Identification and functional reconstitution of the type 2 inositol 1,4,5-trisphosphate receptor from ventricular cardiac myocytes.
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. InsP3 binding experiments (KD = 23.6 nM and Bmax = 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.
Perez
P J
PJ
Department of Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois 60153, USA.
Ramos-Franco
J
J
Fill
M
M
Mignery
G A
GA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015220:Calcium Channels / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018076:DNA, Complementary; D005289:Ferrets; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism
D015220:Calcium Channels; D018076:DNA, Complementary; D053496:Inositol 1,4,5-Trisphosphate Receptors; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D015544:Inositol 1,4,5-Trisphosphate; D012260:Ribonucleases