Identification and Functional Reconstitution of the Type 2 Inositol 1,4,5-Trisphosphate Receptor from Ventricular Cardiac Myocytes*
- From the Department of Physiology, §Cardiovascular Institute and ‖Program in Molecular Biology, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois 60153 and the‡Department of Physiology, Instituto Nacional de Cardiologia “Ignacio Chavez,” Mexico City 14080, Mexico
Abstract
The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+ release channel that mediates the rise in cytoplasmic calcium in response to receptor-activated production of InsP3. The InsP3R-mediated signaling pathway appears to be ubiquitous and is involved in many cellular processes including cell division, smooth muscle contraction, and neuronal signaling. Different regions of the heart also express InsP3 receptors. We report here that acutely dissociated ventricular myocytes from ferret and rat hearts express significant levels of InsP3R as indicated by immunoblotting with a receptor consensus antibody. InsP3binding experiments (K D = 23.6 nm andB max = 0.46 pmol/mg) suggest the myocytes contain the high affinity type 2 InsP3 receptor. Exhaustive mRNA screening by polymerase chain reaction, RNase protection, and subsequent DNA sequencing positively identify the InsP3R as type 2. The type 2 receptor from ferret heart was then incorporated into planar lipid bilayers and formed Ca2+-selective, InsP3-activated, heparin-blocked ion channels. We conclude that the predominant InsP3 receptor isoform expressed in cardiac myocytes is type 2 and that it forms a functional InsP3-gated Ca2+ channel when reconstituted in planar lipid bilayers.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ Supported by the American Heart Association and National Institutes of Health R29 AR41197. American Heart Association Established Investigator.
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↵** Supported by the American Heart Association and National Institutes of Health Grant R29 MH53367. To whom correspondence should be addressed: Dept. of Physiology, Loyola University Chicago, 2160 South First Ave., Maywood, IL 60153.
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↵1 The abbreviations used are: InsP3,d-myoinositol 1,4,5-trisphosphate; InsP3R,d-myoinositol 1,4,5-trisphosphate receptor; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; RyR, ryanodine receptor.
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↵2 J. Ramos-Franco, G. Mignery, and M. Fill, manuscript in preparation.
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- Received June 4, 1997.
