Gautam Bhave

cAMP-Dependent Protein Kinase Regulates Desensitization of the Capsaicin Receptor (VR1) by Direct Phosphorylation

The capsaicin receptor, VR1 (also known as TRPV1), is a ligand-gated ion channel expressed on nociceptive sensory neurons that responds to noxious thermal and chemical stimuli. Capsaicin responses in sensory neurons exhibit robust potentiation by cAMP-dependent protein kinase (PKA). In this study, we demonstrate that PKA reduces VR1 desensitization and directly phosphorylates VR1. In vitro phosphorylation, phosphopeptide mapping, and protein sequencing of VR1 cytoplasmic domains delineate several candidate PKA phosphorylation sites. Electrophysiological analysis of phosphorylation site mutants clearly pinpoints Ser116 as the residue responsible for PKA-dependent modulation of VR1. Given the significant roles of VR1 and PKA in inflammatory pain hypersensitivity, VR1 phosphorylation at Ser116 by PKA may represent an important molecular mechanism involved in the regulation of VR1 function after tissue injury.

Protein kinase C phosphorylation sensitizes but does not activate the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1)

Protein kinase C (PKC) modulates the function of the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1). This modulation manifests as increased current when the channel is activated by capsaicin. In addition, studies have suggested that phosphorylation by PKC might directly gate the channel, because PKC-activating phorbol esters induce TRPV1 currents in the absence of applied ligands. To test whether PKC both modulates and gates the TRPV1 function by direct phosphorylation, we used direct sequencing to determine the major sites of PKC phosphorylation on TRPV1 intracellular domains. We then tested the ability of the PKC-activating phorbol 12-myristate 13-acetate (PMA) to potentiate capsaicin-induced currents and to directly gate TRPV1. We found that mutation of S800 to alanine significantly reduced the PMA-induced enhancement of capsaicin-evoked currents and the direct activation of TRPV1 by PMA. Mutation of S502 to alanine reduced PMA enhancement of capsaicin-evoked currents, but had no effect on direct activation of TRPV1 by PMA. Conversely, mutation of T704 to alanine had no effect on PMA enhancement of capsaicin-evoked currents but dramatically reduced direct activation of TRPV1 by PMA. These results, combined with pharmacological studies showing that inactive phorbol esters also weakly activate TRPV1, suggest that PKC-mediated phosphorylation modulates TRPV1 but does not directly gate the channel. Rather, currents induced by phorbol esters result from the combination of a weak direct ligand-like activation of TRPV1 and the phosphorylation-induced enhancement of the TRPV1 function. Furthermore, modulation of the TRPV1 function by PKC appears to involve distinct phosphorylation sites depending on the mechanism of channel activation.

Peripheral group I metabotropic glutamate receptors modulate nociception in mice

The metabotropic glutamate receptors (mGluRs) are found throughout the central nervous system, where they modulate neuronal excitability and synaptic transmission. Here we report the presence of phospholipase C-coupled group I mGluRs (mGluR1 and mGluR5) outside the central nervous system on peripheral unmyelinated sensory afferents. Given their localization on predominantly nociceptive afferents, we investigated whether these receptors modulate nociceptive signaling, and found that agonist-induced activation of peripheral group I mGluRs leads to increased sensitivity to noxious heat, a phenomenon termed thermal hyperalgesia. Furthermore, group I mGluR antagonists not only prevent, but also attenuate established formalin-induced pain. Taken together, these results suggest that peripheral mGluRs mediate a component of hyperalgesia and may be therapeutically targeted to prevent and treat inflammatory pain.

Bromine Is an Essential Trace Element for Assembly of Collagen IV Scaffolds in Tissue Development and Architecture

Bromine is ubiquitously present in animals as ionic bromide (Br(-)) yet has no known essential function. Herein, we demonstrate that Br(-) is a required cofactor for peroxidasin-catalyzed formation of sulfilimine crosslinks, a posttranslational modification essential for tissue development and architecture found within the collagen IV scaffold of basement membranes (BMs). Bromide, converted to hypobromous acid, forms a bromosulfonium-ion intermediate that energetically selects for sulfilimine formation. Dietary Br deficiency is lethal in Drosophila, whereas Br replenishment restores viability, demonstrating its physiologic requirement. Importantly, Br-deficient flies phenocopy the developmental and BM defects observed in peroxidasin mutants and indicate a functional connection between Br(-), collagen IV, and peroxidasin. We establish that Br(-) is required for sulfilimine formation within collagen IV, an event critical for BM assembly and tissue development. Thus, bromine is an essential trace element for all animals, and its deficiency may be relevant to BM alterations observed in nutritional and smoking-related disease. PAPERFLICK:

Peroxidasin forms sulfilimine chemical bonds using hypohalous acids in tissue genesis

Collagen IV comprises the predominant protein network of basement membranes, a specialized extracellular matrix, which underlie epithelia and endothelia. These networks assemble through oligomerization and covalent crosslinking to endow mechanical strength and shape cell behavior through interactions with cell-surface receptors. A recently discovered sulfilimine (S=N) bond between a methionine sulfur and hydroxylysine nitrogen reinforces the collagen IV network. We demonstrate that peroxidasin, an enzyme found in basement membranes, catalyzes formation of the sulfilimine bond. Drosophila peroxidasin mutants have disorganized collagen IV networks and torn visceral muscle basement membranes, pointing to a critical role for the enzyme in tissue biogenesis. Peroxidasin generates hypohalous acids as reaction intermediates, suggesting a paradoxically anabolic role for these usually destructive oxidants. This work highlights sulfilimine bond formation as what is to our knowledge the first known physiologic function for peroxidasin, a role for hypohalous oxidants in tissue biogenesis, and a possible role for peroxidasin in inflammatory diseases.

High-Throughput Screening Reveals a Small-Molecule Inhibitor of the Renal Outer Medullary Potassium Channel and Kir7.1

The renal outer medullary potassium channel (ROMK) is expressed in the kidney tubule and critically regulates sodium and potassium balance. The physiological functions of other inward rectifying K+ (Kir) channels expressed in the nephron, such as Kir7.1, are less well understood in part due to the lack of selective pharmacological probes targeting inward rectifiers. In an effort to identify Kir channel probes, we performed a fluorescence-based, high-throughput screen (HTS) of 126,009 small molecules for modulators of ROMK function. Several antagonists were identified in the screen. One compound, termed VU590, inhibits ROMK with submicromolar affinity, but has no effect on Kir2.1 or Kir4.1. Low micromolar concentrations inhibit Kir7.1, making VU590 the first small-molecule inhibitor of Kir7.1. Structure-activity relationships of VU590 were defined using small-scale parallel synthesis. Electrophysiological analysis indicates that VU590 is an intracellular pore blocker. VU590 and other compounds identified by HTS will be instrumental in defining Kir channel structure, physiology, and therapeutic potential.

A unique covalent bond in basement membrane is a primordial innovation for tissue evolution

Significance The evolution of multicellular animals from single-celled ancestors was one of the most significant transitions of life on earth. The emergence of larger, more complex animals able to resist predation and colonize new environments was enabled, in part, by a collagen scaffold, which anchors cells together to form tissues and organs. Here, we show that a unique chemical bond, a link between sulfur and nitrogen atoms called a sulfilimine bond, arose over 500 Mya, binding this scaffold together and enabling tissues to withstand mechanical forces. Peroxidasin forms the bond by generating hypohalous acids as strong oxidants, a form of bleach, which normally function as antimicrobial agents. These understandings may lead to approaches for targeting tumors and treatment of other diseases. Basement membrane, a specialized ECM that underlies polarized epithelium of eumetazoans, provides signaling cues that regulate cell behavior and function in tissue genesis and homeostasis. A collagen IV scaffold, a major component, is essential for tissues and dysfunctional in several diseases. Studies of bovine and Drosophila tissues reveal that the scaffold is stabilized by sulfilimine chemical bonds (S = N) that covalently cross-link methionine and hydroxylysine residues at the interface of adjoining triple helical protomers. Peroxidasin, a heme peroxidase embedded in the basement membrane, produces hypohalous acid intermediates that oxidize methionine, forming the sulfilimine cross-link. We explored whether the sulfilimine cross-link is a fundamental requirement in the genesis and evolution of epithelial tissues by determining its occurrence and evolutionary origin in Eumetazoa and its essentiality in zebrafish development; 31 species, spanning 11 major phyla, were investigated for the occurrence of the sulfilimine cross-link by electrophoresis, MS, and multiple sequence alignment of de novo transcriptome and available genomic data for collagen IV and peroxidasin. The results show that the cross-link is conserved throughout Eumetazoa and arose at the divergence of Porifera and Cnidaria over 500 Mya. Also, peroxidasin, the enzyme that forms the bond, is evolutionarily conserved throughout Metazoa. Morpholino knockdown of peroxidasin in zebrafish revealed that the cross-link is essential for organogenesis. Collectively, our findings establish that the triad—a collagen IV scaffold with sulfilimine cross-links, peroxidasin, and hypohalous acids—is a primordial innovation of the ECM essential for organogenesis and tissue evolution.

Association of gadolinium based magnetic resonance imaging contrast agents and nephrogenic systemic fibrosis.

PURPOSE We investigated the recently discovered association between gadolinium based magnetic resonance imaging contrast agents and the development of nephrogenic systemic fibrosis in patients with chronic kidney disease or acute kidney injury. MATERIALS AND METHODS A systematic review of the PubMed database and publicly available patient databases was performed to characterize nephrogenic systemic fibrosis and its possible association with exposure to gadolinium based magnetic resonance imaging contrast agents. RESULTS Data from case series reports, nephrogenic systemic fibrosis patient databases, nephrogenic systemic fibrosis case reporting to the Food and Drug Administration after gadolinium contrast agent exposure and retrospective case control studies suggest a strong association between the use of gadolinium based magnetic resonance imaging contrast agents and the subsequent development of nephrogenic systemic fibrosis in patients with renal disease. These data also suggest that the risk of nephrogenic systemic fibrosis depends on the degree of renal dysfunction, dose of contrast agent, gadolinium contrast agent stability and severity of concomitant illness. Thus, the occurrence of nephrogenic systemic fibrosis after gadolinium contrast agent exposure may vary from negligible up to 2% to 5% in select high risk clinical situations. CONCLUSIONS Magnetic resonance imaging using gadolinium based contrast agents must be performed judiciously in patients with renal dysfunction, carefully weighing on a case by case basis the benefits of magnetic resonance imaging and the risk of nephrogenic systemic fibrosis as well as the disadvantages of undergoing alternative or foregoing imaging studies.

Development of a Selective Small-Molecule Inhibitor of Kir1.1, the Renal Outer Medullary Potassium Channel

The renal outer medullary potassium (K+) channel, ROMK (Kir1.1), is a putative drug target for a novel class of loop diuretic that would lower blood volume and pressure without causing hypokalemia. However, the lack of selective ROMK inhibitors has hindered efforts to assess its therapeutic potential. In a high-throughput screen for small-molecule modulators of ROMK, we previously identified a potent and moderately selective ROMK antagonist, 7,13-bis(4-nitrobenzyl)-1,4,10-trioxa-7,13-diazacyclopentadecane (VU590), that also inhibits Kir7.1. Because ROMK and Kir7.1 are coexpressed in the nephron, VU590 is not a good probe of ROMK function in the kidney. Here we describe the development of the structurally related inhibitor 2,2′-oxybis(methylene)bis(5-nitro-1H-benzo[d]imidazole) (VU591), which is as potent as VU590 but is selective for ROMK over Kir7.1 and more than 65 other potential off-targets. VU591 seems to block the intracellular pore of the channel. The development of VU591 may enable studies to explore the viability of ROMK as a diuretic target.

Metabotropic glutamate receptors on peripheral sensory neuron terminals as targets for the development of novel analgesics.

Metabotropic glutamate receptors on peripheral sensory neuron terminals as targets for the development of novel analgesics