Abstract
A rapid assay for high affinity [3H]ryanodine binding to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-solubilized recombinant or native Ca2+ release channel proteins (ryanodine receptor, RyR) was devised. The key to preservation of high affinity [3H]ryanodine binding sites in the presence of increasing concentrations of CHAPS was the addition of phosphatidylcholine. This assay was used to characterize the equilibrium and kinetic properties of [3H]ryanodine binding to recombinant skeletal (RyR1) and cardiac (RyR2) Ca2+ release channels and the effects on binding of physiological modulators including ATP, Ca2+, and Mg2+. Both RyR1 and RyR2 had a single high affinity ryanodine binding site and low affinity sites, but [3H]ryanodine binding to recombinant RyR2 was not sensitive to ATP activation or Ca2+ inactivation and was less sensitive to Mg2+ inhibition. The [3H]ryanodine binding assay was used to estimate the expression level of recombinant RyR2 and RyR1, and to show that RyR2 can be expressed at very high levels in HEK-293 cells. Analysis of the properties of recombinant RyR2 and RyR1 by measurement of intracellular Fura-2 fluorescence revealed that the different properties of RyR2 and RyR1 are retained in the recombinant expressed proteins.
Highlights
A major goal in studies of ryanodine receptors is to understand structure/function relationships through expression and analysis of mutant forms of both skeletal muscle (RyR1) and cardiac (RyR2) isoforms
In order to carry out such studies, a series of useful assays for ryanodine receptors expressed in homologous cell culture have been developed, including planar bilayer assays (7, 8), Ca2ϩ photometry (9), and Ca2ϩ imaging (10)
We describe the expression of a rabbit cardiac ryanodine receptor (RyR2) clone in HEK-293 cells and we characterize some of its properties through use of the [3H]ryanodine binding assay and through Ca2ϩ photometry
Summary
Materials—Restriction endonucleases, and other DNA modifying enzymes were from Boehringer Mannheim, New England Biolabs, Promega, and Amersham Pharmacia Biotech; Fura-2 acetoxymethyl ester (AM) was from Molecular Probes; caffeine, AMPPCP, and protease inhibitors were from Sigma; [3H]Ryanodine was from NEN Life Science Products; unlabeled ryanodine and thapsigargin were from Calbiochem; CHAPS was from Bio-Rad; and phosphatidylcholine (PC) was from Avanti Polar Lipids. Measurement of [3H]Ryanodine Equilibrium Binding—Aliquots of 25 l of solubilized proteins were diluted into binding buffer B (a total of 0.25 ml) containing various concentrations of [3H]ryanodine, 0.5 M KCl, 1 mM ATP, 20 M free Ca2ϩ, 0.2 mM EGTA, 50 mM Hepes-Tris, pH 7.1, and the protease inhibitor mix. Measurement of Association and Dissociation Kinetics for [3H]Ryanodine Binding—The rate of association of [3H]ryanodine binding was measured by terminating the reaction by rapid filtration at times ranging from 30 s to 300 min after addition of solubilized proteins into binding buffer B with 10 nM [3H]ryanodine and incubation at 37 °C. Dissociation of [3H]ryanodine from the equilibrium complex was determined by equilibrating 10 nM [3H]ryanodine with solubilized proteins in buffer B for 2 h at 37 °C, followed by the addition of aliquots of the incubation mixture into a 50-fold excess of buffer B, without added ATP and Ca2ϩ or with 40 M unlabeled ryanodine. EC50 or IC50 values were obtained by fitting the curves with an equation for logistic dose response using Microcal Origin software
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
