InsP3 Receptor Projects

Introduction

The inositol 1,4,5-trisphosphate receptor (InsP3R) is activated when a ligand binds to its receptor (a hormone in this diagram). The binding of the hormone stimulates the trimeric G-protein to activate an enzyme, phospholipase C (PLC), which, in turn degrades the membrane lipid PIP2 into InsP3 and diacylglycerol (DAG). InsP3 binds to its receptor, opens the channel, and allows calcium to flow from the lumen of the endoplasmic reticulum to the cytoplasm. We are interested in how the InsP3R works, as a molecule, as a channel complex, and as a component of a complex signaling pathway.

Structure of the InsP3R

Three-dimensional reconstruction of InsP3R type 1

A 3D structure of the InsP3R can provide a foundation for understanding structure-function studies and for predicting regulatory sites. We chose to start with single particle reconstruction from cryo-EM images of purified receptors to avoid the artifacts seen in negative-stain images. Thousands of images of detergent-solubilized receptors frozen in random orientations in vitreous ice were obtained by cryo-EM, and combined together by single particle reconstruction to yield a 24 Å resolution structure. The structure is consistent with the predicted topology for the InsP3R, and predicts a possible pathway for calcium flow across the membrane.

Regulation of the InsP3R

The InsP3R can be regulated by a number of cellular factors, including calcium, ATP, and a variety of associated proteins. Many sites on the cytoplasmic side of the InsP3R have been identified as regulatory sites. Although the luminal side is much smaller, this region of the InsP3R also is important for regulation of channel activity.

Regulation from the cytoplasmic side of the InsP3R

Bezprozvanny, Watras, and Ehrlich. Nature (1991); Kaftan, Ehrlich, and Watras J Gen Physiol (1997)

One of the many compounds which regulate the InsP3R from the cytoplasmic side is calcium. Free calcium exerts both stimulatory and inhibitory effects on the InsP3R type I. This regulation of the InsP3R by cytosolic calcium is highly concentration dependent where small elevations in cytosolic calcium concentrations (<300nM) increase channel open probability, however at higher free calcium concentrations, inhibition of InsP3R channel activity occurs. The cytoplasmic calcium-sensitivity of single InsP3R type II and type III channels is quite different from that described for the InsP3R type I where the calcium-dependency is sigmoidal with substantial channel activity occurring at calcium concentrations in the micromolar range. The combination of such isoform-specific properties with the sub-cellular distribution of the different isoforms may contribute to cellular mechanisms for coordinating calcium signals within the cell.

Neuronal calcium sensor 1 (NCS-1) is a Ca2+ storage protein found in most cells (the name reflects the location where it was first found in mammalian cells). In the presence of NCS-1, but with no added Ca2+, the InsP3-induced channel open probability doubled. In the presence of NCS-1 and Ca2+ the open probability increased 5-fold. This effect of NCS-1 on single channel currents is also seen in intact cells, where the expression of NCS-1 increases the magnitude of the Ca2+ increase after stimulation by extracellular agonists.

Regulation from the luminal side of the InsP3R

Chromogranins A and B (CGA and CGB) are Ca2+ storage proteins found in secretory granules of neuroendocrine cells. They associate with InsP3R located in the granule membrane (CGA and CGB) and ER membrane (CGB). In the presence of luminal CGA or CGB and a luminal pH of 5.5 the InsP3-induced channel open probability increased by 16-fold and the mean open time by 25-fold. Changing the luminal pH to 7.5 caused the channel activity to return to control levels with CGA, but not with CGB. Thus, CGB may participate in the regulation of InsP3-mediated calcium in the ER where the intraluminal pH is 7.5, as well as in the secretory granules where both CGA and CGB are found. This difference in the ability of CGA and CGB to regulate the InsP3R could influence the pattern of InsP3-mediated Ca2+ release as CGA/CGB ratios vary in regions of the cell.

Future Directions

Chromogranin A and B bind to the InsP3R Effect of chromogranin on InsP3-gated channels

The long term goals of the laboratory are to understand the molecular machinery of the receptor, as a purified protein and with its regulatory partners. The challenge will be to determine who the critical partners are, the proximity, specificity, and sensitivity within signaling microdomains, and the dynamics of these patterns which are used to generate complex intracellular signals.