lipid Bilayers; D009132:Muscles / Q000378:metabolism*; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000187:drug effects / Q000378:metabolism*; D012433:Ryanodine / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology
D000227:Adenine Nucleotides; D000345:Affinity Labels; D015220:Calcium Channels; D008051:Lipid Bilayers; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D012965:Sodium Chloride; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium