Lung endothelium forms a semi-permeable barrier that restricts water, solute and macromolecular access to tissue compartments. Inflammatory agents promote calcium influx across the endothelial cell plasma membrane, which triggers cytoskeletal reorganization, induces inter-cellular gaps, and increases permeability. However, endothelial cells express many different ion channels, bringing into question exactly which channel(s) provides the calcium source responsible for this cytoskeletal response. We have recently identified an endogenously expressed channel that is activated by inflammatory mediators, and provides a calcium source that induces inter-endothelial cell gaps. This channel is comprised of three different transient receptor potential proteins from the canonical sub-family (TRPC), including one TRPC1, one TRPC3 and two TRPC4 subunits. Patch clamp studies reveal that activation of the TRPC1/3/4 channel results in a calcium selective current, referred to as ISOC. The mechanism necessary to confer calcium selectivity to the TRPC1/3/4 channel is unknown. Our preliminary data indicate that Orai1 functions as an ancillary subunit, and is necessary to confer calcium selectivity to this channel. TRPC1/3/4 calcium selectivity may represent a physiologically important feature of the channel, as it impacts how much calcium permeates the channel and reaches the cortical actin rim. The TRPC4 subunits within this channel bind to protein 4.1, and protein 4.1 tethers the channel to the spectrin membrane skeleton. Spectrin directly binds to actin, and this interaction is necessary for proper alignment of the cortical actin rim. Indeed, increased calcium disrupts the spectrin-actin interaction, suggesting calcium that permeates the TRPC1/3/4 channel may disrupt spectrin-actin binding and catalyze gap formation. Thus, this proposal tests the overall HYPOTHESIS that Orai1 interacts with TRPC1/3/4 and establishes the channel's calcium selectivity, which is necessary to increase membrane calcium to concentrations that disrupt the spectrin-actin interaction causing inter-endothelial cell gap formation.
Specific aims test the related hypotheses that:  Orai1 constitutively interacts with TRPC1/3/4, and contributes to channel calcium selectivity; activation of the TRPC1/3/4 channel requires an intact protein 4.1-TRPC4-Orai1 interaction; calcium permeation through the TRPC1/3/4 channel disrupts the f-actin- spectrin interaction resulting in cytoskeleton reorganization and increased permeability. Studies proposed in this application address three fundamental, yet poorly understood areas in calcium signaling and endothelial cell biology. First, we will address whether Orai1 interacts with the TRPC1/3/4 channel. Second, we will address the mechanism responsible for determining TRPC1/3/4 calcium selectivity. Third, we will determine the physiological importance of TRPC1/3/4 calcium selectivity.
Inflammation causes gaps to form between endothelial cells, where fluid can escape from the circulation into the tissues. In the lung, such fluid leak compromises the ability to oxygenate blood, and can be life threatening. The mechanism(s) responsible for endothelial cell gap formation are incompletely understood. This application tests whether activation of a TRPC1/3/4 calcium channel plays an important role in regulating endothelial cell gap formation.
|Morrow, K Adam; Frank, Dara W; Balczon, Ron et al. (2017) The Pseudomonas aeruginosa Exoenzyme Y: A Promiscuous Nucleotidyl Cyclase Edema Factor and Virulence Determinant. Handb Exp Pharmacol 238:67-85|
|Balczon, Ron; Morrow, K Adam; Zhou, Chun et al. (2017) Pseudomonas aeruginosa infection liberates transmissible, cytotoxic prion amyloids. FASEB J 31:2785-2796|
|Francis, Michael; Xu, Ningyong; Zhou, Chun et al. (2016) Transient Receptor Potential Channel 4 Encodes a Vascular Permeability Defect and High-Frequency Ca(2+) Transients in Severe Pulmonary Arterial Hypertension. Am J Pathol 186:1701-9|
|Morrow, K Adam; Ochoa, Cristhiaan D; Balczon, Ron et al. (2016) Pseudomonas aeruginosa exoenzymes U and Y induce a transmissible endothelial proteinopathy. Am J Physiol Lung Cell Mol Physiol 310:L337-53|
|Zhou, Chun; Townsley, Mary I; Alexeyev, Mikhail et al. (2016) Endothelial hyperpermeability in severe pulmonary arterial hypertension: role of store-operated calcium entry. Am J Physiol Lung Cell Mol Physiol 311:L560-9|
|Xu, Ningyong; Cioffi, Donna L; Alexeyev, Mikhail et al. (2015) Sodium entry through endothelial store-operated calcium entry channels: regulation by Orai1. Am J Physiol Cell Physiol 308:C277-88|
|Gopal, Sandeep; Søgaard, Pernille; Multhaupt, Hinke A B et al. (2015) Transmembrane proteoglycans control stretch-activated channels to set cytosolic calcium levels. J Cell Biol 210:1199-211|
|Morrow, K A; Seifert, R; Kaever, V et al. (2015) Heterogeneity of pulmonary endothelial cyclic nucleotide response to Pseudomonas aeruginosa ExoY infection. Am J Physiol Lung Cell Mol Physiol 309:L1199-207|
|Xu, Ningyong; Francis, Michael; Cioffi, Donna L et al. (2014) Studies on the resolution of subcellular free calcium concentrations: a technological advance. Focus on ""detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells"". Am J Physiol Cell Physiol 306:C636-8|
|Stevens, Trevor C; Ochoa, Cristhiaan D; Morrow, K Adam et al. (2014) The Pseudomonas aeruginosa exoenzyme Y impairs endothelial cell proliferation and vascular repair following lung injury. Am J Physiol Lung Cell Mol Physiol 306:L915-24|
Showing the most recent 10 out of 27 publications