Murali Prakriya, PhD Assistant Professor Molecular Pharmacology and Biological Chemistry Intracellular Calcium Signaling Curricula: Cell Biology Neurobiology Pharmacology and Toxicology E-mail:   m-prakriya@northwestern.edu

Research in our laboratory is focused on the molecular and cellular mechanisms of intracellular calcium (Ca²⁺) signaling. Ca²⁺ is one of the most ubiquitous intracellular signaling messengers, mediating many essential functions including gene expression, chemotaxis, and neurotransmitter release. Cellular Ca²⁺ signals generally arise from the opening of Ca²⁺ permeable ion channels, a diverse family of membrane proteins. We are studying Ca²⁺ signals arising from the opening of a Ca²⁺ channel sub-type known as the store-operated Ca²⁺ channel (SOC). SOCs are found in the plasma membranes of virtually all mammalian cells and are activated through a decrease in the calcium concentration ([Ca²⁺]) in the endoplasmic reticulum (ER), a vast membranous network within the cell that serves as a reservoir for stored calcium. SOC activity is stimulated by a variety of signals such as hormones, neurotransmitters, and growth factors whose binding to receptors generates IP3 to cause ER Ca²⁺ store depletion.

The best-studied SOC is a sub-type known as the Ca²⁺ release activated Ca²⁺ (CRAC) channel. CRAC channels are widely expressed in immune cells and generate Ca²⁺ signals important for gene expression, proliferation, and the secretion of inflammatory mediators. Loss of CRAC channel function due to mutations in CRAC channel genes leads to a devastating immunodeficiency syndrome in humans. A major effort in our lab is to understand the molecular mechanisms of CRAC channel function: how does depletion of Ca²⁺ in the ER trigger the opening of CRAC channels in the plasma membrane? What are the key molecular and structural features of CRAC channel proteins? How are CRAC channels regulated? Progress in understanding these issues has occurred rapidly with the discoveries of STIM1 (the ER Ca²⁺ sensor) and Orai1 (the CRAC channel pore subunit). We have learnt that STIM1 redistributes from diffuse locations throughout the ER to peripheral sub-regions where it accumulates in discrete puncta at junctional ER sites in close proximity to the plasma membrane. CRAC channels gather simultaneously at the same sites and interact physically with STIM1, enabling them to open. This type of activation process, where the stimulus brings the sensor (STIM1) and the channel together in opposite membranes is unprecedented among ion channels. We are studying the molecular and cellular events of this process by patch-clamp electrophysiology and various live-cell imaging techniques including fluorescence resonance energy transfer (FRET) microscopy, total internal reflection (TIRF) microscopy, and confocal imaging.

Despite the fact that SOCs are found in practically all cells, their properties and functions outside the immune system remain largely unexplored.  In order to fill this gap, we have begun investigation of SOC properties and their functions in two major organ systems: in the brain and the lung.

Store-operated Ca²⁺ channels in neural stem cells. 
The development of the nervous system involves extensive proliferation of cells in the neuroepithelium.  In this process, uncommitted neural stem cells (NSCs) divide furiously to generate committed progenitors, which in turn divide, migrate, and ultimately differentiate into billions of neurons. As these events unfold, rhythmic bursts of intracellular Ca²⁺ signals in the NSCs are crucial for influencing their ultimate developmental fates, encoding information in a kind of biological Morse code. Yet, the mechanisms by which Ca²⁺ signals are generated in the NSCs remains mysterious. Classical voltage-gated Ca²⁺ entry channels, which emerge later in development, are absent in the NSCs raising an interesting question: what pathways do these cells employ to generate Ca²⁺ signals? Our recent findings provide an answer. We have discovered that NSCs express SOCs. Moreover, robust Ca²⁺ signals arise from their activation, indicating that SOCs are a major route of Ca²⁺ entry in NSCs. Intriguingly, the opening of SOCs in these cells powerfully activates a program of Ca²⁺-dependent gene expression mediated by the transcription factor, NFAT. The ability of SOCs to turn on gene expression in NSCs portends a multitude of ways in which this route of Ca²⁺ entry could influence early brain development.  We are using numerous approaches including knock-out and transgenic mice to unlock the full significance of these findings and understand the functions of SOCs for the biology of stem cells and neural development.

Store-operated Ca²⁺ channels in the epithelium of the lung airway. 
The epithelial cells present on the surface of the airways of the lung are directly exposed to inhaled air and form the first line of defense against inhaled allergens and pathogens. Epithelial cells do not merely comprise a passive barrier but play an active role in orchestrating inflammatory responses, tuning both innate and adaptive immune reactions through the production of a wide array of secreted factors and through their interactions with various cells of the immune system. Epithelial cells produce cytokines via the Ca²⁺-responsive transcriptional regulators, NFAT and NF-kB. Although it is known that airway epithelial cells express these transcription factors, the nature and sources of Ca²⁺ entry that regulate them are unknown. Our findings indicate that CRAC channels serve as a major route of Ca²⁺ entry in lung epithelial cells. Moreover, CRAC channel activation leads to robust activation of NFAT and the production of proinflammatory cytokines. We are taking a multi-faceted approach to understand how CRAC channels in epithelial cells orchestrate inflammatory responses in the airway with the long-term goal of illuminating their role in airway diseases such as asthma.

Publications:

McNally B, Yamashita M, Engh, AE, Prakriya, M. (2009) Structural determinants of ion permeation in CRAC channels. Proc. Natl. Acad. Sci. USA.  106: 22516-22521.

Prakriya, M. The molecular physiology of CRAC channels. (2009) Immunological Reviews  231: 88-98.

Navarro-Borelly L, Somasundaram A, Yamashita M, Ren D, Miller RJ & Prakriya M. (2008) STIM1-ORAI1 interactions and ORAI1 conformational changes revealed by live-cell FRET microscopy. Journal of Physiology 586, 5383-5401.

Oh-hora, M., Yamashita, M, Hogan, P.G., Sharma, S., Lamperi, E., Chung, W., Prakriya, M., Feske, S., and Rao, A (2008) Dual functions for the endoplamic reticulum calcium sensors STIM1 and STIM2 in T cell activation and tolerance.  Nature Immunology 9:432-443. 

Gwack Y, Srikanth S, Oh-Hora M, Hogan PG, Lamperti ED, Yamashita M, Gelinas C, Neems DS, Sasaki Y, Feske S, Prakriya M, Rajewsky K & Rao A. (2008) Hair loss and defective T- and B-cell function in mice lacking ORAI1. Mol Cell Biol 28, 5209-5222.

Yamashita, M., Navarro-Borelly, L., McNally, B., and Prakriya, M. (2007) Orai1 mutations alter ion permeation and Ca²⁺-dependent fast inactivation of CRAC channels: evidence for coupling of permeation and gating. Journal of General Physiology 130: 525-540. 

Prakriya, M., Feske, S., Gwack Y., Srikanth, S., Rao, A., and Hogan, P.G.  (2006) Orai1 is an essential pore subunit of the CRAC channel.  Nature 443:230-233.

View Publications by Murali Prakriya listed in the National Library of Medicine (PubMed).

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