Role of Neurogranin in the regulation of calcium binding to Calmodulin

Role of Neurogranin in the regulation of calcium binding to Calmodulin Anuja Chandrasekar, B.S Advisor: M. Neal Waxham, Ph.D The overall goal of my project was to gain a quantitative understanding of how the interaction between two proteins neurogranin (RC3) and calmodulin (CaM) alters a fundamental property of CaM. CaM, has been extensively studied for more than four decades due to its seminal role in almost all biological functions as a calcium signal transducer. Calcium signals in cardiac and neuronal cells are exquisitely precise and enable activation of some processes while down-regulating others. CaM, with its four calcium binding sites, serves as a central component of calcium signaling in these cells. It is aided in this role as a regulatory hub that differentially activates targets in response to a calcium flux by proteins that alter its calcium binding properties. Neurogranin, also known as RC3, is a member of a family of small neuronal IQ (SNIQ) domain proteins that was originally thought to play a ‘capacitive’ role by sequestering CaM until a calcium influx of sufficient intensity arrived. However, based on earlier work in our lab on neurogranin, we believe that this protein plays a more nuanced role in neurons than simply acting as a CaM buffer. We believe that neurogranin is one of the proteins which, by altering the kinetics of calcium binding allow CaM to decode a variety of signals with fine precision. To quantify the interaction between CaM, neurogranin and calcium, I used biophysical techniques and computational simulations. From my results, I conclude that neurogranin finely regulates the proportion of calcium-saturated CaM and thereby directs CaM’s target specificity.

Evaluating statistical methods used to estimate the number of postsynaptic receptors.

Calcium levels in spines play a significant role in determining the sign and magnitude of synaptic plasticity. The magnitude of calcium influx into spines is highly dependent on influx through N-methyl D-aspartate (NMDA) receptors, and therefore depends on the number of postsynaptic NMDA receptors in each spine. We have calculated previously how the number of postsynaptic NMDA receptors determines the mean and variance of calcium transients in the postsynaptic density, and how this alters the shape of plasticity curves. However, the number of postsynaptic NMDA receptors in the postsynaptic density is not well known. Anatomical methods for estimating the number of NMDA receptors produce estimates that are very different than those produced by physiological techniques. The physiological techniques are based on the statistics of synaptic transmission and it is difficult to experimentally estimate their precision. In this paper we use stochastic simulations in order to test the validity of a physiological estimation technique based on failure analysis. We find that the method is likely to underestimate the number of postsynaptic NMDA receptors, explain the source of the error, and re-derive a more precise estimation technique. We also show that the original failure analysis as well as our improved formulas are not robust to small estimation errors in key parameters.

Rapid effects of 1alpha,25-dihydroxyvitamin D3 on osteoblasts: Studies of membrane transducers that affect calcium signals

1,25-dihydroxyvitamin D3 [1,25(OH)2D 3] exerts pleiotropic effects on osteoblasts via both long-term nuclear receptor-mediated and rapid membrane-initiated pathways during bone remodeling and mineral homeostasis. This study explored the membrane transducers that mediate rapid effects of 1,25(OH)2D3 on osteoblasts, including sphingomyelinase (SMase) and L-type voltage sensitive calcium channels (VSCCs). ^ It was previously demonstrated that 1,25(OH)2D3 stimulates transmembrane influx of Ca2+ through VSCCs in ROS 17/2.8 osteoblasts, however the molecular identity of 1,25(OH)2D 3-regulated VSCC has not been known. In this study, on the basis of in vitro tests of three unique ribozymes specifically cleaving a1C mRNA, I transfected ROS 17/2.8 cells with vectors coding recombinant ribozyme modified with U1 snRNA structure, and successfully selected stable clonal cells in which the expression of a1C was strikingly reduced. Ca2+ influx studies in these cells compared to control transfectants showed selective attenuation of depolarization- and 1,25(OH)2D3-regulated Ca2+ responses. These results allow us to conclude that the cardiac ( a1C ) subtype of the L-type VSCC is the major membrane transducer of Ca 2+ influx in osteoblasts. ^ I also demonstrated that 1,25(OH)2D3 induces a rapid hydrolysis of membrane sphingomyelin (SM) in ROS 17/2.8 cells, with the concomitant generation of ceramide, detectable at 15 minute, and maximal at 1 hour after addition. Sphingosine, sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPC), downstream products of SM hydrolysis, but not ceramide, elicit Ca 2+ release from intracellular stores. Considering ceramide, sphingosine, and SPP as second messengers modulating intracellular kinases or phosphatases, these findings implicate sphingolipid-signaling pathways in transducing rapid effects of 1,25(OH)2D3 on osteoblasts. In structure/function analyses of sphingolipid signaling, it was observed that psychosine elicits Ca2+ release from intracellular stores. This challenges the dogma that sphingosine phosphorylation permits mobilization of Ca2+ , because psychosine is a sphingosine analog galactosylated at 1-carbon, preventing phosphorylation at that site. Psychosine is the pathological metabolite found in patients with Krabbe's disease, suggesting that psychosine disrupts the physiological sphingolipid signaling by chronic release of Ca2+ from intracellular stores. ^ Slower SM turnover than Ca2+ influx through VSCCs in response to 1,25(OH)2D3 demonstrates ceramide does not mediate the 1,25(OH)2D3-induced Ca2+ signaling, a conclusion endorsed further by the failure of ceramide to induce Ca 2+ signaling. ^

Role of TRP Channels in Mediating the Calcium Signaling Response of Brain Endothelial Cells to Mechanical Stretch

Traumatic brain injury (TBI) often results in disruption of the blood brain barrier (BBB), which is an integral component to maintaining the central nervous system homeostasis. Recently cytosolic calcium levels ([Ca2+]i), observed to elevate following TBI, have been shown to influence endothelial barrier integrity. However, the mechanism by which TBI-induced calcium signaling alters the endothelial barrier remains unknown. In the present study, an in vitro BBB model was utilized to address this issue. Exposure of cells to biaxial mechanical stretch, in the range expected for TBI, resulted in a rapid cytosolic calcium increase. Modulation of intracellular and extracellular Ca2+ reservoirs indicated that Ca2+ influx is the major contributor for the [Ca2+]i elevation. Application of pharmacological inhibitors was used to identify the calcium-permeable channels involved in the stretch-induced Ca2+ influx. Antagonist of transient receptor potential (TRP) channel subfamilies, TRPC and TRPP, demonstrated a reduction of the stretch-induced Ca2+ influx. RNA silencing directed at individual TRP channel subtypes revealed that TRPC1 and TRPP2 largely mediate the stretch-induced Ca2+ response. In addition, we found that nitric oxide (NO) levels increased as a result of mechanical stretch, and that inhibition of TRPC1 and TRPP2 abolished the elevated NO synthesis. Further, as myosin light chain (MLC) phosphorylation and actin cytoskeleton rearrangement are correlated with endothelial barrier disruption, we investigated the effect mechanical stretch had on the myosin-actin cytoskeleton. We found that phosphorylated MLC was increased significantly by 10 minutes post-stretch, and that inhibition of TRP channel activity or NO synthesis both abolished this effect. In addition, actin stress fibers formation significantly increased 2 minutes post-stretch, and was abolished by treatment with TRP channel inhibitors. These results suggest that, in brain endothelial cells, TRPC1 and TRPP2 are activated by TBI-mechanical stress and initiate actin-myosin contraction, which may lead to disruption of the BBB.