Donald L. Gill, PhD
Chairperson, Department of Biochemistry
Professor, Cardiovascular Research Center
Professor, Fels Institute for Cancer Research and Molecular Biology
Telephone: 215-707- 3979
Department of Biochemistry
Independence Blue Cross Cardiovascular Research Center
Fels Institute for Cancer Research and Molecular Biology
Calcium Signal Transduction:
Calcium is one of the most fundamental signaling agents in all animal cells. Cells have evolved to precisely control Ca2+ in the cytoplasm at levels that are 10,000-fold lower than outside cells. This is accomplished by Ca2+ pumps in the plasma membrane (PM) and endoplasmic reticulum (ER). We study the signals of Ca2+ which occur as a result of control of specific channels in the PM and ER membrane which allow Ca2+ to flow into the cytosol. A slight elevation in the resting cytosolic Ca2+ level is enough to trigger rapid cellular responses such as contraction, secretion or changes in the function of key metabolic enzymes. More sustained Ca2+ signals mediate crucial longer term responses including cell growth, cell division, and cell death (apoptosis). Our lab studies signal transduction, meaning that we study how cells transduce external signals into Ca2+ signals. Cells sense many different external signals through specific receptors for chemical agents such as growth factors, neurotransmitters, and hormones, as well as receptors for temperature, pressure, stretch, sound, and light. The cell converts the message received by receptors into Ca2+ signals by precisely controlling the opening of Ca2+ channels. We use a combination of molecular biology, biochemistry, cell biology, and single cell physiological approaches to understand how the Ca2+ channels are controlled. We use molecular biology to mutate the channel proteins, create expression vectors, and to modify channel expression using gene silencing approaches. We follow real-time Ca2+ signals in cells using sophisticated single cell ratiometric fluorescence imaging technology. And we measure the precise biophysical properties of channels using state-of the-art electrophysiological methods. The work centers on the analysis of several distinct types of membrane channels including members of the now widely recognized TRP family of channel proteins involved in transducing a remarkable array of external signals. More recently, we have focused on understanding the mechanisms by which STIM and Orai proteins are involved in the controlling Ca2+ signals. Our work draws together molecular and cellular approaches to understand the basic function and physiological role of these channels which are critical to mediating essential cellular responses. Some of our more recent advances and directions are described in the information below.
STIM and Orai Proteins – fascinating dynamic control of Ca2+ signals in cells
Ca2+ signals controlling a vast array of cell functions involve both Ca2+ store release and external Ca2+ entry. These two events are coordinated through a dynamic intermembrane coupling between two distinct membrane proteins, STIM and Orai. STIM proteins are ER luminal Ca2+ sensors undergoing a profound redistribution into discrete junctional ER domains closely juxtaposed with the plasma membrane. Orai proteins are PM Ca2+ channels that migrate and become tethered by STIM within the ER-PM junctions where they mediate exceedingly selective Ca2+ entry. We describe new understanding on the nature of the proteins and how they function to mediate this remarkable intermembrane signaling process controlling Ca2+ signals.
Calcium Signaling: the role of Store-Operated Channels: Cellular Ca2+ homeostasis and Ca2+ signaling are closely entwined processes. Cytoplasmic Ca2+ is tightly controlled around 100 nM; elevations to 300-500 nM constitute powerful signals controlling a spectrum of cellular functions ranging from short-term contractile, secretory or metabolic responses, to longer term regulation of transcription, growth and cell division. The ER has a special role in Ca2+ signaling, accumulating high (~ 500 µM) luminal free Ca2+ levels. The luminal Ca2+ serves two roles – maintaining a correct protein folding environment, and serving as the major source of Ca2+ for signaling. Cell surface receptors coupled to PLC and InsP3 production, induce rapid Ca2+ signals by releasing ER-stored Ca2+ through InsP3Rs. This triggers a second Ca2+ signaling pathway through activation of “store-operated” channels (SOCs). These PM Ca2+ entry channels are activated by decreased ER luminal Ca2+, involving an intricate ER-PM coupling process. SOCs carry a small but highly Ca2+-selective current, termed the Ca2+ release activated Ca2+ current, or ICRAC. This movement of Ca2+ ions can be viewed as a tightly regulated “trickle” of Ca2+ into cells, crucial in mediating longer-term control of both cytoplasmic and ER luminal Ca2+. Since the first description of SOCs (9), the ER-PM coupling has been considered to involve direct protein interactions occurring at close junctions between ER and PM (10,11). The function of the newly discovered STIM and Orai proteins fulfills this prediction.
STIM and Orai – the Machinery of Store-Operated Channels: Recent high through-put RNAi screens identified two protein families as being essential for SOC activation – STIM in the ER and Orai in the PM. STIM proteins are highly dynamic membrane proteins located mostly in the ER, able to sense luminal Ca2+ changes and undergo rapid translocation into discrete junctional areas of ER, closely juxtaposed with the PM (7). Orai proteins are PM Ca2+ channels that translocate within the PM to the same ER junctions and become activated through coupling with STIM proteins. Although the function of SOCs has been best recognized in hematopoietic cells, STIM and Orai proteins are widely expressed among tissues, representing potentially crucial pharmacological targets for controlling an array of cell functions.
STIM Proteins – Dynamic SOC Intermediaries: The discovery of STIM1 transformed the store-operated hypothesis into an authentic mechanistic paradigm. STIM1 was originally identified as a surface membrane protein in stromal cells. Highly homologous STIM proteins are expressed in species ranging from Drosophila to C. Elegans. Vertebrates also express a second gene product STIM2. The ubiquitously expressed STIM1 and STIM2 proteins are highly similar varying only at the extreme N and C termini (see diagrams below). Both STIM1 and STIM2 are predominantly in the ER . While some STIM1 is also in the PM where it can influence SOC activation, it primarily functions in the ER, coupling to activate SOCs by transfer into ER-PM junctions. Store-depletion is reported to increase PM STIM1, however, SOC activation does not require PM-insertion of STIM1. STIM1 is normally widely distributed through the ER but rapidly oligomerizes and moves into PM-junctional regions seconds after emptying stores. The N-terminal Ca2+-sensing domain of STIM1 is a tightly clustered group of short α-helices comprising EF-hand and sterile α motif (SAM) domains. The cytoplasmic C-terminal region contains more extensive α-helical regions sufficiently long to span much of the ER-PM junctional gap, estimated to be 10-20 nm, and couple with PM Orai channels.
Calcium Signals – STIM Dynamics Mediate Spatially Unique Oscillations
Receptor-induced Ca2+ oscillations provide “digitized” signals conferring great precision in the activation of downstream targets. In recent work, luminal ER Ca2+-sensing STIM proteins are revealed to cyclically translocate during oscillations, transiently coupling to activate cell surface Ca2+ entry channels. The entering Ca2+ provides a spatially unique signal selectively triggering immediate-early gene expression. Calcium signals are crucial in controlling a plethora of cellular functions, and involve extraordinary spatial and temporal precision within cells. In most cells, physiological receptor-activation induces repetitive “oscillations” of cytosolic Ca2+ mediated by cyclic release of Ca2+ from endoplasmic reticulum (ER) stores. These “digital” Ca2+ signals confer unique specificity, sensitivity and accuracy in the activation of downstream target functions. As ER Ca2+ stores empty, Ca2+ enters through highly specific “store-operated channels” (SOCs) in the plasma membrane (PM) which are controlled by the STIM proteins, sensors of ER luminal Ca2+ levels. Rather than merely “replenishing” depleted stores we now beleive that Ca2+ entry through SOCs contributes crucially to the spatial signature of Ca2+ oscillations. Indeed, the STIM1 protein is now shown to cyclically translocate in and out of ER-PM junctions during each Ca2+ oscillatory spike. This STIM-mediated Ca2+ entry component of the digitized Ca2+ signals appears crucial for the Ca2+-induced control of gene expression.
Calcium Signaling by STIM and Orai: Understanding the Intimate Coupling Details
The two recently identified protein families, STIM and Orai, play remarkable and dynamic roles in mediating cellular Ca2+ signals. STIM proteins are sensors of Ca2+ stored within the endoplasmic reticulum (ER). Orai proteins are plasma membrane (PM) channels with almost unparalleled ionic selectivity allowing only Ca2+ ions to flow into cells. Although in separate membranes, the two proteins undergo profound reorganization to end up participating in an exquisite pas-de-deux within small junctional regions between the ER and PM. Before these proteins embrace, STIM undergoes an important activation process triggered by Ca2+ store-depletion. During its union with Orai, STIM induces the channel pore within Orai to open, allowing Ca2+ ions to flow through the PM and provide crucial intracellular signals. Recent studies on the activation of STIM and its coupling to Orai provide valuable new insights into the liaison between the two proteins and the intricate mechanism by which activation of Ca2+ signals occurs.
The calcium store-sensor, STIM1, reciprocally controls Orai and CaV1.2 channels
New information emerging from the lab has provided important information revealing that the control of signals through STIM proteins is much broader than we had previously believed. Thus, the STIM proteins are involved coupling to L-type channels (or CaV1.2 channels) which are a major class of voltage-operated channels in excitable cells
Ca2+ entry channels, crucial in providing cellular Ca2+ signals, are controlled by sensing mechanisms including membrane voltage, surface receptors, and Ca2+-sensing STIM proteins in the endoplasmic reticulum (ER). The coordinated operation of these transduction processes is key to controlling cell function. STIM proteins are dynamic ER Ca2+-sensors which aggregate when ER is depleted of Ca2+, then rapidly translocate into ER-plasma membrane (PM) junctions where they interact with and activate the highly Ca2+-selective Orai family of PM channels. We determined that STIM proteins also mediate inhibitory control of voltage-activated CaV1.2 channels. This action was independent of Orai channel function or changes in cytosolic Ca2+, and mediated by a direct action of STIM1 on the CaV1.2 α1C-subunit. Thus, STIM1 reciprocally controls Orai and CaV1.2 channels, indicating a hitherto unknown and potentially crucial regulatory link between receptor-induced Ca2+ store-depletion and control of voltage-activated Ca2+ signals.
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