title: Characterization of ryanodine receptor type 1 single channel activity using "on-nucleus" patch clamp.
abstract: In this study, we provide the first description of the biophysical and pharmacological properties of ryanodine receptor type 1 (RyR1) expressed in a native membrane using the on-nucleus configuration of the patch clamp technique. A stable cell line expressing rabbit RyR1 was established (HEK-RyR1) using the FLP-in 293 cell system. In contrast to untransfected cells, RyR1 expression was readily demonstrated by immunoblotting and immunocytochemistry in HEK-RyR1 cells. In addition, the RyR1 agonists 4-CMC and caffeine activated Ca(2+) release that was inhibited by high concentrations of ryanodine. On nucleus patch clamp was performed in nuclei prepared from HEK-RyR1 cells. Raising the [Ca(2+)] in the patch pipette resulted in the appearance of a large conductance cation channel with well resolved kinetics and the absence of prominent subconductance states. Current versus voltage relationships were ohmic and revealed a chord conductance of ∼750pS or 450pS in symmetrical 250mM KCl or CsCl, respectively. The channel activity was markedly enhanced by caffeine and exposure to ryanodine resulted in the appearance of a subconductance state with a conductance ∼40% of the full channel opening with a Po near unity. In total, these properties are entirely consistent with RyR1 channel activity. Exposure of RyR1 channels to cyclic ADP ribose (cADPr), nicotinic acid adenine dinucleotide phosphate (NAADP) or dantrolene did not alter the single channel activity stimulated by Ca(2+), and thus, it is unlikely these molecules directly modulate RyR1 channel activity. In summary, we describe an experimental platform to monitor the single channel properties of RyR channels. We envision that this system will be influential in characterizing disease-associated RyR mutations and the molecular determinants of RyR channel modulation.
authors: Department of Pharmacology and Physiology, University of Rochester, 601 Elmwood Ave, Rochester, NY 14642, United States.
authors: Larry E
authors: LE
authors: Wagner
authors: Department of Pharmacology and Physiology, University of Rochester, 601 Elmwood Ave, Rochester, NY 14642, United States.
authors: Linda A
authors: LA
authors: Groom
authors: Department of Pharmacology and Physiology, University of Rochester, 601 Elmwood Ave, Rochester, NY 14642, United States.
authors: Robert T
authors: RT
authors: Dirksen
authors: Department of Pharmacology and Physiology, University of Rochester, 601 Elmwood Ave, Rochester, NY 14642, United States. Electronic address: David_Yule@urmc.rochester.edu.
authors: David I
authors: DI
authors: Yule
mesh_terms: D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D002586:Cesium / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D018408:Patch-Clamp Techniques; D011189:Potassium Chloride / Q000494:pharmacology; D011817:Rabbits; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014466:Ultraviolet Rays
chemical_list: D002120:Calcium Channel Agonists; D002712:Chlorides; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002586:Cesium; D002110:Caffeine; D011189:Potassium Chloride; C028019:cesium chloride; D002118:Calcium
keywords: Inositol 1,4,5-trisphosphate receptor; Ryanodine receptor; Single channel measurement