Browsing by Author "Demaree, Isaac S."

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    Dihydroceramides Derived from Bacteroidetes Species Sensitize TRPV1 Channels
    (MDPI, 2023-01-03) Ludwig, Nora; Demaree, Isaac S.; Yamada, Chiaki; Nusbaum, Amilia; Nichols, Frank C.; White, Fletcher A.; Movila, Alexandru; Obukhov, Alexander G.; Anatomy, Cell Biology and Physiology, School of Medicine
    Bacterial colonization of open wounds is common, and patients with infected wounds often report significantly elevated pain sensitivity at the wound site. Transient Receptor Potential Vanilloid Type 1 (TRPV1) channels are known to play an important role in pain signaling and may be sensitized under pro-inflammatory conditions. Bacterial membrane components, such as phosphoethanolamine dihydroceramide (PEDHC), phosphoglycerol dihydroceramide (PGDHC), and lipopolysaccharide (LPS), are released in the environment from the Gram-negative bacteria of the Bacteroidetes species colonizing the infected wounds. Here, we used intracellular calcium imaging and patch-clamp electrophysiology approaches to determine whether bacterially derived PEDHC, PGDHC, or LPS can modulate the activity of the TRPV1 channels heterologously expressed in HEK cells. We found that PEDHC and PGDHC can sensitize TRPV1 in a concentration-dependent manner, whereas LPS treatment does not significantly affect TRPV1 activity in HEK cells. We propose that sensitization of TRPV1 channels by Bacteroidetes-derived dihydroceramides may at least in part underlie the increased pain sensitivity associated with wound infections.
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    Effects of TRPC1’s Lysines on Heteromeric TRPC5-TRPC1 Channel Function
    (MDPI, 2024-12-06) Demaree, Isaac S.; Kumar, Sanjay; Tennessen, Kayla; Hoang, Quyen Q.; White, Fletcher A.; Obukhov, Alexander G.; Anatomy, Cell Biology and Physiology, School of Medicine
    Background: TRPC5 proteins form plasma membrane cation channels and are expressed in the nervous and cardiovascular systems. TRPC5 activation leads to cell depolarization and increases neuronal excitability, whereas a homologous TRPC1 inhibits TRPC5 function via heteromerization. The mechanism underlying the inhibitory effect of TRPC1 in TRPC5/TRPC1 heteromers remains unknown. Methods: We used electrophysiological techniques to examine the roles of subunit stoichiometry and positively charged luminal residues of TRPC1 on TRPC5/TRPC1 function. We also performed molecular dynamics simulations. Results: We found that increasing the relative amount of TRPC1 in TRPC5/TRPC1 heteromers reduced histamine-induced cation influx through the heteromeric channels. Consistently, histamine-induced cation influx was small in cells co-expressing TRPC5-TRPC1 concatemers and TRPC1, and large in cells co-expressing TRPC5-TRPC1 concatemers and TRPC5. Molecular dynamics simulations revealed that the TRPC1 protein has two positively charged lysine residues that are facing the heteromeric channel pore lumen. Substitution of these lysines with asparagines decreased TRPC1's inhibitory effect on TRPC5/TRPC1 function, indicating that these lysines may regulate cation influx through TRPC5/TRPC1 heteromers. Additionally, we established that extracellular Mg2+ inhibits cation influx through TRPC5/TRPC1, contributing to channel regulation. Conclusions: We revealed that the inhibitory effect of TRPC1 on heteromeric TRPC5/TRPC1 function likely involves luminal lysines of TRPC1.
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    Transient Receptor Potential Canonical (TRPC) Channels: Then and Now
    (MDPI, 2020-08-28) Chen, Xingjuan; Sooch, Gagandeep; Demaree, Isaac S.; White, Fletcher A.; Obukhov, Alexander G.; Anesthesia, School of Medicine
    Twenty-five years ago, the first mammalian Transient Receptor Potential Canonical (TRPC) channel was cloned, opening the vast horizon of the TRPC field. Today, we know that there are seven TRPC channels (TRPC1–7). TRPCs exhibit the highest protein sequence similarity to the Drosophila melanogaster TRP channels. Similar to Drosophila TRPs, TRPCs are localized to the plasma membrane and are activated in a G-protein-coupled receptor-phospholipase C-dependent manner. TRPCs may also be stimulated in a store-operated manner, via receptor tyrosine kinases, or by lysophospholipids, hypoosmotic solutions, and mechanical stimuli. Activated TRPCs allow the influx of Ca2+ and monovalent alkali cations into the cytosol of cells, leading to cell depolarization and rising intracellular Ca2+ concentration. TRPCs are involved in the continually growing number of cell functions. Furthermore, mutations in the TRPC6 gene are associated with hereditary diseases, such as focal segmental glomerulosclerosis. The most important recent breakthrough in TRPC research was the solving of cryo-EM structures of TRPC3, TRPC4, TRPC5, and TRPC6. These structural data shed light on the molecular mechanisms underlying TRPCs’ functional properties and propelled the development of new modulators of the channels. This review provides a historical overview of the major advances in the TRPC field focusing on the role of gene knockouts and pharmacological tools.