Single-channel characterization of the rabbit recombinant RyR2 reveals a novel inactivation property of physiological concentrations of ATP.

The Journal of Membrane Biology
Richard StewartR Sitsapesan

Abstract

Ryanodine receptor 2 (RyR2) cDNA has been available for more than 15 years; however, due to the complex nature of ligand gating in this channel, many aspects of recombinant RyR2 function have been unresearched. We established a stable, inducible HEK 293 cell line expressing full-length rabbit RyR2 cDNA and assessed the single-channel properties of the recombinant RyR2, with particular reference to ligand regulation with Ca2+ as the permeant ion. We found that the single-channel conductances of recombinant RyR2 and RyR2 isolated from cardiac muscle are essentially identical, as is irreversible modification by ryanodine. Although it is known that RyR2 expressed in HEK 293 cells is not associated with FKBP12.6, we demonstrate that these channels do not exhibit any discernable disorganized gating characteristics or subconductance states. We also show that the gating of recombinant RyR2 is indistinguishable from that of channels isolated from cardiac muscle when activated by cytosolic Ca2+, caffeine or suramin. The mechanisms underlying ATP activation are also similar; however, the experiments highlighted a novel effect of ATP at physiologically relevant concentrations of 5-10 mM. With Ca2+ as permeant ion, 5-10 mM ATP consistently ...Continue Reading

References

Jun 1, 1984·The Journal of General Physiology·A M Garcia, C Miller
Jun 1, 1994·The American Journal of Physiology·R CoronadoD M Vaughan
Feb 1, 1994·The Journal of Membrane Biology·R Sitsapesan, A J Williams
Aug 23, 1996·The Journal of Biological Chemistry·A P TimermanS Fleischer
Sep 1, 1996·The Journal of Membrane Biology·R Sitsapesan, A J Williams
Jul 1, 1997·Biophysical Journal·J A CopelloS Fleischer
Jun 9, 2001·Circulation Research·S O MarxA R Marks
Jun 8, 2002·Biochemical and Biophysical Research Communications·Duncan J WestAlan J Williams

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Citations

Apr 25, 2012·Naunyn-Schmiedeberg's Archives of Pharmacology·Aimo KanntHeinz Gögelein
Jan 29, 2011·The Journal of Membrane Biology·Simon CarterRebecca Sitsapesan
Jul 18, 2012·The Journal of General Physiology·Saptarshi MukherjeeAlan J Williams
Dec 24, 2010·Cardiovascular Therapeutics·Elisa VenturiRebecca Sitsapesan
Feb 25, 2014·Biophysical Journal·Elisa VenturiRebecca Sitsapesan
Jun 5, 2015·The Journal of Biological Chemistry·Jason WoodierSamantha J Pitt

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