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  • Mechanism of calcium gating in small-conductance calcium-activated potassium channels. 9774106

    The slow afterhyperpolarization that follows an action potential is generated by the activation of small-conductance calcium-activated potassium channels (SK channels). The slow afterhyperpolarization limits the firing frequency of repetitive action potentials (spike-frequency adaptation) and is essential for normal neurotransmission. SK channels are voltage-independent and activated by submicromolar concentrations of intracellular calcium. They are high-affinity calcium sensors that transduce fluctuations in intracellular calcium concentrations into changes in membrane potential. Here we study the mechanism of calcium gating and find that SK channels are not gated by calcium binding directly to the channel alpha-subunits. Instead, the functional SK channels are heteromeric complexes with calmodulin, which is constitutively associated with the alpha-subunits in a calcium-independent manner. Our data support a model in which calcium gating of SK channels is mediated by binding of calcium to calmodulin and subsequent conformational alterations in the channel protein.
    Document Type:
    Reference
    Product Catalog Number:
    05-173
    Product Catalog Name:
    Anti-Calmodulin Antibody

  • The role of AMPA receptor gating in the development of high-fidelity neurotransmission at the calyx of Held synapse. 14715951

    During early postnatal development of auditory synapses, the decay time course of AMPA receptor (AMPAR) EPSCs accelerates markedly, but the mechanisms underlying this process remain uncertain. Using the developing calyx of Held synapse in the mouse auditory brainstem, we have examined presynaptic and postsynaptic elements that may regulate decay kinetics of AMPAR EPSCs. We found that the decay time kinetics was voltage dependent in both immature and mature synapses, being slower at positive potentials than negative potentials. By recording evoked miniature events in extracellular Ca2+ or Sr2+, we revealed a significant decrease in decay time constants of EPSCs as maturation progresses. On the basis of internal and external polyamine block of AMPAR EPSCs and immunohistochemistry assays with subunit-specific antibodies, we demonstrated that the glutamate receptor (GluR) 2 subunit is virtually absent at all developmental ages. Antibody staining patterns suggest a gradual shift in subunit composition from GluR1- to GluR3/4-dominant phenotypes. Kinetic analyses of deactivation, desensitization, and recovery from desensitization in outside-out patches in response to ultrafast application of glutamate lend supportive evidence that such a shift in the gating phenotype likely accounts for the accelerated time course throughout development. Finally, by pharmacologically manipulating AMPAR gating and using simulated EPSCs to evoke action potentials, we demonstrated that rapid decay kinetics of AMPAR EPSCs is essential for this synapse to accommodate high-frequency firing without compromising spike amplitude. Hence, developmental alterations in the subunit composition likely dictate changes in the time course of AMPAR EPSCs and play an indispensable role in the refinement of high-fidelity neurotransmission at the calyx of Held synapse.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Involvement of lysine residues in the gating of the ryanodine receptor/Ca2+-release channel of skeletal muscle sarcoplasmic reticulum. 9288920

    In this study, the modification of skeletal muscle ryanodine receptor (RyR)/Ca2+-release channel with 7-chloro-4-nitrobenzo-2-oxa-1,3,-diazole (Nbd-Cl) demonstrates that lysyl residues are involved in the channel gating. Nbd-Cl was found to have a dual effect: stimulation and inhibition of ryanodine binding and single channel activities. Nbd-Cl, in a time-dependent manner, first stimulated and subsequently inhibited ryanodine binding to both membrane-bound and purified RyR. Incubation of sacroplasmic reticulum membranes with Nbd-Cl for 5-20 s resulted in enhanced ryanodine-binding activity by 2-4-fold due, to an increased binding affinity by about tenfold, with no effect on the total binding sites (Bmax). However, under prolonged incubation (5-20 min), Nbd-Cl strongly inhibited ryanodine binding by decreasing the Bmax with no effect on the binding affinity. Similar effects of stimulation and inhibition by Nbd-Cl were obtained with single channel activity of RyR reconstituted into planar lipid bilayer. Nbd-Cl initially (within a few seconds) activated the channel to a highly open state, then (within a few minutes) inactivated it to the completely closed state. Nbd-Cl-modified protein, as assayed by ryanodine binding or single channel activities, was stable against thiolysis by dithiothreitol, suggesting Nbd-Cl modification of lysyl residues. Evidence from absorption and fluorescence excitation and emission spectra also demonstrated that lysyl residues in RyR were modified by Nbd-Cl. Spectrophotometric data were used to estimate a ratio of up to 1 mol Nbd bound/mol RyR (tetramer) and up to 4 mol Nbd bound per mol RyR (tetramer) for Nbd-Cl stimulated and inhibited RyR activities, respectively. The results clearly indicate the involvement of two classes of lysyl residues in RyR activity. Modification by Nbd-Cl of the fast-reacting group led to stimulation of ryanodine binding and single channel activities, while modification of the slow-reacting group resulted in inhibition of these activities. Thus, the involvement of lysine residues in the gating of the RyR channel is proposed.
    Document Type:
    Reference
    Product Catalog Number:
    17-191
    Product Catalog Name:
    MAP Kinase/Erk Assay Kit, non-radioactive

  • Temporal lobe epilepsy induces intrinsic alterations in Na channel gating in layer II medial entorhinal cortex neurons. 20946956

    Temporal lobe epilepsy (TLE) is the most common form of adult epilepsy involving the limbic structures of the temporal lobe. Layer II neurons of the entorhinal cortex (EC) form the major excitatory input into the hippocampus via the perforant path and consist of non-stellate and stellate neurons. These neurons are spared and hyper-excitable in TLE. The basis for the hyper-excitability is likely multifactorial and may include alterations in intrinsic properties. In a rat model of TLE, medial EC (mEC) non-stellate and stellate neurons had significantly higher action potential (AP) firing frequencies than in control. The increase remained in the presence of synaptic blockers, suggesting intrinsic mechanisms. Since sodium (Na) channels play a critical role in AP generation and conduction we sought to determine if Na channel gating parameters and expression levels were altered in TLE. Na channel currents recorded from isolated mEC TLE neurons revealed increased Na channel conductances, depolarizing shifts in inactivation parameters and larger persistent (I(NaP)) and resurgent (I(NaR)) Na currents. Immunofluorescence experiments revealed increased staining of Na(v)1.6 within the axon initial segment and Na(v)1.2 within the cell bodies of mEC TLE neurons. These studies provide support for additional intrinsic alterations within mEC layer II neurons in TLE and implicate alterations in Na channel activity and expression, in part, for establishing the profound increase in intrinsic membrane excitability of mEC layer II neurons in TLE. These intrinsic changes, together with changes in the synaptic network, could support seizure activity in TLE.
    Document Type:
    Reference
    Product Catalog Number:
    MAB377
    Product Catalog Name:
    Anti-NeuN Antibody, clone A60

  • The effects of glycine transporter I inhibitor, N-methylglycine (sarcosine), on ketamine-induced alterations in sensorimotor gating and regional brain c-Fos expression in ... 19944746

    Reduced N-methyl-D-aspartate receptor (NMDAR) function may contribute to the pathogenesis of schizophrenia. Sarcosine, a potent glycine transporter inhibitor, can increase synaptic glycine and then promote NMDAR function. We assessed the antipsychotic potential of sarcosine by comparing the abilities of sarcosine and clozapine to restore the prepulse inhibition (PPI) deficit, hyperlocomotion and regional brain c-Fos expression changes caused by an NMDAR antagonist, ketamine. Four groups of rats were given acute injections, including saline+saline, saline+30 mg/kg ketamine, 100mg/kg sarcosine+30 mg/kg ketamine, and 15 mg/kg clozapine+30 mg/kg ketamine. Both sarcosine and clozapine reversed the ketamine-induced PPI deficit and hyperlocomotion. They both did not change ketamine-induced increase in c-Fos expression in the prefrontal cortex and nucleus accumbens. However, in the olfactory bulb, sarcosine, but not clozapine, significantly reduced the ketamine-induced increase in c-Fos expression. Our animal study demonstrated that sarcosine may have antipsychotic potential.
    Document Type:
    Reference
    Product Catalog Number:
    05-100

  • Heterotypic docking of Cx43 and Cx45 connexons blocks fast voltage gating of Cx43. 11509355

    Immunohistochemical co-localization of distinct connexins (Cxs) in junctional areas suggests the formation of heteromultimeric channels. To determine the docking effects of the heterotypic combination of Cx43 and Cx45 on the voltage-gating properties of their channels, we transfected DNA encoding Cx43 or Cx45 into N2A neuroblastoma or HeLa cells. Using a double whole-cell voltage-clamp technique, we determined macroscopic and single-channel gating properties of the intercellular channels formed. Cx43-Cx45 heterotypic channels had rectifying properties where Cx45 connexons inactivated rapidly upon hyperpolarizing voltage pulses applied to the Cx45-expressing cell. During depolarizing pulses to the Cx45-expressing cell, Cx43 connexons inactivated with substantially reduced kinetics as compared with homotypic Cx43 channels. Similar slow kinetics was observed for homotypic Cx43M257 (truncation mutant). Heterotypic channels had a main conductance whose value was predicted by the sum of corresponding homomeric connexon conductances; it was not voltage dependent and had no detectable residual conductance. The voltage-gating kinetics of heterotypic channels and their single-channel behavior implicate a role for the Cx43 carboxyl-terminal domain in the fast gating mechanism and in the establishment of residual conductance. Our results also suggest that heterotypic docking may lead to conformational changes that inhibit this action of the Cx43 carboxyl-terminal domain.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3100
    Product Catalog Name:
    Anti-Connexin 45 Antibody, near CT, cytoplasmic, clone 8A11.2

  • Cornichon-2 modulates AMPA receptor-transmembrane AMPA receptor regulatory protein assembly to dictate gating and pharmacology. 21543622

    Neuronal AMPA receptor complexes comprise a tetramer of GluA pore-forming subunits as well as accessory components, including transmembrane AMPA receptor regulatory proteins (TARPs) and cornichon-2/3 (CNIH-2/3). The mechanisms that control AMPA receptor complex assembly remain unclear. AMPA receptor responses in neurons differ from those in cell lines transfected with GluA plus TARPs γ-8 or γ-7, which show unusual resensitization kinetics and non-native AMPA receptor pharmacologies. Using tandem GluA/TARP constructs to constrain stoichiometry, we show here that these peculiar kinetic and pharmacological signatures occur in channels with four TARP subunits per complex. Reducing the number of TARPs per complex produces AMPA receptors with neuron-like kinetics and pharmacologies, suggesting a neuronal mechanism controls GluA/TARP assembly. Importantly, we find that coexpression of CNIH-2 with GluA/TARP complexes reduces TARP stoichiometry within AMPA receptors. In both rat and mouse hippocampal neurons, CNIH-2 also associates with AMPA receptors on the neuronal surface in a γ-8-dependent manner to dictate receptor pharmacology. In the cerebellum, however, CNIH-2 expressed in Purkinje neurons does not reach the neuronal surface. In concordance, stargazer Purkinje neurons, which express CNIH-2 and γ-7, display AMPA receptor kinetics/pharmacologies that can only be recapitulated recombinantly by a low γ-7/GluA stoichiometry. Together, these data suggest that CNIH-2 modulates neuronal AMPA receptor auxiliary subunit assembly by regulating the number of TARPs within an AMPA receptor complex to modulate receptor gating and pharmacology.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Selective Neuronal and Brain Regional Expession of IL-2 in IL2P 8-GFP Transgenic Mice: Relation to Sensorimotor Gating. 24563821

    Brain-derived interleukin-2 (IL-2) has been implicated in diseases processes that arise during CNS development (e.g., autism) to neurodegenerative alterations involving neuroinflammation (e.g., Alzheimer's disease). Progress has been limited, however, because the vast majority of current knowledge of IL-2's actions on brain function and behavior is based on the use exogenously administered IL-2 to make inferences about the function of the endogenous cytokine. Thus, to identify the cell-type(s) and regional circuitry that express brain-derived IL-2, we used B6.Cg-Tg/ IL2-EGFP17Evr (IL2p8-GFP) transgenic mice, which express green fluorescent protein (GFP) in peripheral immune cells known to produce IL-2. We found that the IL2-GFP transgene was localized almost exclusively to NeuN-positive cells, indicating that the IL-2 is produced primarily by neurons. The IL2-GFP transgene was expressed in discrete nuclei throughout the rostral-caudal extent of the brain and brainstem, with the highest levels found in the cingulate, dorsal endopiriform nucleus, lateral septum, nucleus of the solitary tract, magnocellular/gigantocellular reticular formation, red nucleus, entorhinal cortex, mammilary bodies, cerebellar fastigial nucleus, and posterior interposed nucleus. Having identified IL-2 gene expression in brain regions associated with the regulation of sensorimotor gating (e.g., lateral septum, dorsal endopiriform nucleus, entorhinal cortex, striatum), we compared prepulse inhibition (PPI) of the acoustic startle response in congenic mice bred in our lab that have selective loss of the IL-2 gene in the brain versus the peripheral immune system, to test the hypothesis that brain-derived IL-2 plays a role in modulating PPI. We found that congenic mice devoid of IL-2 gene expression in both the brain and the peripheral immune system, exhibited a modest alteration of PPI. These finding suggest that IL2p8-GFP transgenic mice may be a useful tool to elucidate further the role of brain-derived IL-2 in normal CNS function and disease.
    Document Type:
    Reference
    Product Catalog Number:
    MAB377
    Product Catalog Name:
    Anti-NeuN Antibody, clone A60

  • Deletion of selenoprotein P results in impaired function of parvalbumin interneurons and alterations in fear learning and sensorimotor gating. 22640876

    One of the primary lines of defense against oxidative stress is the selenoprotein family, a class of proteins that contain selenium in the form of the 21st amino acid, selenocysteine. Within this class of proteins, selenoprotein P (Sepp1) is unique, as it contains multiple selenocysteine residues and is postulated to act in selenium transport. Recent findings have demonstrated that neuronal selenoprotein synthesis is required for the development of parvalbumin (PV)-interneurons, a class of GABAergic neurons involved in the synchronization of neural activity. To investigate the potential influence of Sepp1 on PV-interneurons, we first mapped the distribution of the Sepp1 receptor, ApoER2, and parvalbumin in the mouse brain. Our results indicate that ApoER2 is highly expressed on PV-interneurons in multiple brain regions. Next, to determine whether PV-interneuron populations are affected by Sepp1 deletion, we performed stereology on several brain regions in which we observed ApoER2 expression on PV-interneurons, comparing wild-type and Sepp1(-/-) mice. We observed reduced numbers of PV-interneurons in the inferior colliculus of Sepp1(-/-) mice, which corresponded with a regional increase in oxidative stress. Finally, as impaired PV-interneuron function has been implicated in several neuropsychiatric conditions, we performed multiple behavioral tests on Sepp1(-/-) mice. Our behavioral results indicate that Sepp1(-/-) mice have impairments in contextual fear extinction, latent inhibition, and sensorimotor gating. In sum, these findings demonstrate the important supporting role of Sepp1 on ApoER2-expressing PV-interneurons.
    Document Type:
    Reference
    Product Catalog Number:
    MAB5406
    Product Catalog Name:
    Anti-GAD67 Antibody, clone 1G10.2