Altered expression and localization of hippocampal A-type potassium channel subunits in the pilocarpine-induced model of temporal lobe epilepsy.

Altered ion channel expression and/or function may contribute to the development of certain human epilepsies. In rats, systemic administration of pilocarpine induces a model of human temporal lobe epilepsy, wherein a brief period of status epilepticus (SE) triggers development of spontaneous recurrent seizures that appear after a latency of 2-3 weeks. Here we investigate changes in expression of A-type voltage-gated potassium (Kv) channels, which control neuronal excitability and regulate action potential propagation and neurotransmitter release, in the pilocarpine model of epilepsy. Using immunohistochemistry, we examined the expression of component subunits of somatodendritic (Kv4.2, Kv4.3, KChIPl and KChIP2) and axonal (Kv1.4) A-type Kv channels in hippocampi of pilocarpine-treated rats that entered SE. We found that Kv4.2, Kv4.3 and KChIP2 staining in the molecular layer of the dentate gyrus changes from being uniformly distributed across the molecular layer to concentrated in just the outer two-thirds. We also observed a loss of KChIP1 immunoreactive interneurons, and a reduction of Kv4.2 and KChIP2 staining in stratum radiatum of CA1. These changes begin to appear 1 week after pilocarpine treatment and persist or are enhanced at 4 and 12 weeks. As such, these changes in Kv channel distribution parallel the acquisition of recurrent spontaneous seizures as observed in this model. We also found temporal changes in Kv1.4 immunoreactivity matching those in Timm's stain, being expanded in stratum lucidum of CA3 and in the inner third of the dentate molecular layer. Among pilocarpine-treated rats, changes were only observed in those that entered SE. These changes in A-type Kv channel expression may contribute to hyperexcitability of dendrites in the associated hippocampal circuits as observed in previous studies of the effects of pilocarpine-induced SE.

Identification of a trafficking determinant localized to the Kv1 potassium channel pore.

The repertoire of Kv1 potassium channels expressed in presynaptic terminals of mammalian central neurons is shaped by intrinsic trafficking signals that determine surface-expression efficiencies of homomeric and heteromeric Kv1 channel complexes. Here, we show that a determinant controlling surface expression of Kv1 channels is localized to the highly conserved pore region. Point-mutation analysis revealed two residues as critical for channel trafficking, one in the extracellular "turret" domain and one in the region distal to the selectivity filter. Interestingly, these same residues also form the binding sites for polypeptide neurotoxins. Our findings demonstrate a previously uncharacterized function for the channel-pore domain as a regulator of channel trafficking.

Experimental localization of Kv1 family voltage-gated K+ channel alpha and beta subunits in rat hippocampal formation.

In the mammalian hippocampal formation, dendrotoxin-sensitive voltage-gated K(+) (Kv) channels modulate action potential propagation and neurotransmitter release. To explore the neuroanatomical basis for this modulation, we used in situ hybridization, coimmunoprecipitation, and immunohistochemistry to determine the subcellular localization of the Kv channel subunits Kv1.1, Kv1.2, Kv1.4, and Kvbeta2 within the adult rat hippocampus. Although mRNAs encoding all four of these Kv channel subunits are expressed in the cells of origin of each major hippocampal afferent and intrinsic pathway, immunohistochemical staining suggests that the encoded subunits are associated with the axons and terminal fields of these cells. Using an excitotoxin lesion strategy, we explored the subcellular localization of these subunits in detail. We found that ibotenic acid lesions of the entorhinal cortex eliminated Kv1.1 and Kv1.4 immunoreactivity and dramatically reduced Kv1.2 and Kvbeta2 immunoreactivity in the middle third of the dentate molecular layer, indicating that these subunits are located on axons and terminals of entorhinal afferents. Similarly, ibotenic acid lesions of the dentate gyrus eliminated Kv1.1 and Kv1.4 immunoreactivity in the stratum lucidum of CA3, indicating that these subunits are located on mossy fiber axons. Kainic acid lesions of CA3 dramatically reduced Kv1.1 immunoreactivity in the stratum radiatum of CA1-CA3, indicating that Kv1.1 immunoreactivity in these subfields is associated with the axons and terminals of the Schaffer collaterals. Together with the results of coimmunoprecipitation analyses, these data suggest that action potential propagation and glutamate release at excitatory hippocampal synapses are directly modulated by Kv1 channel complexes predominantly localized on axons and nerve terminals.

Kinetic modulation of Kv4-mediated A-current by arachidonic acid is dependent on potassium channel interacting proteins.

The Kv4 subfamily of voltage-gated potassium channels is responsible for the transient A-type potassium current that operates at subthreshold membrane potentials to control membrane excitability. Arachidonic acid was shown recently to modulate both the peak amplitude and kinetics of the hippocampal A-current. However, in Xenopus oocytes, arachidonic acid only inhibited the peak amplitude of Kv4 current without modifying its kinetics. These results suggest the existence of Kv4 auxiliary subunit(s) in native cells. We report here a K-channel interacting protein (KChIP)-dependent kinetic modulation of Kv4.2 current in Chinese hamster ovary cells and Kv4.2 and Kv4.3 currents in Xenopus oocytes by arachidonic acid at physiological concentrations. This concentration-dependent effect of arachidonic acid resembled that observed in cerebellar granule neurons and was fully reversible. Other fatty acids, including a nonhydrolyzable inhibitor of both lipooxygenase and cyclooxygenase, 5,8,11,14-eicosatetraynoic acid (ETYA), also mimicked arachidonic acid in modulating Kv4.3 and Kv4.3/KChIP1 currents. Compared with another transient potassium current formed by Kv1.1/Kvbeta1, Kv4.3/KChIP1 current was much more sensitive to arachidonic acid. Association between KChIP1 and Kv4.2 or Kv4.3 was not altered in the presence of 10 microm ETYA as measured by immunoprecipitation and association-dependent growth in yeast. Our data suggest that the KChIP proteins represent a molecular entity for the observed difference between arachidonic acid effects on A-current kinetics in heterologous cells and in native cells and are consistent with the notion that KChIP proteins modulate the subthreshold A-current in neurons.

beta -Amyloid peptide-induced apoptosis regulated by a novel protein containing a g protein activation module.

Degeneration of neurons in Alzheimer's disease is mediated by beta-amyloid peptide by diverse mechanisms, which include a putative apoptotic component stimulated by unidentified signaling events. This report describes a novel beta-amyloid peptide-binding protein (denoted BBP) containing a G protein-coupling module. BBP is one member of a family of three proteins containing this conserved structure. The BBP subtype bound human beta-amyloid peptide in vitro with high affinity and specificity. Expression of BBP in cell culture induced caspase-dependent vulnerability to beta-amyloid peptide toxicity. Expression of a signaling-deficient dominant negative BBP mutant suppressed sensitivity of human Ntera-2 neurons to beta-amyloid peptide mediated toxicity. These findings suggest that BBP is a target of neurotoxic beta-amyloid peptide and provide new insight into the molecular pathophysiology of Alzheimer's disease.

Modulation of A-type potassium channels by a family of calcium sensors.

In the brain and heart, rapidly inactivating (A-type) voltage-gated potassium (Kv) currents operate at subthreshold membrane potentials to control the excitability of neurons and cardiac myocytes. Although pore-forming alpha-subunits of the Kv4, or Shal-related, channel family form A-type currents in heterologous cells, these differ significantly from native A-type currents. Here we describe three Kv channel-interacting proteins (KChIPs) that bind to the cytoplasmic amino termini of Kv4 alpha-subunits. We find that expression of KChIP and Kv4 together reconstitutes several features of native A-type currents by modulating the density, inactivation kinetics and rate of recovery from inactivation of Kv4 channels in heterologous cells. All three KChIPs co-localize and co-immunoprecipitate with brain Kv4 alpha-subunits, and are thus integral components of native Kv4 channel complexes. The KChIPs have four EF-hand-like domains and bind calcium ions. As the activity and density of neuronal A-type currents tightly control responses to excitatory synaptic inputs, these KChIPs may regulate A-type currents, and hence neuronal excitability, in response to changes in intracellular calcium.

Type I and type II Na(+) channel alpha-subunit polypeptides exhibit distinct spatial and temporal patterning, and association with auxiliary subunits in rat brain.

Here we investigate differences in the temporal and spatial patterning, and subunit interactions of two of the major Na(+) channel alpha-subunit isoforms in mammalian brain, the type I and type II Na(+) channels. By using subtype-specific antibodies, we find that both isoforms are abundant in adult rat brain, where both interact with the covalently bound beta2 auxiliary subunit. Immunoblot analysis reveals complementary levels of type I and type II in different brain regions, with the highest levels of type I in brainstem, cortex, substantia nigra, and caudate, where it is found predominantly on the soma of neurons, and the highest levels of type II in globus pallidus, hippocampus and thalamus, where it is preferentially localized to axons. Developmentally, type I Na(+) channel polypeptide expression in brain increases dramatically during the third postnatal week, peaks at the end of the first postnatal month, and then decreases such that adult levels are approximately 50% of those at peak. Type II Na(+) channel polypeptide expression in brain also undergoes large increases in the third postnatal week, but levels continue to increase such that peak expression levels are maintained in adult animals. Type I Na(+) channels are found associated with the auxiliary beta2 subunit at all ages, whereas free type II Na(+) channels exist during the first two postnatal weeks. Thus, although expression of these two Na(+) channel alpha subunits in heterologous systems yields currents with very similar electrophysiological and pharmacological properties, their distinct spatial and temporal patterning, and association with auxiliary subunits in brain, suggest that they perform distinct, nonoverlapping functions in situ.

Immunohistochemical localization of subtype 4a metabotropic glutamate receptors in the rat and mouse basal ganglia.

Recent studies suggest that metabotropic glutamate receptors (mGluRs) may play a significant role in regulating basal ganglia functions. In this study, we investigated the localization of mGluR4a protein in the mouse and rat basal ganglia. Polyclonal antibodies that specifically react with the metabotropic glutamate receptor subtype mGluR4a were produced and characterized by Western blot analysis. These antibodies recognized a native protein in wild-type mouse brain with a molecular weight similar to the molecular weight of the band from a mGluR4a-transfected cell line. The immunoreactivity was absent in brains of knockout mice deficient in mGluR4. mGluR4a immunoreactivity was most intense in the molecular layer of the cerebellum. We also found a striking mGluR4a immunoreactivity in globus pallidus, and moderate staining in substantia nigra pars reticulata and entopeduncular nucleus. Moderate to low mGluR4a immunoreactivity was present in striatum and other brain regions, including hippocampus, neocortex, and thalamus. Double labeling with mGluR4a antibodies and antibodies to either a dendritic marker or a marker of presynaptic terminals suggest a localization of mGluR4a on presynaptic terminals. Immunocytochemistry at electron microscopy level confirmed these results, revealing that in the globus pallidus, mGluR4a is mainly localized in presynaptic sites in axonal elements. Finally, quinolinic acid lesion of striatal projection neurons decreased mGluR4a immunoreactivity in globus pallidus, suggesting a localization of mGluR4a on striatopallidal terminals. These data support the hypothesis that mGluR4a serves as a presynaptic heteroreceptor in the globus pallidus, where it may play an important role in regulating g-amino-n-butyric acid (GABA) release from striatopallidal terminals.

Structure-activity relationships of the Kvbeta1 inactivation domain and its putative receptor probed using peptide analogs of voltage-gated potassium channel alpha- and beta-subunits.

Certain beta-subunits exert profound effects on the kinetics of voltage-gated (Kv) potassium channel inactivation through an interaction between the amino-terminal "inactivation domain" of the beta-subunit and a "receptor" located at or near the cytoplasmic mouth of the channel pore. Here we used a bacterial random peptide library to examine the structural requirements for this interaction. To identify peptides that bind Kv1.1 we screened the library against a synthetic peptide corresponding to the predicted S4-S5 cytoplasmic loop of the Kv1.1 alpha-subunit (residues 313-328). Among the highest affinity interactors were peptides with significant homology to the amino terminus of Kvbeta1. We performed a second screen using a peptide from the amino terminus of Kvbeta1 (residues 2-31) as "bait" and identified peptide sequences with significant homology to the S4-S5 loop of Kv1.1. A series of synthetic peptides containing mutations of the wild-type Kvbeta1 and Kv1.1 sequences were examined for their ability to inhibit Kvbeta1/Kv1.1 binding. Amino acids Arg20 and Leu21 in Kvbeta1 and residues Arg324 and Leu328 in Kv1.1 were found to be important for the interaction. Taken together, these data provide support for the contention that the S4-S5 loop of the Kv1.1 alpha subunit is the likely acceptor for the Kvbeta1 inactivation domain and provide information about residues that may underlie the protein-protein interactions responsible for beta-subunit mediated Kv channel inactivation.

Distribution of heterotrimeric G-protein beta and gamma subunits in the rat brain.

Heterotrimeric G-proteins, comprising alpha, beta and gamma subunits, have been shown to play a central role in coupling multiple receptors to a variety of enzymes and ion channels. In vitro studies have demonstrated the existence of selective interactions between various alpha, beta and gamma subunits, as well as between specific heterotrimers and target receptor and effector proteins. However, little is known of the physiological relevance of such associations, and the determinants of specificity in G-protein signaling within the brain remain largely unidentified. To investigate the possibility that specific heterotrimeric interactions result from discrete localizations of the G-protein subunits within the brain, we have used the technique of in situ hybridization to map the distribution of G-protein beta and gamma subunits in the rat brain. Beta1, beta2, beta3 and beta5 subunits were found to be widely expressed throughout the rat brain, whilst beta4 and the G-protein gamma subunit messenger RNAs generally showed more discrete expression patterns. The expression patterns for these subunits suggest that individual beta and gamma subunits may be co-expressed in certain cell types and brain regions; a particularly intriguing and striking co-localization was observed in the case of beta4 and gamma2 subunit messenger RNAs in layer VI of the occipital cortex. The localizations of the G-protein beta and gamma subunits, and their potential coupling to various receptor/effector systems, are discussed.

Association and colocalization of the Kvbeta1 and Kvbeta2 beta-subunits with Kv1 alpha-subunits in mammalian brain K+ channel complexes.

The differential expression and association of cytoplasmic beta-subunits with pore-forming alpha-subunits may contribute significantly to the complexity and heterogeneity of voltage-gated K+ channels in excitable cells. Here we examined the association and colocalization of two mammalian beta-subunits, Kvbeta1 and Kvbeta2, with the K+ channel alpha-subunits Kv1.1, Kv1.2, Kv1.4, Kv1.6, and Kv2.1 in adult rat brain. Reciprocal coimmunoprecipitation experiments using subunit-specific antibodies indicated that Kvbeta1 and Kvbeta2 associate with all the Kv1 alpha-subunits examined, and with each other, but not with Kv2.1. A much larger portion of the total brain pool of Kv1-containing channel complexes was found associated with Kvbeta2 than with Kvbeta1. Single- and multiple-label immunohistochemical staining indicated that Kvbeta1 codistributes extensively with Kv1.1 and Kv1.4 in cortical interneurons, in the hippocampal perforant path and mossy fiber pathways, and in the globus pallidus and substantia nigra. Kvbeta2 codistributes extensively with Kv1.1 and Kv1.2 in all brain regions examined and was strikingly colocalized with these alpha-subunits in the juxtaparanodal region of nodes of Ranvier as well as in the axons and terminals of cerebellar basket cells. Taken together, these data provide a direct demonstration that Kvbeta1 and Kvbeta2 associate and colocalize with Kv1 alpha-subunits in native tissues and provide a biochemical and neuroanatomical basis for the differential contribution of Kv1 alpha- and beta-subunits to electrophysiologically diverse neuronal K+ currents.

Selective aggregation of endogenous beta-amyloid peptide and soluble amyloid precursor protein in cerebrospinal fluid by zinc.

Zinc added to buffered solutions of synthetic beta-amyloid peptide (A beta) has been reported to induce accelerated formation of insoluble aggregates. This observation suggests that zinc may play a role in the formation of senile plaques, which contain A beta, in Alzheimer's disease. To test this hypothesis under conditions more representative of the brain, we investigated the ability of zinc to induce aggregation of A beta in freshly drawn canine CSF, which contains the same sequence as human A beta. Aggregates were separated from CSF by ultracentrifugation before and after incubation with zinc and assayed by quantitative western blotting and ELISA. We found that zinc induced the rapid aggregation of endogenous A beta in CSF, with an EC50 of 120-140 microM. The reaction was specific, because most (> or = 95%) CSF protein remained soluble under conditions where most A beta was insoluble, as assayed by scanning densitometry of Coomassie-stained gels. Staining of the precipitated material resulted in the visualization of punctate regions that were thioflavin positive or birefringent when stained with Congo red, suggesting the formation of amyloid-related structures. These results suggest that zinc could play a role in amyloid deposition, because there is overlap between the regions of the brain where zinc concentrations are highest and regions with the highest amyloid content. It is surprising that zinc induced the aggregation of endogenous soluble APP at lower concentrations than required for A beta (EC50 80 microM). The possibility that zinc-induced aggregation of APP may precede the deposition of A beta into plaques is discussed. Investigation of aggregation of A beta in CSF will aid in assessing the biological relevance of other agents that have been reported to accelerate amyloid formation.

Identification of a cytoplasmic domain important in the polarized expression and clustering of the Kv2.1 K+ channel.

The voltage-sensitive K+ channel Kv2.1 has a polarized and clustered distribution in neurons. To investigate the basis for this localization, we expressed wild-type Kv2.1 and two COOH-terminal truncation mutants, delta C318 and delta C187, in polarized epithelial MDCK cells. These functional channel proteins had differing subcellular localization, in that while both wild-type Kv2.1 and delta C187 localized to the lateral membrane in high density clusters, delta C318 was expressed uniformly on both apical and lateral membranes. A chimeric protein containing the hemagglutinin protein from influenza virus and the region of Kv2.1 that differentiates the two truncation mutants (amino acids 536-666) was also expressed in MDCK cells, where it was found in high density clusters similar to those observed for Kv2.1. Polarized expression and clustering of Kv2.1 correlates with detergent solubility, suggesting that interaction with the detergent insoluble cytoskeleton may be necessary for proper localization of this channel.

Voltage-gated K+ channel beta subunits: expression and distribution of Kv beta 1 and Kv beta 2 in adult rat brain.

Recent cloning of K+ channel beta subunits revealed that these cytoplasmic polypeptides can dramatically alter the kinetics of current inactivation and promote efficient glycosylation and surface expression of the channel-forming alpha subunits. Here, we examined the expression, distribution, and association of two of these beta subunits, Kv beta 1 and Kv beta 2, in adult rat brain. In situ hybridization using cRNA probes revealed that these beta-subunit genes are heterogeneously expressed, with high densities of Kv beta 1 mRNA in the striatum, CA1 subfield of the hippocampus, and cerebellar Purkinje cells, and high densities of Kv beta 2 mRNA in the cerebral cortex, cerebellum, and brainstem. Immunohistochemical staining using subunit-specific monoclonal and affinity-purified polyclonal antibodies revealed that the Kv beta 1 and Kv beta 2 polypeptides frequently co-localize and are concentrated in neuronal perikarya, dendrites, and terminal fields, and in the juxtaparanodal region of myelinated axons. Immunoblot and reciprocal co-immunoprecipitation analyses indicated that Kv beta 2 is the major beta subunit present in rat brain membranes, and that most K+ channel complexes containing Kv beta 1 also contain Kv beta 2. Taken together, these data suggest that Kv beta 2 is a component of almost all K+ channel complexes containing Kv 1 alpha subunits, and that individual channels may contain two or more biochemically and functionally distinct beta-subunit polypeptides.

Beta subunits promote K+ channel surface expression through effects early in biosynthesis.

Voltage-gated K+ channels are protein complexes composed of ion-conducting integral membrane alpha subunits and cytoplasmic beta subunits. Here, we show that, in transfected mammalian cells, the predominant beta subunit isoform in brain, Kv beta 2, associates with the Kv1.2 alpha subunit early in channel biosynthesis and that Kv beta 2 exerts multiple chaperone-like effects on associated Kv1.2 including promotion of cotranslational N-linked glycosylation of the nascent Kv1.2 polypeptide, increased stability of Kv beta 2/Kv1.2 complexes, and increased efficiency of cell surface expression of Kv1.2. Taken together, these results indicate that while some cytoplasmic K+ channel beta subunits affect the inactivation kinetics of alpha subunits, a more general, and perhaps more fundamental, role is to mediate the biosynthetic maturation and surface expression of voltage-gated K+ channel complexes. These findings provide a molecular basis for recent genetic studies indicating that beta subunits are key determinants of neuronal excitability.

Selective interaction of voltage-gated K+ channel beta-subunits with alpha-subunits.

To begin to study the molecular bases that determine the selective interaction of the beta-subunits of voltage-gated K+ channels with alpha-subunits observed in situ, we have expressed these polypeptides in transfected mammalian cells. Analysis of the specificity of alpha/bet a-subunit interaction indicates that both the Kvbeta1 and Kvbeta2 beta-subunits display robust and selective interaction with the five members of the Shaker-related (Kv1) alpha-subunit subfamily tested. The interaction of these beta-subunits with Kv1 alpha-subunits does not require the beta-subunit N-terminal domains. Thus, the previously observed failure of N-terminal mutants of Kv beta1 to modulate inactivation kinetics of Kv1 family members is not simply due to a lack of subunit interaction. Interaction of these beta-subunits with members of two other subfamilies (Shab- and Shaw-related) could not be detected. Somewhat surprisingly, a member of the Shal-related subfamily was found to interact with beta-subunits; however, this interaction had biochemical characteristics distinct from the beta-subunit interaction with Kv1 family members. In all cases, Kvbeta1 and Kvbeta2 exhibited indistinguishable alpha-subunit selectivity. These studies point to a selective interaction between K+ channel alpha- and beta-subunits mediated through conserved domains in the respective subunits.

Generation and characterization of subtype-specific monoclonal antibodies to K+ channel alpha- and beta-subunit polypeptides.

Molecular characterization of mammalian voltage-sensitive K+ channel genes and their expression became possible with the cloning of the Shaker locus of Drosophila. However, analysis of the expression patterns and subunit composition of native K+ channel protein complexes requires immunological probes specific for the individual K+ channel gene products expressed in excitable tissue. Here, we describe the generation and characterization of monoclonal antibodies (mAbs) against eight distinct mammalian K+ channel polypeptides; the Kv1.1, Kv1.2, Kv1.4, Kv1.5 and Kv1.6 Shaker-related alpha-subunits, the Kv2.1 Shab-related alpha-subunit, and the Kv beta 1 and Kv beta 2 beta-subunits. We characterized the subtype-specificity of these mAbs against native K+ channels in mammalian brain and against recombinant K+ channels expressed in transfected mammalian cells. In addition, we used these mAbs to investigate the cellular and subcellular distribution of the corresponding polypeptides in rat cerebral cortex, as well as their expression levels across brain regions.

Association and colocalization of K+ channel alpha- and beta-subunit polypeptides in rat brain.

Recent cloning of auxiliary subunits associated with voltage-gated ion channels and their subsequent coexpression with the channel forming alpha-subunits has revealed that the expression level, gating and conductance properties of the expressed channels can be profoundly affected by the presence of an auxiliary subunit polypeptide. In the present study, we raised antibodies against the beta-subunit associated with the bovine dendrotoxin sensitive K(+)-channel complex and used these antibodies to characterize the related beta-subunit polypeptides in rat brain. The anti-beta-subunit antibodies displayed a specific reaction on immunoblots of rat brain membranes with a major 38 kDa polypeptide, and a minor 41 kDa polypeptide, which correspond closely to the predicted sizes of the Kv beta 2 and Kv beta 1 beta-subunit polypeptides, respectively, recently cloned from rat brain. Reciprocal coimmunoprecipitation experiments revealed that the beta-subunit polypeptides are associated with Kv1.2 and Kv1.4, but not Kv2.1, alpha-subunits. Immunohistochemical staining revealed that the beta-subunit polypeptides were widely distributed in adult rat brain. Moreover, the cellular distribution of beta-subunit immunoreactivity corresponded closely with immunoreactivity for Kv1.2, and to a lesser extent Kv1.4, but not with Kv2.1. These results suggest that neuronal mechanisms may exist to direct the selective interaction of K+ channel alpha- and beta-subunit polypeptides, and that the properties of K+ channels in specific subcellular domains may be regulated by the formation of heteromultimeric K+ channel complexes containing specific combinations of alpha- and beta-subunits.