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Quercetin as a fluorescent probe for the ryanodine receptor activity in Jurkat cells.

Baran I, Katona E, Ganea C.

Pflugers Arch. 2013 Mar 9.

Department of Biophysics, “Carol Davila” University of Medicine and Pharmacy, 8 Eroii Sanitari, 050474, Bucharest, Romania, [email protected].

 

Abstract

Because channels of intracellular organelles are not directly accessible to the patch-clamp technique, the activity (open probability) of intracellular ion channels in intact cells has so far eluded direct examination. Here, we present strong evidence that the ratio F380/F440 of the quercetin-specific cellular fluorescence emitted at 540 nm upon excitation at 380/440 nm reflects the open probability of an endoplasmic reticulum Ca2+ release channel, the ryanodine receptor (RyR), in both intact and permeabilized Jurkat cells. The time course of the Ca2+ release signal induced by high levels of quercetin in intact cells and that of F380/F440 were strongly correlated. The RyR specific inhibitor, ryanodine, the RyR type 3 and 1 but not type 2 specific inhibitor, dantrolene, as well as the non-specific RyR inhibitor, ruthenium red, depressed consistently the quercetin-induced Ca2+transient. Confocal microscopy confirmed that the dual fluorescent signal emitted by quercetin colocalizes with the endoplasmic reticulum, not the mitochondria. A novel regulatory mechanism was identified whereby RyR activity under physiological conditions is partially suppressed (hindered channel), whereas the channel becomes nearly fully activated after exposure to millimolar concentrations of bulk cytosolic Ca2+ and subsequent chelation of Ca2+ (rectified channel). Upon rectification, the dependence of F380/F440 on the cytosolic Ca2+ concentration was remarkably similar to that of the open probability of the RyR type 3, not 1 or 2, reported from bilayer experiments. So, quercetin appears to be a semi-specific fluorescentprobe for the activity of ryanodine receptors, which in our Jurkat (clone E6.1) cell preparations probably reports the type 3 RyR activity.

 

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Additional Information: 

We advance the idea that conventional spectrofluorimetry affords the possibility to measure directly and non-invasively the activity (open probability) of an intracellular ionic channel, the ryanodine receptor (RyR)/calcium release channel, and to characterize its regulation by various ligands in intact and permeabilized human leukemia Jurkat T cells. Central to this methodology is the finding that the flavonoid quercetin (3,3’,4’,5,7-pentahydroxyflavone; QC) can be used, at least in Jurkat cells, as an efficient fluorescent probe to distinguish with high sensitivity between the open and the closed conformation of the RyR channel.

The main findings supporting this idea are as follows:

1) the kinetics of the Ca2+ release signal and that of the fluorescence ratio Q = F380/F440 observed after extracellular addition of 50 mM quercetin were highly correlated; however, the Q signal evoked by quercetin was unaffected when the Ca2+ transient was abolished by exposure to ionomycin;

2) the RyR specific inhibitor, ryanodine, the RyR3 and RyR1 but not RyR2 specific inhibitor, dantrolene, as well as the non-specific RyR inhibitor, ruthenium red, depressed consistently the QC-induced Ca2+ transient;

3) both QC fluorescent species, detected by excitation at 380 nm and 440 nm, colocalized with the endoplasmic reticulum, not the mitochondrial compartment;

4) the F380/F440 ratio displayed a bell-shaped Ca2+ dependence, which is a highly distinctive characteristic of calcium release channels;

5) the bell-shaped Ca2+ dependence of the F380/F440 ratio was obtained in permeabilized cells where the IP3 level was virtually depleted, hence the IP3 receptors were inactive;

6) upon rectification, the dependence of the F380/F440 ratio on the cytosolic Ca2+ concentration was remarkably similar to that of the open probability of RyR3, not RyR1 or RyR2, observed in bilayer experiments;

7) at a fixed concentration of cytosolic Ca2+, dantrolene decreased substantially the F380/F440 ratio, irrespective of the concentration of quercetin.

In conclusion, we propose a simple, but robust and sensitive fluorimetric method to assess the functional and molecular properties of the RyR calcium channel in situ with the use of quercetin as a RyR fluorescent probe. The future development of this promising assay can bring significant contributions to our understanding of the RyR function in physiological and pathological conditions. This point is of considerable interest, having in view that to date, the exclusive means to assess directly RyR activity has been to record the ionic current transported by receptors incorporated into artificial lipid bilayers, under voltage-clamp conditions in a controlled environment.

 

Quercetin as a fluorescent probe for the ryanodine receptor activity in Jurkat cells