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1.  Regulation of Protein Phosphatase Inhibitor-1 by Cyclin-dependent Kinase 5* 
The Journal of biological chemistry  2007;282(22):16511-16520.
Inhibitor-1, the first identified endogenous inhibitor of protein phosphatase 1 (PP-1), was previously reported to be a substrate for cyclin-dependent kinase 5 (Cdk5) at Ser67. Further investigation has revealed the presence of an additional Cdk5 site identified by mass spectrometry and confirmed by site-directed mutagenesis as Ser6. Basal levels of phospho-Ser6 inhibitor-1, as detected by a phosphorylation state-specific antibody against the site, existed in specific regions of the brain and varied with age. In the striatum, basal in vivo phosphorylation and dephosphorylation of Ser6 were mediated by Cdk5, PP-2A, and PP-1, respectively. Additionally, calcineurin contributed to dephosphorylation under conditions of high Ca2+. In biochemical assays the function of Cdk5-dependent phosphorylation of inhibitor-1 at Ser6 and Ser67 was demonstrated to be an intramolecular impairment of the ability of inhibitor-1 to be dephosphorylated at Thr35; this effect was recapitulated in two systems in vivo. Dephosphorylation of inhibitor-1 at Thr35 is equivalent to inactivation of the protein, as inhibitor-1 only serves as an inhibitor of PP-1 when phosphorylated by cAMP-dependent kinase (PKA) at Thr35. Thus, inhibitor-1 serves as a critical junction between kinase- and phosphatase-signaling pathways, linking PP-1 to not only PKA and calcineurin but also Cdk5.
PMCID: PMC4296900  PMID: 17400554
2.  Isoflurane Reversibly Destabilizes Hippocampal Dendritic Spines by an Actin-Dependent Mechanism 
PLoS ONE  2014;9(7):e102978.
General anesthetics produce a reversible coma-like state through modulation of excitatory and inhibitory synaptic transmission. Recent evidence suggests that anesthetic exposure can also lead to sustained cognitive dysfunction. However, the subcellular effects of anesthetics on the structure of established synapses are not known. We investigated effects of the widely used volatile anesthetic isoflurane on the structural stability of hippocampal dendritic spines, a postsynaptic structure critical to excitatory synaptic transmission in learning and memory. Exposure to clinical concentrations of isoflurane induced rapid and non-uniform shrinkage and loss of dendritic spines in mature cultured rat hippocampal neurons. Spine shrinkage was associated with a reduction in spine F-actin concentration. Spine loss was prevented by either jasplakinolide or cytochalasin D, drugs that prevent F-actin disassembly. Isoflurane-induced spine shrinkage and loss were reversible upon isoflurane elimination. Thus, isoflurane destabilizes spine F-actin, resulting in changes to dendritic spine morphology and number. These findings support an actin-based mechanism for isoflurane-induced alterations of synaptic structure in the hippocampus. These reversible alterations in dendritic spine structure have important implications for acute anesthetic effects on excitatory synaptic transmission and synaptic stability in the hippocampus, a locus for anesthetic-induced amnesia, and have important implications for anesthetic effects on synaptic plasticity.
PMCID: PMC4113311  PMID: 25068870
3.  Regional differences in the effects of isoflurane on neurotransmitter release 
Neuropharmacology  2011;61(4):699-706.
Stimulus evoked neurotransmitter release requires that Na+ channel-dependent nerve terminal depolarization be transduced into synaptic vesicle exocytosis. Inhaled anesthetics block presynaptic Na+ channels and selectively inhibit glutamate over GABA release from isolated nerve terminals, indicating mechanistic differences between excitatory and inhibitory transmitter release. We compared the effects of isoflurane on depolarization-evoked [3H]glutamate and [14C]GABA release from isolated nerve terminals prepared from four regions of rat CNS evoked by 4-aminopyridine (4AP), veratridine (VTD), or elevated K+. These mechanistically distinct secretegogues distinguished between Na+ channel- and/or Ca2+ channel-mediated presynaptic effects. Isoflurane completely inhibited total 4AP-evoked glutamate release (IC50=0.42 ± 0.03 mM) more potently than GABA release (IC50=0.56 ± 0.02 mM) from cerebral cortex (1.3-fold greater potency), hippocampus and striatum, but inhibited glutamate and GABA release from spinal cord terminals equipotently. Na+ channel-specific VTD-evoked glutamate release from cortex was also significantly more sensitive to inhibition by isoflurane than was GABA release. Na+ channel-independent K+-evoked release was insensitive to isoflurane at clinical concentrations in all four regions, consistent with a target upstream of Ca2+ entry. Isoflurane inhibited Na+ channel-mediated (tetrodotoxin-sensitive) 4AP-evoked glutamate release (IC50=0.30 ± 0.03 mM) more potently than GABA release (IC50=0.67 ± 0.04 mM) from cortex (2.2-fold greater potency). The magnitude of inhibition of Na+ channel-mediated 4AP-evoked release by a single clinical concentration of isoflurane (0.35 mM) varied by region and transmitter: Inhibition of glutamate release from spinal cord was greater than from the three brain regions and greater than GABA release for each CNS region. These findings indicate that isoflurane selectively inhibits glutamate release compared to GABA release via Na+ channel-mediated transduction in the four CNS regions tested, and that differences in presynaptic Na+ channel involvement determine differences in anesthetic pharmacology.
PMCID: PMC3130078  PMID: 21651920
Na+ channels; glutamate; GABA; nerve terminal; tetrodotoxin; rat
4.  Activation of brain protein phosphatase-1I following cardiac arrest and resuscitation involving an interaction with 14-3-3γ 
Journal of neurochemistry  2008;105(5):10.1111/j.1471-4159.2008.05300.x.
The intracellular signaling mechanisms that couple transient cerebral ischemia to cell death and neuroprotective mechanisms provide potential therapeutic targets for cardiac arrest. Protein phosphatase (PP)-1 is a major serine/threonine phosphatase that interacts with and dephosphorylates critical regulators of energy metabolism, ionic balance, and apoptosis. We report here that PP-1I, a major regulated form of PP-1, is activated in brain by approximately twofold in vivo following cardiac arrest and resuscitation in a clinically relevant pig model of transient global cerebral ischemia and reperfusion. PP-1I purified to near homogeneity from either control or ischemic pig brain consisted of the PP-1 catalytic subunit, the inhibitor-2 regulatory subunit, as well as the novel constituents 14-3-3γ, Rab GDP dissociation protein β, PFTAIRE kinase, and C-TAK1 kinase. PP-1I purified from ischemic brain contained significantly less 14-3-3γ than PP-1I purified from control brain, and purified 14-3-3γ directly inhibited the catalytic subunit of PP-1 and reconstituted PP-1I. These findings suggest that activation of brain PP-1I following global cerebral ischemia in vivo involves dissociation of 14-3-3γ, a novel inhibitory modulator of PP-1I. This identifies modulation of PP-1I by 14-3-3 in global cerebral ischemia as a potential signaling mechanism-based approach to neuroprotection.
PMCID: PMC3872065  PMID: 18284617
apoptosis; inhibitor-2; protein phosphorylation
5.  Regional differences in nerve terminal Na+ channel subtype expression and Na+ channel-dependent glutamate and GABA release in rat central nervous system 
Journal of neurochemistry  2010;113(6):1611-1620.
We tested the hypothesis that expression of presynaptic voltage-gated Na+ channel (Nav) subtypes coupled to neurotransmitter release differs between transmitter types and CNS regions in a nerve terminal-specific manner. Nav coupling to transmitter release was determined by measuring the sensitivity of 4-aminopyridine (4AP)-evoked [3H]glutamate and [14C]GABA release to the specific Nav blocker tetrodotoxin (TTX) for nerve terminals isolated from rat cerebral cortex, hippocampus, striatum and spinal cord. Expression of various Nav subtypes was measured by immunoblotting using subtype-specific antibodies. Potencies of TTX for inhibition of glutamate and GABA release were similar between CNS regions. However, the efficacies of TTX for inhibition of 4AP-evoked glutamate release were greater than for inhibition of GABA release in all regions except spinal cord. The relative nerve terminal expression of total Nav subtypes as well as of specific subtypes varied considerably between CNS regions. The region-specific potencies of TTX for inhibition of 4AP-evoked glutamate release correlated with greater relative expression of total nerve terminal Nav and Nav1.2. Nerve terminal-specific differences in the expression of specific Nav subtypes contribute to transmitter-specific and regional differences in pharmacological sensitivities of transmitter release.
PMCID: PMC2914626  PMID: 20374421
6.  Comparative Effects of Halogenated Inhaled Anesthetics on Voltage-gated Na+ Channel Function 
Anesthesiology  2009;110(3):582-590.
Inhibition of voltage-gated Na+ channels (Nav) is implicated in the synaptic actions of volatile anesthetics. We studied the effects of the major halogenated inhaled anesthetics (halothane, isoflurane, sevoflurane, enflurane and desflurane) on Nav1.4, a well characterized pharmacological model for Nav effects.
Na+ currents (INa) from rat Nav1.4 α-subunits heterologously expressed in Chinese hamster ovary cells were analyzed by whole cell voltage-clamp electrophysiological recording.
Halogenated inhaled anesthetics reversibly inhibited Nav1.4 in a concentration- and voltage-dependent manner at clinical concentrations. At equi-anesthetic concentrations, peak INa was inhibited with a rank order of desflurane > halothane ≈ enflurane > isoflurane ≈ sevoflurane from a physiological holding potential (−80 mV). This suggests that the contribution of Na+ channel block to anesthesia might vary in an agent-specific manner. From a hyperpolarized holding potential that minimizes inactivation (−120 mV), peak INa was inhibited with a rank order of potency for tonic inhibition of peak INa of halothane > isoflurane ≈ sevoflurane > enflurane > desflurane. Desflurane produced the largest negative shift in voltage-dependence of fast inactivation consistent with its more prominent voltage-dependent effects. A comparison between isoflurane and halothane showed that halothane produced greater facilitation of current decay, slowing of recovery from fast inactivation, and use-dependent block than isoflurane.
Five halogenated inhaled anesthetics all inhibit a voltage-gated Na+ channel by voltage- and use-dependent mechanisms. Agent-specific differences in efficacy for Na+ channel inhibition due to differential state-dependent mechanisms creates pharmacologic diversity that could underlie subtle differences in anesthetic and nonanesthetic actions.
PMCID: PMC2699670  PMID: 19225394
7.  Thiazolidinedione insulin sensitizers alter lipid bilayer properties and voltage-dependent sodium channel function: implications for drug discovery 
The Journal of General Physiology  2011;138(2):249-270.
The thiazolidinediones (TZDs) are used in the treatment of diabetes mellitus type 2. Their canonical effects are mediated by activation of the peroxisome proliferator–activated receptor γ (PPARγ) transcription factor. In addition to effects mediated by gene activation, the TZDs cause acute, transcription-independent changes in various membrane transport processes, including glucose transport, and they alter the function of a diverse group of membrane proteins, including ion channels. The basis for these off-target effects is unknown, but the TZDs are hydrophobic/amphiphilic and adsorb to the bilayer–water interface, which will alter bilayer properties, meaning that the TZDs may alter membrane protein function by bilayer-mediated mechanisms. We therefore explored whether the TZDs alter lipid bilayer properties sufficiently to be sensed by bilayer-spanning proteins, using gramicidin A (gA) channels as probes. The TZDs altered bilayer elastic properties with potencies that did not correlate with their affinity for PPARγ. At concentrations where they altered gA channel function, they also altered the function of voltage-dependent sodium channels, producing a prepulse-dependent current inhibition and hyperpolarizing shift in the steady-state inactivation curve. The shifts in the inactivation curve produced by the TZDs and other amphiphiles can be superimposed by plotting them as a function of the changes in gA channel lifetimes. The TZDs’ partition coefficients into lipid bilayers were measured using isothermal titration calorimetry. The most potent bilayer modifier, troglitazone, alters bilayer properties at clinically relevant free concentrations; the least potent bilayer modifiers, pioglitazone and rosiglitazone, do not. Unlike other TZDs tested, ciglitazone behaves like a hydrophobic anion and alters the gA monomer–dimer equilibrium by more than one mechanism. Our results provide a possible mechanism for some off-target effects of an important group of drugs, and underscore the importance of exploring bilayer effects of candidate drugs early in drug development.
PMCID: PMC3149818  PMID: 21788612
8.  Localization of DARPP-32 and inhibitor-1 in area 9 of Macaca mulatta prefrontal cortex 
Neuroscience  2010;167(2):428-438.
The actions of dopamine D1 family receptors (D1R) depend upon a signal transduction cascade that modulates the phosphorylation state of important effector proteins, such as glutamate receptors and ion channels. This is accomplished both through activation of protein kinase A (PKA) and the inhibition of protein phosphatase-1 (PP1). Inhibition of PP1 occurs through PKA-mediated phosphorylation of DARPP-32 or the related protein inhibitor-1 (I-1), and the availability of DARPP-32 is essential to the functional outcome of D1R activation in the basal ganglia. While D1R activation is critical for prefrontal cortex (PFC) function, especially working memory, the functional role played by DARPP-32 or I-1 is less clear. In order to examine this more thoroughly, we have utilized immunoelectron microscopy to quantitatively determine the localization of DARPP-32 and I-1 in the neuropil of the rhesus monkey PFC. Both were distributed widely in the different components of the neuropil, but were enriched in dendritic shafts. I-1 label was more frequently identified in axon terminals than was DARPP-32, and DARPP-32 label was more frequently identified in glia than was I-1. We also quantified the extent to which these proteins were found in dendritic spines. DARPP-32 and I-1 were present in small subpopulations of dendritic spines, (4.4 and 7.7% and respectively), which were substantially smaller than observed for D1R in our previous studies (20%). Double-label experiments did not find evidence for colocalization of D1R and DARPP-32 or I-1 in spines or terminals. Thus, at the least, not all prefrontal spines which contain D1R also contain I-1 or DARPP-32, suggesting important differences in D1R signaling in the PFC compared to the striatum.
PMCID: PMC2863358  PMID: 20156529
electron microscopy; dendritic spines; D1; D5; protein phosphatase-1; dopamine
9.  Neurotoxicity of General Anesthetics: Cause for Concern? 
Anesthesiology  2009;111(6):1365-1371.
PMCID: PMC2784653  PMID: 19934883
10.  Isoflurane Inhibits the Tetrodotoxin-resistant Voltagegated Sodium Channel Nav1.8 
Anesthesiology  2009;111(3):591-599.
Voltage-gated sodium channels (Nav) mediate neuronal action potentials. Tetrodotoxin inhibits all Nav isoforms, but Nav1.8 and Nav1.9 are relatively tetrodotoxin-resistant (TTX-r) compared to other isoforms. Nav1.8 is highly expressed in dorsal root ganglion neurons and is functionally linked to nociception, but the sensitivity of TTX-r isoforms to inhaled anesthetics is unclear.
The sensitivities of heterologously expressed rat TTX-r Nav1.8 and endogenous tetrodotoxin-sensitive (TTX-s) Nav to the prototypic inhaled anesthetic isoflurane were tested in mammalian ND7/23 cells using patch-clamp electrophysiology.
From a holding potential of −70 mV, isoflurane (0.53±0.06 mM, ~1.8 MAC at 24°C) reduced normalized peak Na+ current (INa) of Nav1.8 to 0.55±0.03 and of endogenous TTX-s Nav to 0.56±0.06. Isoflurane minimally inhibited INa from a holding potential of −140 mV. Isoflurane did not affect voltage-dependence of activation, but significantly shifted voltage-dependence of steady-state inactivation by −6 mV for Nav1.8 and by −7 mV for TTX-s Nav. IC50 values for inhibition of peak INa were 0.67±0.06 mM for Nav1.8 and 0.66±0.09 mM for TTX-s Nav; significant inhibition occurred at clinically relevant concentrations as low as 0.58 MAC. Isoflurane produced use-dependent block of Nav1.8; at a stimulation frequency of 10 Hz, 0.56±0.08 mM isoflurane reduced INa to 0.64±0.01 vs. 0.78±0.01 for control.
Isoflurane inhibited the tetrodotoxin-resistant isoform Nav1.8 with potency comparable to that for endogenous tetrodotoxin-sensitive Nav isoforms, indicating that sensitivity to inhaled anesthetics is conserved across diverse Nav family members. Block of Nav1.8 in dorsal root ganglion neurons could contribute to the effects of inhaled anesthetics on peripheral nociceptive mechanisms.
PMCID: PMC2756082  PMID: 19672182
11.  Is a New Paradigm Needed to Explain How Inhaled Anesthetics Produce Immobility? 
Anesthesia and analgesia  2008;107(3):832-848.
A paradox arises from present information concerning the mechanism(s) by which inhaled anesthetics produce immobility in the face of noxious stimulation. Several findings, such as additivity, suggest a common site at which inhaled anesthetics act to produce immobility. However, two decades of focused investigation have not identified a ligand- or voltage-gated channel that alone is sufficient to mediate immobility. Indeed, most putative targets provide minimal or no mediation. For example, opioid, 5-HT3, gamma-aminobutyric acid type A and glutamate receptors, and potassium and calcium channels appear to be irrelevant or play only minor roles. Furthermore, no combination of actions on ligand- or voltage-gated channels seems sufficient. A few plausible targets (e.g., sodium channels) merit further study, but there remains the possibility that immobilization results from a nonspecific mechanism.
PMCID: PMC2653203  PMID: 18713892
12.  Intrathecal Veratridine Administration Increases MAC in Rats 
Anesthesia and analgesia  2008;107(3):875-878.
Results from several studies point to sodium channels as potential mediators of the immobility produced by inhaled anesthetics. We hypothesized that the intrathecal administration of veratridine, a drug that enhances the activity or effect of sodium channels, should increase MAC.
We measured the change in isoflurane MAC caused by intrathecal infusion of various concentrations of veratridine into the lumbothoracic subarachnoid space of rats. We compared these result to those obtained from intracerebroventricular infusion.
As predicted, intrathecal infusion of veratridine increased MAC. The greatest infused concentration (25 μM) also produced neuronal injury in the hind limbs of two rats and decreased the peak effect on MAC. A concentration of 1.6 μM produced the greatest (21%) increase in MAC. Intraventricular infusion of 1.6 and 6.4 μM veratridine did not alter MAC. Rats given 25 μM died.
Intrathecal administration of veratradine increases MAC of isoflurane, a finding consistent with a role for sodium channels as potential mediators of the immobility produced by inhaled anesthetics.
Intrathecal administration of veratridine can increase MAC, presumably by an effect on sodium channels.
PMCID: PMC2587212  PMID: 18713899
Anesthetics; inhaled; isoflurane; Anesthetic potency; MAC; ECso; Catecholamines; Mechanisms of anesthetic action; Sodium channels; Veratridine
13.  Differential regulation of protein phosphatase-1I by neurabin 
Neurabin is a brain-specific actin and protein phosphatase-1 (PP-1) binding protein that inhibits the purified catalytic subunit of protein phosphatase-1 (PP-1C). However, endogenous PP-1 exists primarily as multimeric complexes of PP-1C bound to various regulatory proteins that determine its activity, substrate specificity, subcellular localization and function. The major form of endogenous PP-1 in brain is protein phosphatase-1I (PP-1I), a Mg2+/ATP-dependent form of PP-1 that consists of PP-1C, the inhibitor-2 regulatory subunit, an activating protein kinase and other unidentified proteins. We have identified four PP-1I holoenzyme fractions (PP-1IA, PP-1IB, PP-1IC and PP-1ID) in freshly harvested pig brain separable by poly-L-lysine chromatography. Purified recombinant neurabin (amino acid residues 1-485) inhibited PP-1IB (IC50=1.1 μM), PP-1IC (IC50=0.1 μM) and PP-1ID (IC50=0.2 μM), but activated PP-1IA by up to 3-fold (EC50=40 nM). The PP-1IA activation domain was localized to neurabin1-210. Our results indicate a novel mechanism of PP-1 regulation by neurabin as both an inhibitor and an activator of distinct forms of PP-1I in brain.
PMCID: PMC1989152  PMID: 17467665
protein phosphatase-1; neurabin; PP-1 inhibitor; actin binding protein

Results 1-13 (13)