Identification and Functional Reconstitution of the Type 2 Inositol 1,4,5-Trisphosphate Receptor from Ventricular Cardiac Myocytes

Abstract

The inositol 1,4,5-trisphosphate receptor (InsP 3 R) is an intracellular Ca 2؉ release channel that mediates the rise in cytoplasmic calcium in response to receptor-activated production of InsP 3 . The InsP 3 R-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 InsP 3 receptors. We report here that acutely dissociated ventricular myocytes from ferret and rat hearts express significant levels of InsP 3 R as indicated by immunoblotting with a receptor consensus antibody. InsP 3 binding experiments (K D ‫؍‬ 23.6 nM and B max ‫؍‬ 0.46 pmol/mg) suggest the myocytes contain the high affinity type 2 InsP 3 receptor. Exhaustive mRNA screening by polymerase chain reaction, RNase protection, and subsequent DNA sequencing positively identify the InsP 3 R as type 2. The type 2 receptor from ferret heart was then incorporated into planar lipid bilayers and formed Ca 2؉ -selective, InsP 3 -activated, heparin-blocked ion channels. We conclude that the predominant InsP 3 receptor isoform expressed in cardiac myocytes is type 2 and that it forms a functional InsP 3 -gated Ca 2؉ channel when reconstituted in planar lipid bilayers.Inositol 1,4,5-trisphosphate (InsP 3 ) 1 is a well known second messenger mediating the regulated release of intracellular calcium and is produced through the action of phospholipase C. Phospholipase C is activated in response to the stimulation of cell surface receptors coupled to heterotrimeric G-proteins and tyrosine kinases resulting in the hydrolysis of phosphatidylinositol 4,5-bisphosphate to liberate InsP 3 and diacylglycerol. InsP 3 is a readily diffusible compound that binds to specific (InsP 3 R) receptors localized to the endoplasmic reticulum and results in the release of calcium from intracellular stores (see reviews Refs.

Emerging roles of inositol 1,4,5-trisphosphate signaling in cardiac myocytes

“…For example, rat heart contains all three IP 3 R isoforms with the IP 3 R type 1 being the most abundant isoform (more than 60% of total IP 3 R mRNA). Ventricular myocytes, however, do not contain IP 3 R type 1 (less than 2% of total IP 3 R mRNA), but rather express the type 2 (85%) and 3 (14%) IP 3 R [57]. Thus, we have also indicated in Table 1 whether or not IP 3 R expression was confirmed in cardiac myocytes.…”

“…For example, RyR densities amounted to ∼3−4 pmol/mg protein in human ventricle [55,56] and to ∼5.5−7 pmol/mg protein in dog, mouse, rabbit and rat ventricle [56]. IP 3 R densities, on the other hand, were ∼0.09 pmol/mg protein in bovine ventricle [16], 0.66 pmol/mg protein in canine ventricle [32], 0.46 pmol/mg protein in ferret ventricle [57] and 0.35 pmol/mg protein in rat ventricle [58]. Thus, on average there appear to be ∼5−80 times less IP 3 Rs than RyRs in ventricular SR.…”

“…For example, IP 3 has been shown to directly release Ca 2+ from permeabilized/fractionated ventricular cells [41,140,141]. In addition, IP 3 Rs have been purified from ventricular myocytes, incorporated into lipid membranes and shown to open after addition of IP 3 [57]. Although published data have suggested that all three IP 3 R isoforms are expressed in ventricular myocytes, the majority of recent reports concur that type 2 IP 3 Rs constitute the predominant channel isoform [17,18,41,58].…”

Regulation of ryanodine receptor-dependent calcium signaling by polycystin-2

“…Both Pkd2 and RyR2 are expressed in mouse cardiomyocytes starting at embryonic day 9.5 (28,30). Although the InsP 3 R type 2 is present in the cardiomyocytes, its mRNA was shown to be 50 times lower than that of RyR2 in cardiomyocytes (31,32). To further explore the regulation of RyR2 function by PC2, we examined the frequency of spontaneous Ca 2ϩ oscillations of the Pkd2 ϩ/ϩ and Pkd2 Ϫ/Ϫ cardiomyocytes isolated at embryonic day 17.5.…”

Increased InsP 3 Rs in the junctional sarcoplasmic reticulum augment Ca 2+ transients and arrhythmias associated with cardiac hypertrophy

“…Mammals have 3 InsP 3 R isoforms (types 1-3) (8), with InsP 3 R2 being the main isoform in cardiomyocytes (9,10). Although Ca 2ϩ flux via these InsP 3 Rs is relatively small in comparison to the large Ca 2ϩ transients occurring during every heartbeat, recent data suggests that InsP 3 Rs have an important role in cardiac physiology.…”