WWW.JNEUROSCI.ORG
-
The Journal of Neuroscience
 QUICK SEARCH:   [advanced]


     
-


HOME
  |  
SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an eLetter
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (110)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rhodes, K. J.
Right arrow Articles by Trimmer, J. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rhodes, K. J.
Right arrow Articles by Trimmer, J. S.
Right arrowPubmed/NCBI databases
*Substance via MeSH

 Previous Article  |  Next Article 

Volume 16, Number 16, Issue of August 15, 1996 pp. 4846-4860
Copyright ©1996 Society for Neuroscience

Voltage-Gated K+ Channel beta  Subunits: Expression and Distribution of Kvbeta 1 and Kvbeta 2 in Adult Rat Brain

Kenneth J. Rhodes1, Michael M. Monaghan1, Nestor X. Barrezueta1, Stanley Nawoschik1, Zewditu Bekele-Arcuri2, Maria F. Matos2, Kensuke Nakahira2, Lee E. Schechter1, and James S. Trimmer2

1 CNS Disorders, Wyeth-Ayerst Research, Princeton, New Jersey 08543, and 2 Department of Biochemistry and Cell Biology and Institute for Cell and Developmental Biology, State University of New York, Stony Brook, New York 11794

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

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, Kvbeta 1 and Kvbeta 2, in adult rat brain. In situ hybridization using cRNA probes revealed that these beta -subunit genes are heterogeneously expressed, with high densities of Kvbeta 1 mRNA in the striatum, CA1 subfield of the hippocampus, and cerebellar Purkinje cells, and high densities of Kvbeta 2 mRNA in the cerebral cortex, cerebellum, and brainstem. Immunohistochemical staining using subunit-specific monoclonal and affinity-purified polyclonal antibodies revealed that the Kvbeta 1 and Kvbeta 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 Kvbeta 2 is the major beta  subunit present in rat brain membranes, and that most K+ channel complexes containing Kvbeta 1 also contain Kvbeta 2. Taken together, these data suggest that Kvbeta 2 is a component of almost all K+ channel complexes containing Kv1 alpha  subunits, and that individual channels may contain two or more biochemically and functionally distinct beta -subunit polypeptides.

Key words: ion channel; central nervous system; auxiliary subunit; striatum; immunoprecipitation; immunohistochemistry


INTRODUCTION

Voltage-gated K+ channels are composed of pore-forming alpha  subunits and associated cytoplasmic beta -subunit polypeptides (Pongs, 1995). These channels are critical for action potential conduction and neurotransmitter release, and are essential to the control of neuronal excitability (Hille, 1992). Expression of alpha -subunit mRNAs in heterologous cells gives rise to tetrameric channel complexes with electrophysiological characteristics similar to A-type or delayed-rectifier channels (for review, see Pongs, 1992). The large number of alpha -subunit genes, their ability to assemble into heteromultimers (Ruppersberg et al., 1990; Sheng et al., 1993; Wang et al., 1993; Scott et al., 1994a; Rhodes et al., 1995), and their heterogeneous expression in mammalian brain undoubtedly contributes to the diversity of voltage-gated K+ channels in situ (Stühmer et al., 1989; Jan and Jan, 1990). However, the recent discovery of auxiliary subunits associated with ion channels in general (for review, see Isom et al., 1994), and with voltage-gated K+ channels in particular (Parcej et al., 1992; Rettig et al., 1994; Scott et al., 1994b), revealed that their electrophysiological and biochemical properties can be dramatically affected by the presence of beta  subunits (Rettig et al., 1994; Majumder et al., 1995; Morales et al., 1995; Shi et al., 1996). This association of beta  subunits with K+ channels not only increases the potential for diversity, it also indicates that the functional properties of individual channels are governed by the specific combination of alpha  and beta  subunits present in the channel complex.

The beta  subunits of voltage-gated K+ channels were identified as 38-41 kDa polypeptides associated with the dendrotoxin acceptor purified from bovine brain (Parcej and Dolly, 1989, 1992; Scott et al., 1994a,b), and as a 38 kDa polypeptide in immunoprecipitated rat brain K+ channel complexes (Trimmer, 1991). Subsequent cloning of cDNAs encoding a bovine beta  subunit (Scott et al., 1994b); the related Kvbeta 1, Kvbeta 2, and Kvbeta 3 in rat brain (Rettig et al., 1994; Heinemann et al., 1995); and closely related beta  subunits in Drosophila (Chouinard et al., 1995), ferret (Morales et al., 1995), and human tissues (England et al., 1995a,b; Majumder et al., 1995; McCormack et al., 1995) indicated that these beta  subunits are highly conserved and that some can modulate the rate of inactivation of certain alpha  subunits (Rettig et al., 1994; England et al., 1995a,b; Heinemann et al., 1995; Majumder et al., 1995; McCormack et al., 1995; Morales et al., 1995).

We recently reported that Kvbeta 1 and Kvbeta 2 associate with alpha  subunits early in their biosynthesis and exert chaperone-like effects on the alpha  subunits, promoting their efficient glycosylation and stable expression in the plasma membrane (Shi et al., 1996). In addition, we reported that Kvbeta 1 and Kvbeta 2 associate with all members of the Shaker-related (Kv1) alpha -subunit subfamily upon co-expression in transfected mammalian cells (Nakahira et al., 1996). Here, we used riboprobes and antibodies specific for Kvbeta 1 and Kvbeta 2 to examine their expression, subcellular distribution, and co-association in adult rat brain. We observed that these beta  subunits are widely expressed, and that immunoreactivity for Kvbeta 1 and Kvbeta 2 is concentrated in multiple subcellular domains including neuronal somata and dendrites, the paranodal segments of myelinated axons, and in the terminal fields of several cortical and subcortical projection systems. We also observed that Kvbeta 2 is the predominant beta -subunit isoform in rat brain; although Kvbeta 1 mRNA is widely expressed, the Kvbeta 1 polypeptide is not a major component of the total rat brain beta -subunit pool, and almost all of the Kvbeta 1 that is present is in K+ channel complexes that also contain Kvbeta 2. Together, these observations suggest that beta  subunits are integral components of K+ channel complexes, and that the inclusion of Kvbeta 1 in Kvbeta 2-containing complexes may serve to fine tune the electrophysiological properties of channels in specific brain regions.


MATERIALS AND METHODS

Materials. All reagents were molecular biology grade from Sigma (St. Louis, MO) or Boehringer Mannheim (Indianapolis, IN), except where otherwise noted.

Preparation of 35S-labeled cRNA probes. DNA templates for riboprobe synthesis were prepared by the PCR using plasmid clones containing the full-length rat Kvbeta 1 or Kvbeta 2 cDNAs (Nakahira et al., 1996) or a partial Kvbeta 2 cDNA (K. Nakahira, S. Nawoschik, J. Trimmer, unpublished observations) as PCR templates. Two independent riboprobes targeted to unique, nonoverlapping regions of Kvbeta 1 or Kvbeta 2 were used in the present study. The probe sequences were checked versus the Genbank database to ensure that they only recognize the appropriate targets among all deposited sequences. To generate riboprobes for the Kvbeta 1 subunit, one pair of oligonucleotide primers was designed to amplify a 264 bp region spanning nucleotides -46 to 218 of the rat Kvbeta 1 cDNA and, in addition, to add the promoter sequences for T3 RNA polymerase. These primers contained the following sequences: 5'-CGGATCCG- CTGTGCTGTGGGGTTCTGAGAGGAC-3' (forward); 5'-AATTAACC- CTCACTAAAGGGATATTTCATGCCAGTCTGCT-3' (reverse). The forward primer for this Kvbeta 1 probe contained eight nucleotides of vector sequence at the 5' end from the BamHI site used for cloning. A second pair of oligonucleotide primers was designed to amplify a 271 bp sequence spanning nucleotides -321 to -51 of the rat Kvbeta 1 cDNA and, in addition, to add the promoter sequences for T3 RNA polymerase. These primers had the following sequences: 5'-CCTGACCACATGGATCTGGC-3' (forward); 5'-AATTAACCCTCACTAAAGGGCGGCAGAGGGTGAGACGTT-3' (reverse). To generate riboprobes for the Kvbeta 2 subunit, one pair of primers was designed to amplify a 228 bp fragment from the 3' untranslated region (nucleotides 1130-1357) of the rat Kvbeta 2 cDNA and, in addition, to add the promoter sequences for T3 RNA polymerase. These primers had the following sequences: 5'-CCCAGCTCGGACAGTTCCTGGTTCC-3' (forward); 5'-AATTAACCCTCACTAAAGGGGCATCCAGCGAGG AAG- CGGC-3' (reverse). A second riboprobe for Kvbeta 2 was generated using oligonucleotide primers designed to amplify a 300 bp fragment spanning nucleotides 809-1108 of the rat Kvbeta 2 cDNA and, in addition, to add the promoter sequences for T3 RNA polymerase. These primers contained the following sequences: 5'-ACCAGTGGTTGAAGGACAAG-3' (forward); 5'-AATTAACCCTCACTAAAGGGTGACTTAGGATCTATAGTCC-3' (re-verse). All PCR products were gel purified on 1.5% low-melt agarose gels, and bands containing the Kvbeta 1 or Kvbeta 2 products were excised, phenol and phenol-chloroform extracted, and ethanol precipitated. The pellet then was dried and resuspended in 1× TE buffer containing (in mM): 10 Tris/HCl, 1 EDTA, pH 7.4. Fifty nanograms of DNA template were used for transcription reactions using 35S-CTP (New England Nuclear, Boston, MA) and the Riboprobe Gemini System (Promega, Madison, WI). Probes were examined by Northern analysis of rat brain RNA and by Southern analysis of plasmids containing the Kvbeta 1 and Kvbeta 2 cDNAs. Each probe reacted with a single band of appropriate size on Northern blots and with only the appropriate cDNA on the plasmid Southern blots, indicating that these riboprobes specifically recognized Kvbeta 1 and Kvbeta 2 transcripts (data not shown).

In situ hybridization. Eight adult male Sprague-Dawley rats were used for analysis of Kvbeta 1 and Kvbeta 2 mRNA expression by in situ hybridization histochemistry. Animals were killed by asphyxiation with CO2, and the brains were removed, immediately frozen in a bed of pulverized dry ice, and stored at -70°C. Sections were cut at 10 µm on a Hacker-Brights cryostat and thaw mounted onto chilled (-20°C) slides coated with Vectabond reagent (Vector Labs, Burlingame, CA). All solutions were prepared in dH2O treated with 0.1% (v/v) diethylpyrocarbonate and autoclaved. Sections were fixed by immersion in 4% paraformaldehyde in PBS, pH 7.4, then immersed sequentially in 2× SSC (1× SSC is 0.150 M sodium chloride, 0.015 M sodium citrate), dH2O, and 0.1 M triethanolamine, pH 8.0. The sections then were acetylated by immersion in 0.1 M triethanolamine containing 0.25% (v/v) acetic anhydride; washed in 0.2× SSC; dehydrated in 50, 70, and 90% ethanol; and rapidly dried. One ml of prehybridization solution containing 0.9 M NaCl, 1 mM EDTA, 5× Denhardt's solution, 0.25 mg/ml single-stranded herring sperm DNA (Gibco, Gaithersburg, MD), and 50% deionized formamide (EM Sciences, Gibbstown, NJ) in 10 mM Tris, pH 7.6, was pipetted onto each slide, and the slides incubated for 3 hr at 50°C in a humidified box. The sections then were dehydrated by immersion in 50, 70, and 90% ethanol and air dried.

Labeled riboprobes were denatured in a small volume (100 µl) of hybridization solution containing 0.9 M NaCl, 1 mM EDTA, 1× Denhardt's solution, 0.1 mg/ml yeast tRNA, 0.1 mg/ml single-stranded salmon sperm DNA, dextran sulfate (10%), 0.08% BSA, 10 mM DTT (Boehringer Mannheim, Indianapolis, IN), and 50% deionized formamide in 10 mM Tris, pH 7.6, at 95°C (1 min); placed on ice (5 min); and added at a final concentration of 50,000 cpm/µl to 10 ml of prewarmed (to 55°C) hybridization solution. The hybridization solution then was pipetted onto the sections and allowed to hybridize overnight at 55°C in a humidified chamber. The sections were subsequently washed once for 45 min at 37°C in 2× SSC containing 10 mM DTT, once for 30 min at 37°C in 1× SSC containing 50% formamide, and once for 30 min at 37°C in 2× SSC. Single-stranded and nonspecifically hybridized riboprobe was digested by immersion in 10 mM Tris, pH 8.0, containing bovine pancreas RNase A (Boehringer Mannheim) (40 µg/ml), 0.5 M NaCl, and 1 mM EDTA. The sections then were washed in 2× SSC for 1 hr at 60°C, followed by 0.1× SSC containing 0.5% (w/v) sodium thiosulfate for 2 hr at 60°C. The sections then were dehydrated in 50, 70, and 90% ethanol containing 0.3 M ammonium acetate, and dried.

The slides then were loaded into X-ray cassettes and exposed to Hyperfilm beta -Max (Amersham, Arlington Heights, IL) for 3-7 d. Once a satisfactory exposure was obtained, the slides were coated with nuclear-track emulsion (NTB-2) (Eastman Kodak, Rochester, NY) and exposed for 7-21 d at 4°C. The emulsion autoradiograms were developed and fixed according to the manufacturer's instructions, and the underlying tissue sections were stained with hematoxylin.

To assess nonspecific labeling in the in situ hybridization procedure, a control probe was generated from a template provided in the Riboprobe Gemini System kit (Promega catalog #P2651). This vector was linearized using ScaI, and transcribed using T3 RNA polymerase. The resulting transcription reaction generates two products, a 250 bp and a 1525 bp riboprobe, containing only vector sequence. This control probe mixture was labeled as described above and added to the hybridization solution at a final concentration of 50,000 cpm/µl. No specific hybridization was observed in control sections; i.e., these sections gave a very weak uniform hybridization signal that did not follow neuroanatomical landmarks (data not shown).

Production of synthetic peptides and antibodies. Synthetic peptides corresponding to amino acids 7-28 from the N terminus of the rat Kvbeta 1 polypeptide (CTEHNLKSRNGEDRLLSKQSST) (Rettig et al., 1994) and amino acids 1-17 of the rat Kvbeta 2 polypeptide (MYPESTTGSPARLSLRQC) (Rettig et al., 1994) were synthesized (Quality Controlled Biochemicals, Hopkinton, MA) and conjugated to keyhole limpet hemocyanin (KLH) (1 mg peptide/mg carrier protein) using sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide ester (Pierce, Rockford, IL). These peptide/KLH conjugates were injected into rabbits for the production of polyclonal antisera (Pocono Rabbit Farm, Canadensis, PA), and into mice for the production of monoclonal antibodies (mAbs). Production of mAbs and purification of monoclonal immunoglobulins were performed essentially as described previously (Trimmer et al., 1985), and will be described in detail elsewhere (Z. Bekele-Arcuri, M. Matos, J. Trimmer, unpublished observations). For affinity purification of the polyclonal antibodies, the Kvbeta 1 and Kvbeta 2 peptides were conjugated to SulfoLink coupling gel (Pierce) via the terminal cysteine residues. Polyclonal antibodies were affinity purified from immune serum by standard procedures (Harlow and Lane, 1988).

Immunofluorescence. Immunofluorescence staining of transiently transfected cells expressing Kvbeta 1 and Kvbeta 2 was performed essentially as described previously (Shi et al., 1994). Briefly, green monkey fibroblast COS-1 cells were cultured on glass coverslips that had been previously coated with 25 µg/ml poly-L-lysine. For staining, cells were washed three times in ice-cold PBS, then fixed and permeabilized by treatment for 20 min at 4°C with a freshly prepared ice-cold fixative containing 3% paraformaldehyde/0.1% Triton X-100 in PBS. Cells then were washed three times in TBS containing 0.1% Triton X-100 (TBS-T), and nonspecific protein binding sites were blocked by incubation in TBS-T containing 4.5% w/v nonfat dry milk powder (Blotto-T) (Johnson et al., 1984). Cells then were incubated in primary antibodies diluted in Blotto-T for 1 hr at room temperature, washed three times in Blotto-T for 30 min total, and incubated in the appropriate secondary antibodies for 30 min. After three washes in TBS-T for 15 min total, cells were mounted in PBS medium containing 90% glycerol and 1 mg/ml p-phenylenediamine and viewed on a Zeiss Axiophot microscope using epifluorescence illumination.

Brain membrane preparations. A crude synaptosomal membrane fraction was prepared from freshly dissected adult rat brains, essentially as described previously (Trimmer, 1991, 1993). Briefly, brains were homogenized in 0.3 M sucrose, 10 mM sodium phosphate, pH 7.4, and 10 mM sodium fluoride, containing a protease inhibitor cocktail (1 mM phenylmethyl sulfonyl fluoride, 1 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 µg/ml pepstatin). The resultant homogenate was centrifuged at 3,000 × g for 10 min to remove nuclei and cellular debris. The supernatant then was centrifuged at 45,000 × g for 60 min to pellet the membranes. Aliquots of the membrane preparations were suspended in the homogenization buffer, and protein was determined using the BCA method (Pierce).

SDS/polyacrylamide gels and immunoblotting. For immunoblots, 50 µg of membrane protein was added to SDS sample buffer, boiled, and fractionated on 7.5 or 12% SDS/polyacrylamide gels (Maizel, 1971). Disulfide bonds were reduced by the addition of 20 mM 2-mercaptoethanol to the sample buffer. Lauryl sulfate (Sigma) was the SDS source used for all SDS-PAGE (Shi et al., 1994). After electrophoretic transfer to nitrocellulose paper, the resulting blots were blocked in TBS containing 4% low fat milk (Blotto) (Johnson et al., 1984), incubated in affinity-purified antibody diluted 1:50-1:100 in Blotto for 1 hr or undiluted mAb tissue culture supernatants, and washed three times in Blotto for 30 min total. Blots then were incubated in HRP-conjugated secondary antibody (1:2000 dilution in Blotto) (Cappel, West Chester, PA) for 1 hr and then washed in TBS three times for 30 min total. The blots then were incubated in substrate for enhanced chemiluminescence (ECL) (Amersham, Arlington Heights, IL) for 1 min and autoradiographed on preflashed (to OD545 = 0.15) Kodak X-OMAT LS film.

Immunoprecipitation. Immunoprecipitation reactions were performed using detergent lysates of rat brain membranes. All procedures were performed at 4°C. Membranes (1 mg membrane protein/tube) were solubilized to 1 ml final volume/tube in lysis buffer (1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 10 mM sodium azide, 10 mM Tris/HCl, pH 8.0, containing a protease inhibitor cocktail (see above). Affinity-purified antibodies were added and the samples incubated for 2 hr on a rotator, followed by addition of 20 µl of a 50% suspension of protein A sepharose and additional incubation for 45 min. After incubation, protein A sepharose was centrifuged at 10,000 × g for 20 sec, and the resulting pellets were washed by resuspension and centrifugation six times with lysis buffer. The final pellets were resuspended in 50 µl reducing sample buffer, and 20 µl electrophoresed on 12% SDS-PAGE and subjected to immunoblotting as described above.

Immunohistochemistry. Twelve adult male Sprague-Dawley rats were deeply anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and perfused through the ascending aorta with 150 ml of 0.9% saline, followed by 500 ml of fixative containing freshly prepared 4% paraformaldehyde in 0.1 M NaPO4 buffer, pH 7.4 (PB). The brains were removed, cryoprotected for 18-48 hr in 20% sucrose in PB, frozen in a bed of pulverized dry ice, and then cut into 35-µm-thick sections on a sliding microtome. Consecutive 1-in-10 series of sections were collected in 0.05 M PB and processed for light microscopic immunohistochemistry as described previously (Rhodes et al., 1995). Affinity-purified rabbit polyclonal anti-Kvbeta 1 and rabbit anti-Kvbeta 2 antibodies were used at dilutions of 1:1500 and 1:4000, respectively. Mouse mAbs (purified IgG fractions and tissue culture supernatants) raised against Kvbeta 1 (clone K9/40) and Kvbeta 2 (clone K17/70) were used at dilutions of 1:100-1:1500. To verify the specificity of the immunohistochemical reactions, some sections were processed either without addition of the primary antibody or using antibodies incubated previously (1 hr) in vehicle containing an excess of the synthetic peptide/BSA antigen (5-25 µg/ml). No specific staining was observed in these control sections (data not shown).

Analysis of sections processed for in situ hybridization and immunohistochemistry was performed using a Zeiss Axiophot photomicroscope. Low-magnification photographs of immunohistochemically stained sections and autoradiograms generated on film were taken using a Nikon Multiphot macrophotography system.


RESULTS

Localization of Kvbeta 1 and Kvbeta 2 mRNA

General features Analysis of autoradiograms prepared on film indicated that the Kvbeta 1 and Kvbeta 2 mRNAs are widely and heterogeneously expressed in adult rat brain (Table 1). The pair of riboprobes for Kvbeta 1 generated patterns of hybridization signal that were indistinguishable from one another, as did the pair of riboprobes for Kvbeta 2. Taken together with the result of Northern and Southern blotting using these probes, this result strongly suggests that the riboprobes used in the present study specifically reveal the presence of Kvbeta 1 or Kvbeta 2 mRNA.

Table 1. Distribution of Kvbeta 1 and Kvbeta 2 mRNA and protein in rat brain


Region Subfield/lamina/sublamina Kvbeta 1 mRNA Kvbeta 2 mRNA Kvbeta 1 Immunoreactivity Kvbeta 2 Immunoreactivity

Cortex ++ + + +
I ++ +++ ++ +++
II +++ +++ ++ +++
III +++ ++ +++ +++
IV +++ ++ ++ ++
V ++++ ++++ ++++ ++++
VI ++++ ++++ +++ +++
Hippocampus
Dentate gyrus
Infragranular ++ +++ + +
Granule cell +++ ++++ ++ ++
Inner third + + + +
Middle third + + ++ +++
Outer third + + + ++
CA1
S. oriens ++ ++ ++ ++
S. pyramidale ++++ +++++ ++ ++++
S. radiatum ++ ++ ++ +++
S. moleculare + + + +
Striatum
Caudate +++++ ++ ++ ++
Accumbens +++++ ++ ++ ++
Olfactory tubercle +++++ ++ ++ ++
Globus pallidus ++ ++ ++++ ++++
Basal forebrain
Medial septal n. +++ +++ +++ +++
Lateral septal n. + + + +
Diagonal band vert. +++ +++ +++ +++
Diagonal band hor. +++ +++ +++ +++
Nucleus basalis +++ +++ +++ +++
Amygdala
Basolateral n. +++ ++++ ++ ++
Thalamus
Anterior n. +++ ++++ ++ ++
Lateral n. ++ ++ +++ +++
Laterodorsal n. ++ ++ ++ ++
VL +++ +++ +++ ++
VPM +++ ++ +++ ++
VPL +++ +++ +++ ++
Lateral geniculate n. ++ +++ +++ ++
Medial geniculate n. ++ ++ ++ ++
Hypothalamus ++ ++ ++ ++
Habenula
Medial n. + ++ ++ +
Lateral n. ++ ++ ++ +
Midbrain
Sup. colliculus +++ ++ +++ ++
Inf. colliculus ++++ +++ ++ ++
Substantia nigra
Pars compacta ++ ++ ++ ++
Pars reticulata + + ++++ ++++
Red n. ++++ ++++ +++++ +++++
nIII +++ +++ +++ +++
nIV +++ +++ ++++ ++++
Pons
mnV +++ +++ ++++ ++++
nVI +++ +++ ++++ ++++
nVII +++ +++ ++++ ++++
nVIII ++ ++ ++ ++
Medulla
mnIX +++ +++ ++++ ++++
mnX +++ +++ ++++ ++++
mnXI +++ +++ ++++ ++++
mnXII +++ +++ ++++ ++++
Cerebellum
Purkinje cells ++++ ++ +++ +++
Granule cells ++ ++ + +
Interneurons + ++ + ++
Deep nuclei +++ +++ +++ +++

A very high density of Kvbeta 1 expression was observed in the striatum, nucleus accumbens, olfactory tubercle, CA1 subfield of the hippocampus, and entorhinal and posterior cingulate cortices, and in several midbrain and brainstem motor nuclei (Fig. 1). Intermediate levels of Kvbeta 1 expression were observed in the piriform cortex; neocortex; medial septal-diagonal band complex; anterior, mediodorsal, and ventral tier thalamic nuclei; and the cerebellar cortex and deep nuclei. Intermediate levels of Kvbeta 1 expression also were observed in the laterodorsal, ventral posteromedial (VPM), ventral posterolateral (VPL), and dorsal lateral geniculate, and medial geniculate nuclei of the thalamus. Low levels of Kvbeta 1 expression were observed in the globus pallidus and hypothalamus. The highest levels of Kvbeta 2 expression were observed in the piriform cortex, hippocampal formation, and in layer II of the entorhinal cortex, with somewhat lower levels in the neocortex, medial septal-diagonal band complex, and the anterior, VPM, and VPL nuclei of the thalamus. Low and levels of Kvbeta 2 expression were observed in the remaining thalamic nuclei and in the striatum, globus pallidus, and hypothalamus.


Fig. 1. Expression of Kvbeta 1 and Kvbeta 2 mRNA in adult rat brain. Horizontal and coronal sections of rat brain were processed by in situ hybridization histochemistry to localize Kvbeta 1 (A, B, E-G) and Kvbeta 2 (C, D, H-J) mRNA. Areas containing a high density of hybridization signal appear dark in these bright-field images. The autoradiograms in A-D were exposed for 3 d, whereas those in E-J were exposed for 7 d. At the shorter exposure time, subtle differences in expression levels are more easily discernible. For example, there is a comparatively greater density of Kvbeta 1 mRNA in the CA1 subfield of the hippocampus as compared with the adjacent CA3 subfield (B). CA1, Hippocampal subfield; CB, cerebellum; CPu, caudate putamen; EC, entorhinal cortex; RN, red nucleus; ms, medial septal nuclei; AN, anterior thalamic nucleus; MD, mediodorsal thalamic nucleus.
[View Larger Version of this Image (143K GIF file)]

Neocortex and hippocampus Examination of high-resolution emulsion autoradiograms of sections processed to visualize hybridization to Kvbeta 1 and Kvbeta 2 mRNAs indicated that these two transcripts are expressed within cellular profiles with the size and morphology of neurons as opposed to glial cells (Fig. 2). Kvbeta 1 mRNA was expressed in large pyramidal cells in the deep half of layer III and in layers V and VI. Very high levels of Kvbeta 1 mRNA were observed in small to medium interneurons in cortical layers II, III, V, and VI. Interestingly, high levels of Kvbeta 1 mRNA also were observed in small cells juxtaposed to the subcortical white matter (Fig. 1). In virtually all regions of the neocortex, Kvbeta 2 mRNA was highly expressed in pyramidal cells in layers II, III, V, and VI. In proisocortical areas such as the entorhinal and cingulate cortices, Kvbeta 1 and Kvbeta 2 mRNA also were highly expressed in large multipolar neurons in layer II (Fig. 2).
Fig. 2. Cellular localization of Kvbeta 1 and Kvbeta 2 mRNA expression. Emulsion autoradiograms were prepared to localize Kvbeta 1 and Kvbeta 2 mRNA within individual cells. Cells containing a high density of mRNA contain a correspondingly high density of silver grains, which appear as bright spots in these dark-field images. In posterior cingulate cortex (area 23), Kvbeta 1 mRNA (A) is highly expressed in many small cells in layers II and III and larger cells in layer V (arrows), whereas Kvbeta 2 (B) is expressed predominantly in small cells in layer II and larger cells in layer V (arrows). In the entorhinal cortex, Kvbeta 1 mRNA (C) is expressed in large and small cells in layers II, III, V, and VI, whereas Kvbeta 2 mRNA (D) is expressed at high density in layer II cells and with a lower density in cells in the remaining layers of this structure. In the caudate nucleus, there is a very high level of Kvbeta 1 expression (E) and a lower level of Kvbeta 2 expression (F) in virtually all cells (arrows). The bundles of myelinated axons that course through this structure do not contain hybridization signal (arrowheads). In the basal forebrain, Kvbeta 1 (G) and Kvbeta 2 (H) mRNAs are expressed in large cells (arrows) with the distribution and frequency characteristic of the large cholinergic neurons present in this region. The short arrows in G and H mark the midline of the brain.
[View Larger Version of this Image (213K GIF file)]

In the hippocampus, Kvbeta 1 mRNA was expressed in dentate granule cells and in pyramidal cells of all subfields. However, pyramidal cells in the CA1 subfield expressed higher levels of Kvbeta 1 mRNA than the adjacent CA2 subfield and subiculum (Fig. 1). In addition, Kvbeta 1 was expressed in large interneurons located in stratum oriens and radiatum of all subfields. The distribution of Kvbeta 2 mRNA was strikingly similar to that observed for Kvbeta 1, with the exception that there appeared to be a uniformly high density of Kvbeta 2 expression across all subfields, and Kvbeta 2 mRNA did not appear to be expressed at an appreciably greater density in hippocampal interneurons.

Striatum and basal forebrain The greatest density of Kvbeta 1 expression was observed in the caudate putamen, nucleus accumbens, and olfactory tubercle. Virtually all neurons in these structures expressed extremely high levels of Kvbeta 1 mRNA (Figs. 1, 2E). Kvbeta 1 mRNA also was highly expressed in the medial septal nuclei and the vertical and horizontal limbs of the diagonal band of Broca in a distribution that closely resembled the distribution of cholinergic neurons in these structures (Fig. 2G) (Mesulam et al., 1983). In contrast to Kvbeta 1, there was a low level of Kvbeta 2 expression in the caudate putamen, nucleus accumbens, and olfactory tubercle (Figs. 1, 2F). However, there was a high level of Kvbeta 2 expression in the medial septal and diagonal band nuclei in a pattern that overlapped with, but was somewhat more extensive than, that observed for Kvbeta 1 (Figs. 1, 2H). Thalamus and hypothalamus A moderate density of Kvbeta 1 expression was observed throughout the thalamus, with a somewhat greater density in the anterior, mediodorsal, and entopeduncular nuclei (Fig. 1), and a moderate density in the laterodorsal, VPM, and VPL nuclei. A low level of Kvbeta 1 expression was observed in the hypothalamus, but with a somewhat more intense hybridization signal in the ventromedial hypothalamic nucleus. A low to moderate level of Kvbeta 2 expression was observed throughout the thalamus, with a somewhat greater density in the anterior, mediodorsal, paracentral, and ventroposterior nuclei. Low levels of Kvbeta 2 expression also were observed in the hypothalamus, however, as for Kvbeta 1, there was a somewhat greater density of Kvbeta 2 expression in the ventromedial nucleus. Midbrain Several midbrain motor nuclei, including the red nucleus and all cranial nerve nuclei, contained a very high density of Kvbeta 1 and Kvbeta 2 expression (Fig. 1). In addition, cells in the superficial and deep layers of the superior and inferior colliculi, the periaqueductal gray, and the pars compacta and pars reticulata of the substantia nigra expressed intermediate levels of Kvbeta 1 mRNA. Similarly, large neurons in the deep layers of the superior and inferior colliculi expressed moderate levels of Kvbeta 2 mRNA, and cells scattered throughout the substantia nigra pars compacta and pars reticulata expressed moderate to low levels of Kvbeta 2 mRNA (Fig. 1). Cerebellum and brainstem In the cerebellar cortex, Purkinje cells and granule cells displayed high and moderate levels of Kvbeta 1 expression, respectively, and small neurons scattered throughout the molecular layer expressed much lower levels of Kvbeta 1 mRNA. In contrast, Purkinje cells and granule cells displayed intermediate levels of Kvbeta 2 expression, and small neurons with a size and distribution similar to basket cells expressed moderate levels of Kvbeta 2 mRNA. Large neurons in all deep cerebellar nuclei expressed moderate levels of Kvbeta 1 and Kvbeta 2 mRNA (Fig. 1).

In the pons and medulla, all cranial nerve sensory and motor nuclei displayed moderate to high levels of Kvbeta 1 and Kvbeta 2 expression. In addition, mRNA for both beta  subunits was highly expressed in large pontine and medullary reticular neurons and in neurons of the superior and inferior olivary complexes.

Generation and characterization of Kvbeta 1- and Kvbeta 2-specific polyclonal and mAbs

A polyclonal antiserum was produced by the immunization of rabbits with a synthetic Kvbeta 1 peptide immunogen, corresponding to N-terminal amino acids 7-28 of the deduced rat brain Kvbeta 1 sequence (Rettig et al., 1994). This sequence is not present in the deduced sequence of the rat brain Kvbeta 2 beta  subunit, although some overlap is seen to a recently identified rat brain Kvbeta 3 beta  subunit (11/22 positions identical with changes spread throughout the sequences) (Heinemann et al., 1995; Pongs, 1995). Thus, it is likely that antibodies generated to this peptide are specific for Kvbeta 1. Rabbit polyclonal antibodies raised against this peptide exhibited a high titer against the peptide on ELISA assays and displayed a monospecific reaction on immunoblot assays versus rat brain membranes to a 44 kDa polypeptide (Fig. 3). That this peptide was Kvbeta 1 was supported by analysis of Kvbeta 1 and Kvbeta 2 expressed in COS-1 cells, which showed that the expressed recombinant Kvbeta 1 polypeptide shared immunoreactivity and electrophoretic mobility with the putative rat brain Kvbeta 1 polypeptide (Fig. 3). No corresponding immunoreactivity was seen to the recombinant 38 kDa Kvbeta 2 polypeptide expressed in COS-1 cells or present in the crude rat brain membranes on the same immunoblot (Fig. 3). Immunofluorescence staining of transfected COS-1 cells revealed a similar pattern of immunoreactivity in that the anti-Kvbeta 1 antibody exhibited a strong immunofluorescence staining of Kvbeta 1- , but not Kvbeta 2- , transfected cells (Fig. 4).
Fig. 3. Immunoblot analyses of the Kvbeta 1 and Kvbeta 2 beta -subunit polypeptides in rat brain membranes and in transfected COS-1 cells. Crude rat brain membranes (RBM) (50 µg) and the detergent extracts of COS-1 cells transfected with Kvbeta 1/RBG4 (Kvbeta 1), Kvbeta 2/RBG4 (Kvbeta 2), or RBG4 alone (vector) were fractionated on a 12.5% SDS gel and transferred to nitrocellulose, and the resultant immunoblot probed with either rabbit anti-Kvbeta 1 polyclonal antibody at 1:50 (left) or mouse anti-Kvbeta 2 mAb K17/70 tissue culture supernatant neat (right). Signals were visualized using ECL (left, 30 min; right, 1 min). Numbers on left refer to mobility of prestained molecular weight standards.
[View Larger Version of this Image (47K GIF file)]


Fig. 4. Analysis of antibody specificity. Immunofluorescence staining of Kvbeta 1 and Kvbeta 2 beta  subunits expressed in COS-1 cells. COS-1 cells were transfected with Kvbeta 1/RBG4 (A-D) or Kvbeta 2/RBG4 (E-H) cDNAs. Transfected cells then were fixed, permeabilized, and incubated with rabbit anti-Kvbeta 1 polyclonal antibody at 1:100 (A, E), rabbit anti-Kvbeta 2 polyclonal antibody at 1:200 (B, F), mouse anti-Kvbeta 1 mAb K9/40 at 1:2 (C, G), or mouse anti-Kvbeta 2 mAb K17/70 at 1:2 (D, H). Cells then were incubated with Texas Red-conjugated anti-rabbit (A, B, E, F) or anti-mouse (C, D, G, H) secondary antibody.
[View Larger Version of this Image (72K GIF file)]

mAbs generated against the same Kvbeta 1-specific peptide exhibited similar properties. All of the mAbs exhibited strong reactions in ELISA assays against the Kvbeta 1 peptide immunogen and in immunofluorescence staining of COS-1 cells expressing recombinant Kvbeta 1 polypeptide (Fig. 4). Staining was not observed in nontransfected or Kvbeta 2-transfected cells (Fig. 4). Immunoblot analysis of crude rat brain membranes revealed that none of the mAbs isolated recognized the SDS-denatured rat brain Kvbeta 1 polypeptide, although specific immunoreactivity was seen to the recombinant Kvbeta 1 (but not Kvbeta 2) polypeptide present in COS-1 cell extracts, perhaps because of the higher expression levels of Kvbeta 1 in these extracts compared with that observed in rat brain membranes (data not shown). All of these mAbs, and the rabbit polyclonal antibody, selectively immunoprecipitate the recombinant Kvbeta 1 polypeptide present in 35S-methionine labeled COS-1 cell extracts (data not shown). Additional details on the generation and characterization of these mAbs will be described elsewhere.

A polyclonal antiserum was produced by the immunization of rabbits with a synthetic Kvbeta 2 peptide immunogen, corresponding to N-terminal amino acids 1-17 of the deduced rat brain Kvbeta 2 sequence (Rettig et al., 1994). This sequence is not present in the deduced sequence of the rat brain Kvbeta 1 or Kvbeta 3 beta  subunit (Heinemann et al., 1995; Pongs, 1995). Thus, it is likely that antibodies generated to this peptide would be specific for Kvbeta 2. Rabbit polyclonal antibodies raised against this peptide exhibited a high titer against the Kvbeta 2 peptide on ELISA assays and displayed a monospecific reaction on immunoblot assays versus rat brain membranes to a 38 kDa polypeptide (Fig. 3). Additional immunoreactivity was exhibited to a minor, 41 kDa polypeptide (Fig. 3) that is also recognized by the pan-beta -subunit antibody (Rhodes et al., 1995). The molecular identity of this 41 kDa polypeptide is not known; however, the fact that it exhibits immunoreactivity to both the N-terminally directed anti-Kvbeta 2 antibody and the C-terminally directed pan-beta antibody indicates that it is either a post-translational variant of Kvbeta 2 or an alternative splice variant of the Kvbeta 2 gene. That the 38 kDa polypeptide was Kvbeta 2 was supported by analysis of Kvbeta 1 and Kvbeta 2 expressed in COS-1 cells, which showed that the expressed recombinant Kvbeta 2 polypeptide shared immunoreactivity and electrophoretic mobility (Mr = 38 kDa) with the putative rat brain Kvbeta 2 polypeptide (the 41 kDa variant was not detected). No corresponding immunoreactivity was seen to the recombinant 44 kDa Kvbeta 1 polypeptide expressed in COS-1 cells or present in the crude rat brain membranes on the same immunoblot (Fig. 3). Immunofluorescence staining of transfected COS-1 cells revealed a similar pattern of immunoreactivity in that the anti-Kvbeta 2 antibody exhibited a strong immunofluorescence staining of Kvbeta 2- but not Kvbeta 1- transfected cells (Fig. 4).

Anti-Kvbeta 2 mAbs were generated against a glutathione-S-transferase fusion protein containing the entire Kvbeta 2 polypeptide. All of the N-terminally directed mAbs exhibited monospecific immunofluorescence staining of COS-1 cells expressing recombinant Kvbeta 2, but not Kvbeta 1, polypeptide (Fig. 4). No staining to untransfected cells was seen (Fig. 4). Immunoblot analysis of crude rat brain membranes revealed that only the K17/70 mAb recognizes the SDS-denatured rat brain Kvbeta 2 polypeptide and recombinant Kvbeta 2 (but not Kvbeta 1) polypeptide present in COS-1 cell extracts (Fig. 3). Additional details on the generation and characterization of these mAbs will be described elsewhere.

Reciprocal co-immunoprecipitations: subunit association

To determine whether Kvbeta 1 and Kvbeta 2 were present in the same rat brain K+ channel complexes, we performed reciprocal co-immunoprecipitation experiments with the anti-Kvbeta 1 and anti-Kvbeta 2 antibodies. Detergent lysates were prepared from rat brain membranes under conditions shown previously to preserve alpha - or beta -subunit interactions (Sheng et al., 1993; Wang et al., 1993; Rhodes et al., 1995; Nakahira et al., 1996; Shi et al., 1996). These lysates then were used in immunoprecipitation reactions performed with the anti-Kvbeta 1 and anti-Kvbeta 2 polyclonal antibodies and with a pan-beta polyclonal antibody that recognizes both Kvbeta 1 and Kvbeta 2 (Rhodes et al., 1995). Immunoprecipitations also were performed using a polyclonal antibody against the Kv2.1 alpha  subunit, which should not co-immunoprecipitate either of these beta  subunits (Rhodes et al., 1995). Immunoprecipitation reactions then were subjected to immunoblot analyses to assay for the presence of Kvbeta 1 with the anti-Kvbeta 1 polyclonal antibody, for Kvbeta 2 with the K17/70 mAb, and for the beta -subunit-associated alpha  subunit Kv1.2 with the Kv1.2C polyclonal antibody (Rhodes et al., 1995).

As expected, the anti-Kvbeta 1 antibody immunoprecipitated the 44 kDa Kvbeta 1 polypeptide from these rat brain extracts (Fig. 5), as did the anti-pan-beta polyclonal antibody, whereas the anti-Kv2.1 antibody did not immunoprecipitate detectable amounts of Kvbeta 1. That the same amount of rabbit IgG was precipitated in each of these reactions is demonstrated by the ~50 kDa band present in each immunoprecipitation lane that is attributable to immunoreactivity of the rabbit IgG from the immunoprecipitating antibody, with the anti-rabbit secondary antibody used to develop the immunoblots. Surprisingly, levels of Kvbeta 1 comparable to those observed in the anti-Kvbeta 1 reaction were co-immunoprecipitated by the anti-Kvbeta 2 polyclonal antibody. Because these immunoprecipitation reactions were performed under conditions in which antigen is limiting, these data demonstrate that a large proportion of the Kvbeta 1 found in rat brain is present in complexes that also contain the Kvbeta 2 beta  subunit (Fig. 5).


Fig. 5. Presence of Kvbeta 1 and Kvbeta 2 in rat brain K+ channel complexes. Samples of adult rat brain membranes (RBM) (60 µg) and aliquots of products of immunoprecipitation reactions from detergent extracts of 500 µg RBM with polyclonal antibodies specific for both Kvbeta 1 and Kvbeta 2 (pan-beta , 1:100), Kvbeta 1 (anti-Kvbeta 1, 1:200), Kvbeta 2 (anti-Kvbeta 2, 1:500), or the Kv2.1 alpha  subunit (anti-Kv2.1, 1:200) were size fractionated by 12.5% SDS-PAGE. Samples were transferred to nitrocellulose and probed with rabbit anti-Kvbeta 1 polyclonal antibody at 1:50 (left panel), mouse anti-Kvbeta 2 mAb K17/70 neat (middle panel), or rabbit anti-Kv1.2 polyclonal antibody at 1:100 (right panel). Bound antibody detected by ECL-autoradiography for 40 min (left panel) or 3 min (middle and right panels). Arrows point to mobility of the heavy-chain polypeptides of the rabbit immunoglobulins used in the immunoprecipitation reactions and of the respective K+ channel polypeptides; numbers at left of the panels denote Mr of prestained molecular weight standards. Bands at ~50 kDa (left and right panels) are heavy chains of rabbit IgG used for immunoprecipitations, which react with anti-rabbit, but not anti-mouse, secondary antibody.
[View Larger Version of this Image (29K GIF file)]

These same immunoprecipitation reactions then were assayed for the presence of Kvbeta 2; because these immunoblots were performed with the anti-Kvbeta 2 mouse mAb K17/70, the rabbit IgG bands typically seen in the immunoprecipitation reactions are not visualized. The rabbit anti-Kvbeta 2 polyclonal antibody was able to immunoprecipitate the 38 kDa Kvbeta 2 polypeptide present in these samples. Comparable amounts of Kvbeta 2 also were observed in the immunoprecipitation reaction performed with the anti-pan-beta polyclonal antibody (Fig. 5). Low levels of Kvbeta 2 also were observed in the reactions performed with the anti-Kvbeta 1 antibody, confirming the observation above and indicating that individual K+ channel complexes may contain both of these beta -subunit polypeptides. As expected, the anti-Kv2.1 antibody also was unable to co-immunoprecipitate detectable amounts of Kvbeta 2. All of the immunoprecipitation reactions performed with the anti-beta -subunit antibodies contained Kv1.2 (Fig. 5), showing that each of these antibodies could effectively recognize and isolate intact K+ channel complexes.

Immunohistochemical localization of Kvbeta 1 and Kvbeta 2

Analysis of immunohistochemically stained sections indicated that Kvbeta 1 and Kvbeta 2 are present in cell bodies, dendrites, the juxtaparanodal regions of myelinated axons, and terminal fields of several major projection systems. The pattern of immunohistochemical staining observed with mAbs and polyclonal antibodies to the same beta  subunit was indistinguishable. The areal and laminar distribution of labeled cells suggests and comparisons of sections processed by in situ hybridization and immunohistochemistry suggest that cells expressing Kvbeta 1 and Kvbeta 2 mRNA also are immunoreactive for the corresponding proteins. Regions containing moderate to intense immunoreactivity for Kvbeta 1 and Kvbeta 2 were the neocortex, hippocampus, piriform cortex, striatum, thalamus, cerebellum, cranial nerve nuclei, and virtually all major white matter pathways. Although it is beyond the scope of this paper to provide a comprehensive account of beta -subunit immunoreactivity in every brain region, the salient features of the immunohistochemical staining are described below and summarized in Table 1. Neocortex and hippocampus In the neocortex, immunoreactivity for Kvbeta 1 was concentrated within pyramidal cells and was particularly intense in smaller bipolar and multipolar interneurons in layers II, III, V, and VI (Fig. 6A). In each of these cell populations, immunoreactivity for Kvbeta 1 was concentrated within the cell body and proximal portions of the dendritic tree. Immunoreactivity for Kvbeta 2 also was observed in cortical pyramidal cells, with very intense labeling of large pyramidal cells in layer V (Fig. 6C). However, in contrast to immunoreactivity for Kvbeta 1, immunoreactivity for Kvbeta 2 was concentrated throughout the entire apical dendrite of layer V pyramidal cells, including fine branches and apical tufts. Interestingly, the small interneurons that displayed strong immunoreactivity for Kvbeta 1 were not intensely immunoreactive for Kvbeta 2. In addition to the somatodendritic staining described above, in many cortical regions, moderate to intense immunoreactivity for Kvbeta 1 and Kvbeta 2 was observed in axonal profiles coursing throughout the cortical neuropil and in a pattern consistent with labeling of terminal fields. This pattern of labeling was particularly intense in the piriform, posterior cingulate, retrosplenial, and entorhinal cortices. Myelinated axons in the subcortical white matter as well as other regions also contained immunoreactivity for Kvbeta 1 and Kvbeta 2. The staining in these fibers was discontinuous, with a greater density of reaction product in the juxtaparanodal segments at nodes of Ranvier (Shi et al., 1996).
Fig. 6. Immunohistochemical localization of Kvbeta 1 and Kvbeta 2 in the cerebral cortex and striatum. In parietal cortex, immunoreactivity for Kvbeta 1 (K9/40 mAb) (A) is concentrated in large pyramidal cells in layer V and in small interneurons (arrows) concentrated primarily in layers II and III. Immunoreactivity for Kvbeta 2 (affinity-purified polyclonal antibody) (C) is concentrated in the somas and apical dendrites of layer V pyramidal cells. The small interneurons that contain a high density of immunoreactivity for Kvbeta 1 contain a much lower density of immunoreactivity for Kvbeta 2. In the striatum, there is a very high density of immunoreactivity for Kvbeta 1 (B) and Kvbeta 2 (D) in the globus pallidus (GP) and a lower density in the caudate nucleus (CD). In the globus pallidus, immunoreactivity for Kvbeta 1 and Kvbeta 2 is primarily concentrated in terminal fields and is present in both the supra- and subcommissural segments. AC, Anterior commissure.
[View Larger Version of this Image (159K GIF file)]

In the hippocampal formation, there was a heterogeneous distribution of Kvbeta 1 and Kvbeta 2 immunoreactivity with moderate to intense staining of granule and pyramidal cell somata (data not shown). Immunoreactivity for Kvbeta 1 and Kvbeta 2 also was concentrated within interneurons in stratum oriens and stratum radiatum of the CA1-CA3 subfields. In the dentate gyrus, granule cells contained a moderate density of Kvbeta 1 and Kvbeta 2 immunoreactivity. In these cells, staining was concentrated in the cell soma and did not extend into dendritic branches, suggesting that this staining is associated with beta  subunits synthesized within these cells. In the molecular layer of the dentate gyrus, there was a distinct band of Kvbeta 1 and Kvbeta 2 immunoreactivity in the middle third and somewhat less intense labeling in the outer third. As described previously (Rhodes et al., 1995), the location of this band of intense immunoreactivity corresponds closely to the termination zone of the medial perforant path. In the CA subfields and subiculum, immunoreactivity for Kvbeta 1 and Kvbeta 2 was distributed diffusely throughout the neuropil, with a greater density in stratum radiatum and stratum oriens than in stratum moleculare, and immunoreactivity for Kvbeta 2, but not Kvbeta 1, was concentrated throughout the apical dendrites and apical dendritic tufts of hippocampal pyramidal cells. In the CA3 subfield, there was a moderate density of Kvbeta 1 immunoreactivity in the mossy fiber zone.

Striatum and basal forebrain In the caudate nucleus and nucleus accumbens, there was a moderate density of immunoreactivity for Kvbeta 1 and Kvbeta 2 within neurons of all sizes and morphologies and distributed diffusely throughout the neuropil. Interestingly, there was a higher density of immunoreactivity for Kvbeta 1 and Kvbeta 2 in bundles of axons coursing through the ventromedial portion of the caudate nucleus than in similar fiber bundles in the dorsolateral component of this structure. These fibers appeared to originate from cells within the caudate nucleus and could be followed in consecutive sections to their apparent termination throughout the supra- and subcommissural segments of the globus pallidus (Fig. 6B,D). Additional fibers showing intense immunoreactivity for Kvbeta 1 and Kvbeta 2 appeared to continue beyond the globus pallidus and run through the medial forebrain bundle and cerebral peduncle to terminate within the substantia nigra pars reticulata (Fig. 7A,C). Intense terminal-field labeling for Kvbeta 1 and Kvbeta 2 also was observed in the nucleus accumbens and olfactory tubercle, and in the deep half of the molecular layer of pyriform cortex.
Fig. 7. Immunohistochemical localization of Kvbeta 1 and Kvbeta 2 in the midbrain and cerebellum. In the midbrain, immunoreactivity for Kvbeta 1 (K9/40 mAb) (A) and Kvbeta 2 (affinity-purified polyclonal antibody) (C) is concentrated in terminal fields in the pars reticulata of the substantia nigra (SNr) and in large neurons in the red nucleus (RN). In the cerebellar cortex, immunoreactivity for Kvbeta 1 (B) and Kvbeta 2 (D) is concentrated in the cell bodies of Purkinje cells (P) and in terminal fields throughout the molecular layer. Immunoreactivity for Kvbeta 2 also is concentrated in the dendrites of Purkinje cells and in the axon terminals of basket cells, which form a characteristic synaptic plexus (arrows) that terminates on the initial segment of Purkinje cell axons.
[View Larger Version of this Image (162K GIF file)]

In the medial septal and diagonal band nuclei, immunoreactivity for Kvbeta 1 and Kvbeta 1 was present in the somatodendritic compartments of magnocellular neurons. These neurons formed a continuous column of cells extending through the medial septum and vertical and horizontal limbs of the diagonal band and into the magnocellular neurons of the nucleus basalis of Meynert. This pattern of Kvbeta 1 and Kvbeta 2 immunoreactivity was virtually identical to the distribution of Kvbeta 1 and Kvbeta 2 mRNA and is very similar to the distribution and characteristic morphology of basal forebrain cholinergic neurons (Mesulam et al., 1983).

Thalamus Many cells within various thalamic nuclei were immunoreactive for Kvbeta 1 and Kvbeta 2. The distribution of labeled cells corresponded closely to the pattern of expression Kvbeta 1 and Kvbeta 2 mRNA. In virtually all of these cells, a large proportion of the immunoreactivity was concentrated within the somatic and dendritic cytoplasm, suggesting that this staining is associated with the beta -subunit polypeptides not yet transported to a distinct plasma membrane domain. Cerebellum In the cerebellum, immunoreactivity for Kvbeta 1 and Kvbeta 2 was concentrated within the cell bodies and dendrites of Purkinje cells and was distributed diffusely and with moderate intensity throughout the molecular layer (Fig. 7B,D). Immunoreactivity for Kvbeta 2 also was present in the axon terminals of basket cells. These basket-cell terminals form a characteristic plexus surrounding the initial segment of Purkinje cell axons and have been shown previously to contain intense immunoreactivity for Kv1.1 and Kv1.2 alpha  subunits (McNamara et al., 1993; Wang et al., 1993, 1994; Rhodes et al., 1995; Shi et al., 1996). Numerous fine-caliber axons within the granule cell layer also contained immunoreactivity for Kvbeta 1 and Kvbeta 2, as did large neurons within the deep cerebellar nuclei. The axons in the granule cell layer appeared to originate from Purkinje cells and frequently appeared to emerge from the core of labeling of the basket-cell terminal plexus. Large-caliber myelinated axons in the cerebellar white matter also contained intense immunoreactivity for Kvbeta 1 and Kvbeta 2. As described above, this immunoreactivity was strikingly discontinuous, with intense staining in patches juxtaposed to nodes of Ranvier (Shi et al., 1996). Midbrain and brainstem Large motor and sensory neurons within structures such as the red nucleus and all cranial nerve sensory and motor nuclei contained moderate to intense immunoreactivity for Kvbeta 1 and Kvbeta 2 (Fig. 6A,C). In addition, neurons within the substantia nigra pars reticulata and in the deep layers of the superior colliculus were immunoreactive for these two beta -subunit polypeptides. Large neurons within the midbrain and pontine reticular formation and inferior olivary complex were moderately to intensely labeled by both anti-beta -subunit antibodies, as were axons within midbrain and brainstem white matter pathways and cranial nerve efferents.

DISCUSSION

Molecular, immunological, and neuroanatomical techniques were used to examine the expression, distribution, and co-association of two voltage-gated K+ channel beta  subunits in adult rat brain. Although the relative density of Kvbeta 1 and Kvbeta 2 expression and immunoreactivity differs considerably across brain regions, the expression of these two subunits shows substantial overlap, and in many cell populations the respective mRNAs and polypeptides probably co-localize. The riboprobes used in the present study reacted with single bands by Northern and Southern analysis, and their specificity is supported by the observation that two independent riboprobes targeted to nonoverlapping sequences of the target mRNA gave identical hybridization patterns. Moreover, the pattern of immunoreactivity revealed by Kvbeta 1- and Kvbeta 2-specific antibodies is consistent with localization of the respective polypeptides to the cell bodies, dendrites, or terminals of cells expressing the corresponding mRNA. Antibodies raised against unique N-terminal sequences of Kvbeta 1 and Kvbeta 2 reacted with 44 kDa and 38 kDa polypeptides, respectively, in membranes prepared from rat brain and transfected COS cells; in brain, the 38 kDa Kvbeta 2 polypeptide is far more abundant. On the basis of reciprocal co-immunoprecipitation experiments, it appears that almost all channel complexes containing Kvbeta 1 also contain Kvbeta 2.

Using subtype-specific anti-beta -subunit antibodies, we unambiguously identified the Kvbeta 1 and Kvbeta 2 polypeptides in rat brain. We based this identification on their reaction with antibodies made to unique regions of the deduced amino acid sequence and their co-migration on SDS gels with the recombinant protein expressed in transfected cells. Together, these results indicate that in rat brain, Kvbeta 1 is a relatively low-abundance polypeptide of Mr = 44 kDa, whereas Kvbeta 2 is an abundant polypeptide of Mr = 38 kDa. Subsequent analyses of the Mr = 44 kDa Kvbeta 1 and Mr = 38 kDa Kvbeta 2 polypeptide pools in rat brain membranes using a polyclonal antibody raised against a C-terminal peptide conserved in both beta  subunits (``pan-beta '' antibody) (Rhodes et al., 1995) reveal a striking difference in the relative abundance of these two subunits. Quantitative analysis of immunoblots performed using the pan-beta -subunit antibody, which recognizes both Kvbeta 1 and Kvbeta 2, shows that Kvbeta 2 is ~50-fold more abundant than Kvbeta 1 in rat brain membranes, supporting the model we initially proposed (Rhodes et al., 1995) that Kvbeta 2 was by far the more abundant beta  subunit subtype in brain. In addition, most Kvbeta 1 appears to be present in complexes that also contain Kvbeta 2, as evidenced by the fact that Kvbeta 1-containing complexes are co-immunoprecipitated using a Kvbeta 2-specific antibody. A small portion of the total Kvbeta 2 pool in brain is present in these complexes, as evidenced by the small amount of Kvbeta 2 in immunoprecipitations performed using the anti-Kvbeta 1 antibody, thus most of the Kvbeta 2 in brain appears to exist in complexes lacking Kvbeta 1. Conversely, it appears that the small amount of Kvbeta 1 present in brain exists in complexes containing Kvbeta 2; very few ``pure'' Kvbeta 1 complexes are present.

Comparison of immunohistochemical staining patterns observed in the present study with those obtained using the pan-beta antibody (Rhodes et al., 1995) suggests that the immunoreactivity for Kvbeta 2 recapitulates virtually the entire pattern and extent of immunoreactivity observed using the pan-beta antibody. If we assume that pan-beta staining represents the total beta -subunit pool associated with Shaker alpha  subunits, then we can assign Kvbeta 2 as the major component of this pool. In addition, we can assign Kvbeta 1 as a comparatively minor subcomponent, found predominantly in channel complexes that also contain Kvbeta 2 (Fig. 8). On the basis of this analysis, we also can speculate that in brain, the distribution of other beta -subunit isoforms containing this conserved C-terminal epitope (England et al., 1995a,b; Heinemann et al., 1995; Majumder et al., 1995; Morales et al., 1995) will show considerable overlap with that observed using the pan-beta and Kvbeta 2-specific antibodies, and that these other beta  subunits will be present in rat brain K+ channel complexes in association with Kvbeta 2.


Fig. 8. Hypothetical model of beta -subunit associations in rat brain. The total rat brain beta -subunit pool contains at least three beta -subunit isoforms, designated Kvbeta 1, Kvbeta 2, and Kvbeta X in this Venn diagram. Immunoreactivity observed using Kvbeta 2-specific antibodies recapitulates virtually the entire pattern and extent of immunoreactivity revealed using the pan-beta antibody (Rhodes et al., 1995), suggesting that Kvbeta 2 is present in virtually all beta  subunit-containing K+ channel complexes. In contrast, immunoreactivity for Kvbeta 1 is present in a small proportion of pan-beta /Kvbeta 2-immunoreactive structures. Because the C-terminal epitope contained within the pan-beta antibody is present in all cloned Kvbeta subunits, it is likely that these beta -subunit isoforms (designated Kvbeta X) also will associate with a subset of the total brain beta -subunit-containing complexes, i.e., in complexes that also contain Kvbeta 2.
[View Larger Version of this Image (20K GIF file)]

The pattern of Kvbeta 1 expression reported here is virtually identical to that reported for Kvbeta 1 mRNA by Rettig et al. (1994). Their data and ours indicated that Kvbeta 1 is highly expressed in the cortical interneurons; basal ganglia; limbic cortical regions such as the hippocampus, posterior cingulate, and entorhinal cortex; and brainstem and cerebellum. In the majority of regions, immunoreactivity for Kvbeta 1 is concentrated at the terminals of cells expressing Kvbeta 1 mRNA. The very high levels of Kvbeta 1 expression in the caudate putamen corresponded well to the high density of Kvbeta 1 immunoreactivity in terminal fields in the globus pallidus and substantia nigra and, similarly, the high levels of Kvbeta 1 expression in entorhinal cortex correspond to a band of Kvbeta 1 immunoreactivity in the molecular layer of the dentate gyrus (data not shown). One clear exception to this relationship is that numerous cortical and hippocampal interneurons contain high levels of Kvbeta 1 mRNA and also contain a high density of Kvbeta 1 immunoreactivity at their somata and proximal dendrites.

Overall, the pattern of Kvbeta 1 expression corresponds closely to that observed for Kv1.4 (Sheng et al., 1992) and, to a lesser extent, Kv1.1 (Wang et al., 1994). In agreement, the high levels of Kvbeta 1 immunoreactivity in the globus pallidus and pars reticulata of the substantia nigra correspond closely to the patterns reported for Kv1.1 and Kv1.4 in these structures (Sheng et al., 1992; Wang et al., 1994). Similarly, the high levels of Kvbeta 1 immunoreactivity in cortical interneurons corresponds closely to the patterns of expression and immunoreactivity for Kv1.1, Kv1.2, and Kv1.6 alpha  subunits in these cells (Wang et al., 1994) (M. Monaghan, N. Barrezueta, J. Trimmer, K. Rhodes, unpublished observations). This observation suggests that Kv1.1, Kv1.2, Kv1.6, Kvbeta 1, and perhaps Kvbeta 2 may co-localize and co-assemble in these cells. In view of the high spontaneous firing rates and short-duration action potentials reported for cortical (hippocampal) interneurons (Williams et al., 1994), it is tempting to speculate that the high levels of Kvbeta 1 expression and its subsequent effects on associated alpha  subunits (Rettig et al., 1994) in these cells are related to an intrinsic requirement for rapidly inactivating channels.

Although Rettig et al. (1994) did not examine Kvbeta 2 expression in their study, the data reported here indicate that Kvbeta 2 also is widely expressed in rat brain. Cohen and his colleagues (Arai et al., 1992; Cohen et al., 1992) have characterized the expression of a gene, F5 (Sabath et al., 1990), that is highly expressed in activated T lymphocytes and brain. The recent submission of the F5 sequence to the Genbank database identified F5 as the mouse homolog of Kvbeta 2. The pattern of F5 mRNA expression in brain (Arai et al., 1992; Cohen et al., 1992) is qualitatively similar to that reported here for Kvbeta 2, but is described more accurately as a composite of Kvbeta 1 and Kvbeta 2 mRNA expression. This may be attributable to the probe that was used for hybridization, which was derived from a region, which in rat, is highly conserved between Kvbeta 1 and Kvbeta 2 (80% identity at the nucleotide level).

Immunoreactivity for Kvbeta 2 is concentrated in the juxtaparanodal regions of myelinated axons and in terminal fields of cells expressing Kvbeta 2 mRNA. This is clearly evident in the cerebellar cortex, where Kvbeta 2 mRNA is expressed in basket cells and Kvbeta 2 immunoreactivity concentrated in the basket-cell terminal plexus, and in the hippocampus, where Kvbeta 2 mRNA is expressed in layer II cells in entorhinal cortex and Kvbeta 2 immunoreactivity is present in the molecular layer of the dentate gyrus. However, there also are clear examples of Kvbeta 2 immunoreactivity in somatic and dendritic domains. For example, in cortical and hippocampal pyramidal cells, Kvbeta 2 immunoreactivity is concentrated in the cell body and throughout the apical dendritic tree, and in cerebellar Purkinje cells, Kvbeta 2 immunoreactivity is concentrated in somatic and dendritic domains.

In general, the pattern of expression and immunoreactivity for Kvbeta 2 is best approximated by a composite of that reported for the Kv1.1 and Kv1.2 alpha  subunits (Sheng et al., 1994; Wang et al., 1994; Rhodes et al., 1995). In cerebellar basket-cell terminals, the pattern of Kvbeta 2 immunoreactivity corresponds precisely to that observed for Kv1.1 and Kv1.2 (McNamara et al., 1993; Wang et al., 1993, 1994; Sheng et al., 1994; Rhodes et al., 1995; Shi et al., 1996), indicating that Kvbeta 2 is likely to be associated with Kv1.1 and Kv1.2 in these complexes. Similarly, in the juxtaparanodal membrane of myelinated axons, the pattern of Kvbeta 2 immunoreactivity is identical to that observed for Kv1.1 and Kv1.2 (Wang et al., 1993, 1994; Sheng et al., 1994; Shi et al., 1996). In other regions, such as the somatodendritic membranes of cortical pyramidal cells and the cell bodies of midbrain and brainstem motor neurons, the pattern of Kvbeta 2 immunoreactivity also corresponds closely to Kv1.1 and Kv1.2. However, in other regions, such as the apical dendritic trees of cortical and hippocampal pyramidal cells, the pattern of Kvbeta 2 immunoreactivity is matched by that for Kv1.6 (K. Rhodes, M. Monaghan, J. Trimmer, unpublished observations) and Kv4.2 (Sheng et al., 1992), but not by any of the other Kv1 family alpha  subunits. Because Kvbeta 2 (and Kvbeta 1) can associate with all Kv1 alpha  subunits, as well as Kv4.2, in heterologous expression systems (Nakahira et al., 1996), in pyramidal cell dendrites, Kvbeta 2 may be present in channel complexes containing Kv1.6 or Kv4.2. However, this association remains to be confirmed by reciprocal co-immunoprecipitation studies.

The results of the present study indicate that beta  subunits are fundamental components of voltage-gated K+ channels in mammalian brain. Their widespread expression and tight association with alpha  subunits suggests that they play a fundamental role in the function of native channels (Parcej and Dolly, 1989, 1992; Trimmer, 1991; Scott et al., 1994a,b; Rhodes et al., 1995; Shi et al., 1996). As both Kvbeta 1 and Kvbeta 2 are found in both presynaptic (axons and terminals) and postsynaptic (soma and dendrites) plasma membrane domains, it may be assumed that they in themselves do not effectively target K+ channels to specific subcellular compartments (Sheng et al., 1992, 1994). Some beta  subunits, including Kvbeta 1, do exert dramatic effects on the electrophysiological properties of expressed channels (Rettig et al., 1994; England et al., 1995a,b; Majumder et al., 1995; Morales et al., 1995). However, Kvbeta 2, the major beta  subunit in brain, does not, raising questions as to its precise function. We reported recently that Kvbeta 2 exerts chaperone-like effects on the Kv1.2 alpha  subunit, promoting its efficient glycosylation, surface expression, and stability in the plasma membrane (Shi et al., 1996). Similar effects are mediated by Kvbeta 1 (Shi et al., 1996). Interestingly, mutations in a Drosophila beta  subunit result in a reduction in the surface density of K+ channel complexes (Chouinard et al., 1995), supporting the contention that the effects of beta  subunits on the biosynthetic maturation and stability of alpha  subunits is a fundamental role. Further definition of the sites where beta  subunits co-localize and co-associate with individual alpha  subunits will further define the role of Kvbeta 1 and Kvbeta 2 in determining the properties of K+ channel complexes in mammalian brain.


FOOTNOTES

Received Jan. 18, 1996; revised May 9, 1996; accepted May 21, 1996.

  

This work was supported by Wyeth-Ayerst Research and the Center for Biotechnology at Stony Brook, and funded by the New York State Science and Technology Foundation and by National Institutes of Health Grant NS34383 (J.S.T.). This work was done during the tenure of an Established Investigatorship from the American Heart Association (J.S.T.). We thank Drs. James E. Barrett and John A. Moyer for critically reviewing this manuscript.

Correspondence should be addressed to Dr. James S. Trimmer, Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215.



REFERENCES

  • Arai M, Prystowsky MB, Cohen JA (1992) Expression of the T-lymphocyte activation gene, F5, by mature neurons. J Neurosci Res 33:527-537 . [Web of Science][Medline]
  • Chouinard SW, Wilson GF, Schliomgen AK, Ganetsky B (1995) A potassium channel beta  subunit related to the aldo-keto reductase superfamily is encoded by the Drosophila hyperkinetic locus. Proc Natl Acad Sci USA 92:6763-6767 . [Abstract/Free Full Text]
  • Cohen JA, Arai M, Luning Prak E, Brooks SA, Young LH, Prystowsky MB (1992) Characterization of a novel mRNA expressed by neurons in mature brain. J Neurosci Res 31:273-284 . [Web of Science][Medline]
  • England SK, Uebele VN, Kodali J, Bennett PB, Tamkun MM (1995a) A novel K+ channel beta -subunit (hKvbeta 1.3) is produced via alternative mRNA splicing. J Biol Chem 270:28531-28534 . [Abstract/Free Full Text]
  • England SK, Uebele VN, Shear H, Kodali J, Bennett PB, Tamkun MM (1995b) Characterization of a voltage-gated K+ channel beta  subunit expressed in human heart. Proc Natl Acad Sci USA 92:6309-6313 . [Abstract/Free Full Text]
  • Harlow E, Lane D (1988) Antibodies: a laboratory manual. .
  • Heinemann SH, Rettig J, Wunder F, Pongs O (1995) Molecular and functional characterization of a rat brain Kvbeta 3 potassium channel subunit. FEBS Lett 377:383-389 . [Web of Science][Medline]
  • Hille B (1992) Ionic channels of excitable membranes. .
  • Isom LL, De Jongh KS, Catterall WA (1994) Auxiliary subunits of voltage-gated ion channels. Neuron 12:1183-1194 . [Web of Science][Medline]
  • Jan LY, Jan YN (1990) How might the diversity of potassium channels be generated? Trends Neurosci 13:415-419 . [Web of Science][Medline]
  • Johnson DAG, Sportsman JR, Elder JH (1984) Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal Tech 1:3-8.
  • Maizel JV (1971) Polyacrylamide gel electrophoresis of viral proteins. Methods Virol 5:179-246.
  • Majumder K, De Biasi M, Wang Z, Wible BA (1995) Molecular cloning and functional expression of a novel potassium channel beta -subunit from human atrium. FEBS Lett 361:13-16 . [Web of Science][Medline]
  • McCormack K, McCormack T, Tanouye M, Rudy B, Stühmer W (1995) Alternative splicing of the human Shaker K+ channel beta 1 gene and functional expression of a beta 2 gene product. FEBS Lett 370:32-36 . [Web of Science][Medline]
  • McNamara NM, Muniz ZM, Wilkin GP, Dolly JO (1993) Prominent location of a K+ channel containing the alpha subunit Kv1.2 in the basket cell nerve terminals of rat cerebellum. Neuroscience 57:1039-1045 . [Web of Science][Medline]
  • Mesulam M-M, Mufson EJ, Wainer BH, Levey AI (1983) Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Ch1-Ch6). Neuroscience 10:1185-1201 . [Web of Science][Medline]
  • Morales MJ, Castellino RC, Crews AL, Rasmussen RL, Strauss HC (1995) A novel beta  subunit increases the rate of inactivation of specific voltage-gated potassium channel alpha  subunits. J Biol Chem 270:6272-6277 . [Abstract/Free Full Text]
  • Nakahira K, Shi G, Rhodes KJ, Trimmer JS (1996) Selective interaction of voltage-gated K+ channel beta -subunits with alpha -subunits. J Biol Chem 271:7084-7089 . [Abstract/Free Full Text]
  • Parcej DN, Dolly JO (1989) Elegance persists in the purification of K+ channels. Biochem J 264:623-624 . [Web of Science][Medline]
  • Parcej DN, Scott VES, Dolly JO (1992) Oligomeric properties of alpha -dendrotoxin-sensitive potassium channels purified from bovine brain. Biochemistry 31:11084-11088 . [Medline]
  • Pongs O (1992) Molecular biology of voltage-dependent potassium channels. Physiol Rev 72 [Suppl 4]:S69-S88.
  • Pongs O (1995) Regulation of the activity of voltage-gated potassium channels by beta  subunits. Semin Neurosci 7:137-146.
  • Rettig J, Heinemann SH, Wunder F, Lorra C, Parcej DN, Dolly JO, Pongs O (1994) Inactivation properties of voltage-gated K+ channels altered by presence of beta -subunit. Nature 369:289-294 . [Medline]
  • Rhodes KJ, Keilbaugh SA, Barrezueta NX, Lopez KL, Trimmer JS (1995) Association and colocalization of K+ channel alpha - and beta -subunit polypeptides in rat brain. J Neurosci 15:5630-5671.
  • Ruppersberg JP, Schroter KH, Sakmann B, Stocker M, Sewing S, Pongs O (1990) Heteromultimeric channels formed by rat brain potassium-channel protein. Nature 345:535-537 . [Medline]
  • Sabath DE, Podolin PL, Comber PG, Prystowsky MB (1990) cDNA cloning and characterization of interleukin 2-induced genes in a cloned T helper lymphocyte. J Biol Chem 265:12671-12678 . [Abstract/Free Full Text]
  • Scott VE, Muniz ZM, Sewing S, Lichtinghagen R, Parcej DN, Pongs O, Dolly JO (1994a) Antibodies specific for distinct Kv subunits unveil a hetero-oligomeric basis for subtypes of alpha -dendrotoxin-sensitive K+ channels in bovine brain. Biochemistry 33:1617-1623 . [Medline]
  • Scott VE, Rettig J, Parcej DN, Keen JN, Findlay JB, Pongs O, Dolly JO (1994b) Primary structure of a beta -subunit of alpha -dendrotoxin-sensitive K+ channels from bovine brain. Proc Natl Acad Sci USA 91:1637-1641 . [Abstract/Free Full Text]
  • Sheng M, Tsaur ML, Jan YN, Jan LY (1992) Subcellular segregation of two A-type K+ channel proteins in rat central neurons. Neuron 9:271-284 . [Web of Science][Medline]
  • Sheng M, Liao YJ, Jan YN, Jan LY (1993) Presynaptic A-current based on heteromultimeric K+ channels detected in vivo. Nature 365:72-75 . [Medline]
  • Sheng M, Tsaur ML, Jan YN, Jan LY (1994) Contrasting subcellular localization of the Kv1.2 K+ channel subunits in different neurons of rat brain. J Neurosci 14:2408-2417 . [Abstract]
  • Shi G, Kleinklaus A, Marrion N, Trimmer JS (1994) Properties of Kv2.1 K+ channels expressed in transfected mammalian cells. J Biol Chem 269:23204-23211 . [Abstract/Free Full Text]
  • Shi G, Nakahira K, Hammond S, Rhodes KJ, Schechter LE, Trimmer JS (1996) beta -subunits promote K+ channel surface expression through effects early in biosynthesis. Neuron 16:843-852 . [Web of Science][Medline]
  • Stühmer W, Ruppersberg JP, Schroter KH, Sakmann B, Stocker M, Giese KP, Perschke A, Baumann A, Pongs O (1989) Molecular basis of functional diversity of voltage gated potassium channels in mammalian brain. EMBO J 8:3235-3244 . [Web of Science][Medline]
  • Trimmer JS (1991) Immunological identification and characterization of a delayed rectifier K+ channel in rat brain. Proc Natl Acad Sci USA 88:10764-10768 . [Abstract/Free Full Text]
  • Trimmer JS, Trowbridge IS, Vacquier VD (1985) Monoclonal antibody to a membrane glycoprotein inhibits the acrosome reaction and associated Ca2+and H+ fluxes of sea urgin sperm. Cell 40:697-703 . [Web of Science][Medline]
  • Wang H, Kunkel DD, Martin TM, Schwartzkroin PA, Tempel BL (1993) Heteromultimeric K+ channels in terminal and juxtaparanodal regions of neurons. Nature 365:75-79 . [Medline]
  • Wang H, Kunkel DD, Martin TM, Schwartzkroin PA, Tempel BL (1994) Localization of Kv1.1 and Kv1.2, two K channel proteins, to synaptic terminals, somata, and dendrites in the mouse brain. J Neurosci 14:4588-4599 . [Abstract]
  • Williams S, Samulack DD, Beaulieu C, LaCaille JC (1994) Membrane properties and synaptic responses of interneurons located near the stratum lacunosum-moleculare/radiatum border of area CA1 in whole-cell recordings from rat hippocampal slices. J Neurophysiol 71:2217-2235 . [Abstract/Free Full Text]

Copyright ©1996 Society for Neuroscience   0270-6474/1996/164846-15$05.00/0



This article has been cited by other articles:


Home page
J. Neurophysiol.Home page
R. H. Pineda, C. S. Knoeckel, A. D. Taylor, A. Estrada-Bernal, and A. B. Ribera
Kv1 Potassium Channel Complexes In Vivo Require Kv{beta}2 Subunits in Dorsal Spinal Neurons
J Neurophysiol, October 1, 2008; 100(4): 2125 - 2136.
[Abstract] [Full Text] [PDF]


Home page
Physiol. Rev.Home page
H. Vacher, D. P. Mohapatra, and J. S. Trimmer
Localization and Targeting of Voltage-Dependent Ion Channels in Mammalian Central Neurons
Physiol Rev, October 1, 2008; 88(4): 1407 - 1447.
[Abstract] [Full Text] [PDF]


Home page
JGPHome page
K. J. Rhodes and J. S. Trimmer
Antibody-based Validation of CNS Ion Channel Drug Targets
J. Gen. Physiol., May 1, 2008; 131(5): 407 - 413.
[Full Text] [PDF]


Home page
J. Neurosci.Home page
K. J. Rhodes and J. S. Trimmer
Antibodies as Valuable Neuroscience Research Tools versus Reagents of Mass Distraction
J. Neurosci., August 2, 2006; 26(31): 8017 - 8020.
[Full Text] [PDF]


Home page
PNHome page
J. M Schott
Limbic encephalitis: a clinician's guide
Practical Neurology, June 1, 2006; 6(3): 143 - 153.
[Full Text] [PDF]


Home page
J. Physiol.Home page
B. E McKay and R. W Turner
Physiological and morphological development of the rat cerebellar Purkinje cell
J. Physiol., September 15, 2005; 567(3): 829 - 850.
[Abstract] [Full Text] [PDF]


Home page
J. Neurosci.Home page
B. E. McKay, M. L. Molineux, W. H. Mehaffey, and R. W. Turner
Kv1 K+ Channels Control Purkinje Cell Output to Facilitate Postsynaptic Rebound Discharge in Deep Cerebellar Neurons
J. Neurosci., February 9, 2005; 25(6): 1481 - 1492.
[Abstract] [Full Text] [PDF]


Home page
J. Neurosci.Home page
K. J. Rhodes, K. I. Carroll, M. A. Sung, L. C. Doliveira, M. M. Monaghan, S. L. Burke, B. W. Strassle, L. Buchwalder, M. Menegola, J. Cao, et al.
KChIPs and Kv4 {alpha} Subunits as Integral Components of A-Type Potassium Channels in Mammalian Brain
J. Neurosci., September 8, 2004; 24(36): 7903 - 7915.
[Abstract] [Full Text] [PDF]


Home page
Physiol. Rev.Home page
S. G. Birnbaum, A. W. Varga, L.-L. Yuan, A. E. Anderson, J. D. Sweatt, and L. A. Schrader
Structure and Function of Kv4-Family Transient Potassium Channels
Physiol Rev, July 1, 2004; 84(3): 803 - 833.
[Abstract] [Full Text] [PDF]


Home page
BrainHome page
A. Vincent, C. Buckley, J. M. Schott, I. Baker, B.-K. Dewar, N. Detert, L. Clover, A. Parkinson, C. G. Bien, S. Omer, et al.
Potassium channel antibody-associated encephalopathy: a potentially immunotherapy-responsive form of limbic encephalitis
Brain, March 1, 2004; 127(3): 701 - 712.
[Abstract] [Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
O. Sokolova, A. Accardi, D. Gutierrez, A. Lau, M. Rigney, and N. Grigorieff
Conformational changes in the C terminus of Shaker K+ channel bound to the rat Kv{beta}2-subunit
PNAS, October 28, 2003; 100(22): 12607 - 12612.
[Abstract] [Full Text] [PDF]


Home page
J. Physiol.Home page
T. Sacco and F. Tempia
A-Type potassium currents active at subthreshold potentials in mouse cerebellar purkinje cells
J. Physiol., September 1, 2002; 543(2): 505 - 520.
[Abstract] [Full Text] [PDF]


Home page
J. Physiol.Home page
M. A Lazaroff, A. D Taylor, and A. B Ribera
In vivo analysis of Kv{beta}2 function in Xenopus embryonic myocytes
J. Physiol., June 15, 2002; 541(3): 673 - 683.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
K. McCormack, J. X. Connor, L. Zhou, L. L. Ho, B. Ganetzky, S.-Y. Chiu, and A. Messing
Genetic Analysis of the Mammalian K+ Channel beta Subunit Kvbeta 2 (Kcnab2)
J. Biol. Chem., April 5, 2002; 277(15): 13219 - 13228.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
C. R. Campomanes, K. I. Carroll, L. N. Manganas, M. E. Hershberger, B. Gong, D. E. Antonucci, K. J. Rhodes, and J. S. Trimmer
Kvbeta Subunit Oxidoreductase Activity and Kv1 Potassium Channel Trafficking
J. Biol. Chem., March 1, 2002; 277(10): 8298 - 8305.
[Abstract] [Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
M. H. Holmqvist, J. Cao, R. Hernandez-Pineda, M. D. Jacobson, K. I. Carroll, M. A. Sung, M. Betty, P. Ge, K. J. Gilbride, M. E. Brown, et al.
Elimination of fast inactivation in Kv4 A-type potassium channels by an auxiliary subunit domain
PNAS, January 22, 2002; 99(2): 1035 - 1040.
[Abstract] [Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
M. N. Rasband, E. W. Park, T. W. Vanderah, J. Lai, F. Porreca, and J. S. Trimmer
Distinct potassium channels on pain-sensing neurons
PNAS, November 6, 2001; 98(23): 13373 - 13378.
[Abstract] [Full Text] [PDF]


Home page
StrokeHome page
J. S. Bains, M. J. Follwell, K. J. Latchford, J. W. Anderson, and A. V. Ferguson
Slowly Inactivating Potassium Conductance (ID): A Potential Target for Stroke Therapy
Stroke, November 1, 2001; 32(11): 2624 - 2634.
[Abstract] [Full Text] [PDF]


Home page
J. Neurosci.Home page
M. M. Monaghan, J. S. Trimmer, and K. J. Rhodes
Experimental Localization of Kv1 Family Voltage-Gated K+ Channel {alpha} and {beta} Subunits in Rat Hippocampal Formation
J. Neurosci., August 15, 2001; 21(16): 5973 - 5983.
[Abstract] [Full Text] [PDF]


Home page
J. Physiol.Home page
Y. Saito and T. Isa
Voltage-gated transient outward currents in neurons with different firing patterns in rat superior colliculus
J. Physiol., October 1, 2000; 528(1): 91 - 105.
[Abstract] [Full Text] [PDF]


Home page
J. Physiol.Home page
A. Korngreen and B. Sakmann
Voltage-gated K+ channels in layer 5 neocortical pyramidal neurones from young rats: subtypes and gradients
J. Physiol., June 15, 2000; 525(3): 621 - 639.
[Abstract] [Full Text] [PDF]


Home page
J. Neurosci.Home page
A. P. Southan and B. Robertson
Electrophysiological Characterization of Voltage-Gated K+ Currents in Cerebellar Basket and Purkinje Cells: Kv1 and Kv3 Channel Subfamilies Are Present in Basket Cell Nerve Terminals
J. Neurosci., January 1, 2000; 20(1): 114 - 122.
[Abstract] [Full Text] [PDF]


Home page
J. Neurosci.Home page
M. A. Lazaroff, A. D. Hofmann, and A. B. Ribera
Xenopus Embryonic Spinal Neurons Express Potassium Channel Kvbeta Subunits
J. Neurosci., December 15, 1999; 19(24): 10706 - 10715.
[Abstract] [Full Text] [PDF]


Home page
J. Neurosci.Home page
C.-L. Zhang, A. Messing, and S. Y. Chiu
Specific Alteration of Spontaneous GABAergic Inhibition in Cerebellar Purkinje Cells in Mice Lacking the Potassium Channel Kv1.1
J. Neurosci., April 15, 1999; 19(8): 2852 - 2864.
[Abstract] [Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
M. L. Sutherland, S. H. Williams, R. Abedi, P. A. Overbeek, P. J. Pfaffinger, and J. L. Noebels
Overexpression of a Shaker-type potassium channel in mammalian central nervous system dysregulates native potassium channel gene expression
PNAS, March 2, 1999; 96(5): 2451 - 2455.
[Abstract] [Full Text] [PDF]


Home page
J. Neurosci.Home page
I. Vabnick, J. S. Trimmer, T. L. Schwarz, S. R. Levinson, D. Risal, and P. Shrager
Dynamic Potassium Channel Distributions during Axonal Development Prevent Aberrant Firing Patterns
J. Neurosci., January 15, 1999; 19(2): 747 - 758.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
T. Leicher, R. Bahring, D. Isbrandt, and O. Pongs
Coexpression of the KCNA3B Gene Product with Kv1.5 Leads to a Novel A-type Potassium Channel
J. Biol. Chem., December 25, 1998; 273(52): 35095 - 35101.
[Abstract] [Full Text] [PDF]


Home page
Learn. Mem.Home page
K. P. Giese, J. F. Storm, D. Reuter, N. B. Fedorov, L.-R. Shao, T. Leicher, O. Pongs, and A. J. Silva
Reduced K+ Channel Inactivation, Spike Broadening, and After-Hyperpolarization in Kvbeta 1.1-Deficient Mice with Impaired Learning
Learn. Mem., September 1, 1998; 5(4): 257 - 273.
[Abstract] [Full Text]


Home page
J. Neurosci.Home page
A. P. Southan and B. Robertson
Patch-Clamp Recordings from Cerebellar Basket Cell Bodies and Their Presynaptic Terminals Reveal an Asymmetric Distribution of Voltage-Gated Potassium Channels
J. Neurosci., February 1, 1998; 18(3): 948 - 955.
[Abstract] [Full Text] [PDF]


Home page
J. Neurosci.Home page
M. N. Rasband, J. S. Trimmer, T. L. Schwarz, S. R. Levinson, M. H. Ellisman, M. Schachner, and P. Shrager
Potassium Channel Distribution, Clustering, and Function in Remyelinating Rat Axons
J. Neurosci., January 1, 1998; 18(1): 36 - 47.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
E. A. Accili, J. Kiehn, B. A. Wible, and A. M. Brown
Interactions among Inactivating and Noninactivating Kvbeta Subunits, and Kvalpha 1.2, Produce Potassium Currents with Intermediate Inactivation
J. Biol. Chem., November 7, 1997; 272(45): 28232 - 28236.
[Abstract] [Full Text] [PDF]


Home page
J. Neurosci.Home page
K. J. Rhodes, B. W. Strassle, M. M. Monaghan, Z. Bekele-Arcuri, M. F. Matos, and J. S. Trimmer
Association and Colocalization of the Kvbeta 1 and Kvbeta 2 beta -Subunits with Kv1 alpha -Subunits in Mammalian Brain K+ Channel Complexes
J. Neurosci., November 1, 1997; 17(21): 8246 - 8258.
[Abstract] [Full Text] [PDF]


Home page
J. Neurosci.Home page
D. J. Baro, R. M. Levini, M. T. Kim, A. R. Willms, C. C. Lanning, H. E. Rodriguez, and R. M. Harris-Warrick
Quantitative Single-Cell-Reverse Transcription-PCR Demonstrates That A-Current Magnitude Varies as a Linear Function of shal Gene Expression in Identified Stomatogastric Neurons
J. Neurosci., September 1, 1997; 17(17): 6597 - 6610.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
J. Xu and M. Li
Kvbeta 2 Inhibits the Kvbeta 1-mediated Inactivation of K+ Channels in Transfected Mammalian Cells
J. Biol. Chem., May 2, 1997; 272(18): 11728 - 11735.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
L. N. Manganas and J. S. Trimmer
Subunit Composition Determines Kv1 Potassium Channel Surface Expression
J. Biol. Chem., September 15, 2000; 275(38): 29685 - 29693.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
B. A. Firulli, D. B. Hadzic, J. R. McDaid, and A. B. Firulli
The Basic Helix-Loop-Helix Transcription factors dHAND and eHAND Exhibit Dimerization Characteristics That Suggest Complex Regulation of Function
J. Biol. Chem., October 20, 2000; 275(43): 33567 - 33573.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
E. M. Kajkowski, C. F. Lo, X. Ning, S. Walker, H. J. Sofia, W. Wang, W. Edris, P. Chanda, E. Wagner, S. Vile, et al.
beta -Amyloid Peptide-induced Apoptosis Regulated by a Novel Protein Containing a G Protein Activation Module
J. Biol. Chem., May 25, 2001; 276(22): 18748 - 18756.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
L. N. Manganas, S. Akhtar, D. E. Antonucci, C. R. Campomanes, J. O. Dolly, and J. S. Trimmer
Episodic Ataxia Type-1 Mutations in the Kv1.1 Potassium Channel Display Distinct Folding and Intracellular Trafficking Properties
J. Biol. Chem., December 21, 2001; 276(52): 49427 - 49434.
[Abstract] [Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
M. K. Ahlijanian, N. X. Barrezueta, R. D. Williams, A. Jakowski, K. P. Kowsz, S. McCarthy, T. Coskran, A. Carlo, P. A. Seymour, J. E. Burkhardt, et al.
Hyperphosphorylated tau and neurofilament and cytoskeletal disruptions in mice overexpressing human p25, an activator of cdk5
PNAS, March 14, 2000; 97(6): 2910 - 2915.
[Abstract] [Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
L. N. Manganas, Q. Wang, R. H. Scannevin, D. E. Antonucci, K. J. Rhodes, and J. S. Trimmer
Identification of a trafficking determinant localized to the Kv1 potassium channel pore
PNAS, November 20, 2001; 98(24): 14055 - 14059.
[Abstract] [Full Text] [PDF]


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an eLetter
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (110)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rhodes, K. J.
Right arrow Articles by Trimmer, J. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rhodes, K. J.
Right arrow Articles by Trimmer, J. S.
Right arrowPubmed/NCBI databases
*Substance via MeSH

-
-

Home  |   Search  |   Archive  |   Subscribe  |   Contact  |   Help

-
Copyright 2009 by Society for Neuroscience ONLINE ISSN: 1529-2401
-