Biomedical subjects

Charles P Gilman

Publications and source records attributed to Charles P Gilman.

5 recordsLinked to original sources

PubMed2006

Identification of cysteines involved in S-nitrosylation, S-glutathionylation, and oxidation to disulfides in ryanodine receptor type 1.

The skeletal muscle Ca(2+)-release channel (ryanodine receptor type 1 (RyR1)) is a redox sensor, susceptible to reversible S-nitrosylation, S-glutathionylation, and disulfide oxidation. So far, Cys-3635 remains the only cysteine residue identified as functionally relevant to the redox sensing properties of the channel. We demonstrate that expression of the C3635A-RyR1 mutant in RyR1-null myotubes alters the sensitivity of the ryanodine receptor to activation by voltage, indicating that Cys-3635 is involved in voltage-gated excitation-contraction coupling. However, H(2)O(2) treatment of C3635A-RyR1 channels or wild-type RyR1, following their expression in human embryonic kidney cells, enhances [(3)H]ryanodine binding to the same extent, suggesting that cysteines other than Cys-3635 are responsible for the oxidative enhancement of channel activity. Using a combination of Western blotting and sulfhydryl-directed fluorescent labeling, we found that two large regions of RyR1 (amino acids 1-2401 and 3120-4475), previously shown to be involved in disulfide bond formation, are also major sites of both S-nitrosylation and S-glutathionylation. Using selective isotopecoded affinity tag labeling of RyR1 and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, we identified, out of the 100 cysteines in each RyR1 subunit, 9 that are endogenously modified (Cys-36, Cys-315, Cys-811, Cys-906, Cys-1591, Cys-2326, Cys-2363, Cys-3193, and Cys-3635) and another 3 residues that were only modified with exogenous redox agents (Cys-253, Cys-1040, and Cys-1303). We also identified the types of redox modification each of these cysteines can undergo. In summary, we have identified a discrete subset of cysteines that are likely to be involved in the functional response of RyR1 to different redox modifications (S-nitrosylation, S-glutathionylation, and oxidation to disulfides).

Animals↗

PubMed2004

Regulation of ryanodine receptors by FK506 binding proteins.

Ryanodine receptors (RyRs) are the major sarcoplasmic reticulum calcium-release channels required for excitation-contraction coupling in skeletal and cardiac muscle. Mutations in RyRs have been linked to several human diseases. Mutations in the cardiac isoform of RyR2 are associated with catecholaminergic polymorphic ventricular arrhythmias (CPVT), and arrhythmogenic right ventricular dysplasia type 2 (ARVD2), whereas mutations in the skeletal muscle isoform (RyR1) are linked to malignant hyperthermia (MH) and central core disease (CCD). RyRs are modulated by several other proteins, including the FK506 binding proteins (FKBPs), FKBP12 and FKBP12.6. These immunophilins appear to stabilize a closed state of the channel and are important for cooperative interactions among the subunits of RyRs. This review discusses the regulation of RyRs by FKBPs and the possibility that defective modulation of RyR2 by FKBP12.6 could play a role in heart failure, CPVT, and ARVD2. Also discussed are the consequences of FKBP12 depletion to skeletal muscle and the possibility of FKBP12 involvement in certain forms of MH or CCD.

Arrhythmogenic Right Ventricular Dysplasia↗

PubMed2003

Glucagon-like peptide 1 modulates calcium responses to glutamate and membrane depolarization in hippocampal neurons.

Glucagon-like peptide 1 (GLP-1) activates receptors coupled to cAMP production and calcium influx in pancreatic cells, resulting in enhanced glucose sensitivity and insulin secretion. Despite evidence that the GLP-1 receptor is present and active in neurons, little is known of the roles of GLP-1 in neuronal physiology. As GLP-1 modulates calcium homeostasis in pancreatic beta cells, and because calcium plays important roles in neuronal plasticity and neurodegenerative processes, we examined the effects of GLP-1 on calcium regulation in cultured rat hippocampal neurons. When neurons were pre-treated with GLP-1, calcium responses to glutamate and membrane depolarization were attenuated. Whole-cell patch clamp analyses showed that glutamate-induced currents and currents through voltage-dependent calcium channels were significantly decreased in neurons pre-treated with GLP-1. Pre-treatment of neurons with GLP-1 significantly decreased their vulnerability to death induced by glutamate. Acute application of GLP-1 resulted in a transient elevation of intracellular calcium levels, consistent with the established effects of GLP-1 on cAMP production and activation of cAMP response element-binding protein. Collectively, our findings suggest that, by modulating calcium responses to glutamate and membrane depolarization, GLP-1 may play important roles in regulating neuronal plasticity and cell survival.

Animals↗

PubMed2003

p53 is present in synapses where it mediates mitochondrial dysfunction and synaptic degeneration in response to DNA damage, and oxidative and excitotoxic insults.

A form of programmed cell-death called apoptosis occurs in neurons during development of the nervous system, and may also occur in a variety of neuropathological conditions. Here we present evidence obtained in studies of adult mice and neuronal cell cultures showing that p53 protein is present in synapses where its level and amount of phosphorylation are increased following exposure of the cells to the DNA-damaging agent etoposide. We also show that levels of active p53 increase in isolated cortical synaptosomes exposed to oxidative and excitotoxic insults. Increased levels of p53 also precede loss of synapsin I immunoreactive terminals in cultured hippocampal neurons exposed to etoposide. Synaptosomes from p53-deficient mice exhibit increased resistance to oxidative and excitotoxic insults as indicated by stabilization of mitochondrial membrane potential and decreased production of reactive oxygen species. Finally, we show that a synthetic inhibitor of p53 (PFT-alpha) protects synaptosomes from wild-type mice against oxidative and excitotoxic injuries, and preserves presynaptic terminals in cultured hippocampal neurons exposed to etoposide. Collectively, these findings provide the first evidence for a local transcription-independent action of p53 in synapses, and suggest that such a local action of p53 may contribute to the dysfunction and degeneration of synapses that occurs in various neurodegenerative disorders.

Animals↗

PubMed2002

Do apoptotic mechanisms regulate synaptic plasticity and growth-cone motility?

Signals between neurons are transduced primarily by receptors, and second messenger and kinase cascades, located in pre- and postsynaptic terminals. Such synaptic signaling pathways include those activated by neurotransmitters, cytokines, neurotrophic factors, and cell-adhesion molecules. Many of these signaling systems are also localized in the growth cones of axons and dendrites, where they control pathfinding and synaptogenesis during development. Although it has been known for decades that such signaling pathways can affect the survival of neurons, by promoting or preventing a form of programmed cell death known as apoptosis, we have discovered that apoptotic biochemical cascades can exert local actions on the functions and structural dynamics of growth cones and synapses. In this article, we provide a brief background on apoptotic biochemical cascades, and present examples of studies in this laboratory that have identified novel apoptotic and anti-apoptotic signaling mechanisms that are activated and act locally in synapses, growth cones, and dendrites to modify their structure and function. Apoptotic synaptic cascades that may play roles in neuronal plasticity include activation of caspases that can cleave certain types of ionotropic glutamate-receptor subunits and thereby modify synaptic plasticity. Caspases may also cleave cytoskeletal protein substrates in growth cones of developing neurons and may thereby regulate neurite outgrowth. Par-4 and the tumor-suppressor protein p53 are pro-apoptotic proteins that may also function in synaptic and developmental plasticity. Examples of anti-apoptotic signals that regulate the plasticity of growth cones and synapses include neurotrophic factor-activated kinase cascades, calcium-mediated actin depolymerization, and activation of the transcription factor NF-kappaB. The emerging data strongly suggest that many of the signaling mechanisms that control apoptosis are also involved in regulating the structural and functional plasticity of neuronal circuits under physiological conditions.

Animals↗