Two sulfhydryl reagents, N-ethylmaleimide (NEM), an alkylating agent, and diamide, an oxidizing agent, were examined for effects on the skeletal muscle Ca 2؉ release channel. NEM incubated with the channel for increasing periods of time displays three distinct phases in its functional effects on the channel reconstituted into planar lipid bilayers; first it inhibits, then it activates, and finally it again inhibits channel activity. NEM also shows a three-phase effect on the binding of
Bloodborne infections with Candida albicans are an increasingly recognized complication of modern medicine. Here, we present a mouse model of low-grade candidemia to determine the effect of disseminated infection on cerebral function and relevant immune determinants. We show that intravenous injection of 25,000 C. albicans cells causes a highly localized cerebritis marked by the accumulation of activated microglial and astroglial cells around yeast aggregates, forming fungal-induced glial granulomas. Amyloid precursor protein accumulates within the periphery of these granulomas, while cleaved amyloid beta (Aβ) peptides accumulate around the yeast cells. CNS-localized C. albicans further activate the transcription factor NF-κB and induce production of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor (TNF), and Aβ peptides enhance both phagocytic and antifungal activity from BV-2 cells. Mice infected with C. albicans display mild memory impairment that resolves with fungal clearance. Our results warrant additional studies to understand the effect of chronic cerebritis on cognitive and immune function.
This study presents evidence for a close relationship between the oxidation state of the skeletal muscle Ca2+ release channel (RyR1) and its ability to bind calmodulin (CaM). CaM enhances the activity of RyR1 in low Ca2+ and inhibits its activity in high Ca2+. Oxidation, which activates the channel, blocks the binding of125I-labeled CaM at both micromolar and nanomolar Ca2+concentrations. Conversely, bound CaM slows oxidation-induced cross-linking between subunits of the RyR1 tetramer. Alkylation of hyperreactive sulfhydryls (<3% of the total sulfhydryls) on RyR1 with N-ethylmaleimide completely blocks oxidant-induced intersubunit cross-linking and inhibits Ca2+-free125I-CaM but not Ca2+/125I-CaM binding. These studies suggest that 1) the sites on RyR1 for binding apocalmodulin have features distinct from those of the Ca2+/CaM site, 2) oxidation may alter the activity of RyR1 in part by altering its interaction with CaM, and 3) CaM may protect RyR1 from oxidative modifications during periods of oxidative stress.
The skeletal muscle Ca 2؉ release channel (RYR1), which plays a critical role in excitation-contraction coupling, is a homotetramer with a subunit molecular mass of 565 kDa. Oxidation of the channel increases its activity and produces intersubunit cross-links within the RYR1 tetramer (Aghdasi, B., Zhang, J., Wu, Y., Reid, M. B., and Hamilton, S. L. (1997) J. Biol. Chem. 272, 3739 -3748). Alkylation of hyperreactive sulfhydryls on RYR1 with N-ethylmaleimide (NEM) inhibits channel function and blocks the intersubunit cross-linking. We used calpain and tryptic cleavage, two-dimensional SDS-polyacrylamide gel electrophoresis, N-terminal sequencing, sequence-specific antibody Western blotting, and [ 14 C]NEM labeling to identify the domains involved in these effects. Our data are consistent with a model in which 1) diamide, an oxidizing agent, simultaneously produces an intermolecular cross-link between adjacent subunits within the RYR1 tetramer and an intramolecular cross-link within a single subunit; 2) all of the cysteines involved in both cross-links are in either the region between amino acids ϳ2100 and 2843 or the region between amino acids 2844 and 4685; 3) oxidation exposes a new calpain cleavage site in the central domain of the RYR1 (in the region around amino acid 2100); 4) sulfhydryls that react most rapidly with NEM are located in the N-terminal domain (between amino acids 426 and 1396); 5) alkylation of the N-terminal cysteines completely inhibits the formation of both inter-and intrasubunit cross-links. In summary, we present evidence for interactions between the N-terminal region and the putatively cytoplasmic central domains of RYR1 that appear to influence subunit-subunit interactions and channel activity.In skeletal muscle, the Ca 2ϩ release channel (RYR1) of the sarcoplasmic reticulum (SR) 1 responds to T tubule depolarization by releasing lumenal Ca 2ϩ into the myoplasmic space (1), triggering the sequence of events that leads to muscle contraction. Single amino acid changes in RYR1 (2-8) are thought to produce the human diseases malignant hyperthermia and central core disease. Based on a hydropathy analysis of the primary amino acid sequence of RYR1, the monomeric subunit is predicted to have a short cytoplasmic C terminus and between 4 and 10 membrane-spanning regions in the C-terminal onefifth of the molecule (9, 10). The transmembrane regions of the monomers may combine to form the pore of the homotetrameric Ca 2ϩ release channel. The large N-terminal part of the molecule is thought to extend into the cytoplasm as a "foot" structure (11), and this region of the protein plays an important role in regulation of the channel activity of RYR1. Most of the mutations in RYR1 that produce malignant hyperthermia and central core disease have been found in this region and cluster in two cytoplasmic locations (2-8). Also, most modulators of the channel are thought to interact with cytoplasmic domains of RYR1 (12-16).Reactive oxygen intermediates are produced in resting muscle and their production increa...
We used a combination of bioinformatics, electron cryomicroscopy, and biochemical techniques to identify an oxidoreductase-like domain in the skeletal muscle Ca 2؉ release channel protein (RyR1). The initial prediction was derived from sequence-based fold recognition for the N-terminal region (41-420) of RyR1. The putative domain was computationally localized to the clamp domain in the cytoplasmic region of a 22Å structure of RyR1. This localization was subsequently confirmed by difference imaging with a sequence specific antibody. Consistent with the prediction of an oxidoreductase domain, RyR1 binds [ 3 H]NAD ؉ , supporting a model in which RyR1 has a oxidoreductase-like domain that could function as a type of redox sensor. During excitation-contraction coupling in skeletal muscle, Ca 2ϩ is released from the lumen of the sarcoplasmic reticulum (SR) via the Ca 2ϩ release channel, also known as the ryanodine receptor, RyR1. In skeletal muscle, the Ca 2ϩ release channel is physically coupled to the L-type voltage dependent Ca 2ϩ channel dihydropyridine receptor (DHPR), such that a depolarization induced change in the conformation of DHPR induces the opening of ryanodine receptor 1 (RyR1). This leads to an increase of cytoplasmic Ca 2ϩ , triggering a sequence of events that lead to muscle contraction. RyR1 is a homotetramer (1) whose subunits are ϳ565 kDa (e.g., human, 5,038 residues; rabbit, 5,037 residues) (2, 3). Mutations in three domains of this protein, one of which is between amino acids 35 and 614, have been implicated in the pathogenesis of two human diseases, malignant hyperthermia and central core disease (4, 5).The Ca 2ϩ release channel exists in at least two functional states, opened and closed (6), which likely have conformational differences. The low-resolution structures of the Ca 2ϩ release channel in different functional states have been studied extensively by electron cryomicroscopy (7-9). On opening, a number of structural changes occur in several regions of the channel, including both the clamplike domains in the cytoplasmic region and the transmembrane domain. The clamp domains are the most likely candidates for interaction with DHPR (8) and must, therefore, be allosterically coupled to the transmembrane domain in order for DHPR to induce the opening of the Ca 2ϩ permeable pore of RyR1. Here we describe a unique approach for identification of new functional and structural domains of this complex protein. MethodsSequence Analysis. Initial motif searching in the primary sequence of rabbit RyR1 (P11716) was done by using PROSCAN (10) with a threshold of 70%. Subsequently, 500-residue consecutive, serial sequence segments of the RyR1 were submitted to the University of California, Los Angeles-Department of Energy (UCLA-DOE) Fold recognition server (11). Primary sequence alignments were performed by using CLUSTALW (Gonnet weight matrix) with a Gonnet Pam250 positive-value similarities scoring system (12, 13). Additionally, multiple sequence alignments were done with other RyR sequences. As sequenc...
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