Immunological evidence for a relationship between the dendrotoxin‐binding protein and the mammalian homologue of the <i>Drosophila Shaker</i> K<sup>+</sup> channel

Rehm, Hubert; Newitt, Richard; Tempel, Bruce L.

doi:10.1016/0014-5793(89)80628-3

Cited by 41 publications

(11 citation statements)

References 35 publications

(45 reference statements)

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“…Interestingly, the one class of K+ channels amenable to biochemical characterization-those sensitive to the epileptogenic neurotoxins dendrotoxin and p-bungarotoxin-are also formed as a oligomeric complex containing an a-peptide and a 38-kDa protein (19,20 a-peptides expressed in Xenopus oocytes have shown that heterooligomers can be formed with different a-peptides. It is not clear whether such structures exist in situ, although electrophysiological evidence suggests that in heterologous expression systems some K+ channels are formed as homotetramers of a-peptides (23).…”

Section: Resultsmentioning

confidence: 99%

Immunological identification and characterization of a delayed rectifier K+ channel polypeptide in rat brain.

Trimmer

1991

Proc. Natl. Acad. Sci. U.S.A.

251

266

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Antibodies specific for the drkl polypeptide were used to characterize the corresponding protein in rat brain. Recombinant and synthetic immunogens containing fragments of the drkl polypeptide were produced. Antibodies raised to these immunogens display monospecific reactions with the same 130-kDa polypeptide on immunoblots of adult rat brain membranes. Immunoprecipitation of '2SI-labeled brain membranes identifies a 38-kDa peptide in tight association with the drkl polypeptide. Immunohistochemical staining of sections of adult rat cortex shows that drkl protein is restricted to neurons, where staining is present on dendrites and cell bodies but not on axons. These studies point to the value of such immunological reagents to the further characterization of the components of this delayed rectifier K+ channel in the mammalian central nervous system. Recently, a number of cDNAs have been isolated from rat brain that, when expressed in Xenopus oocytes from in vitro-transcribed mRNAs, produce IK-like currents (4-9). Among these in vitro transcription products, those from the drkl cDNA template produce currents in oocytes that most closely resemble neuronal IK in voltage-dependence and kinetics of activation, pharmacological characteristics, and lack of inactivation over tens of milliseconds (6). Thus, probes derived from the drkl cDNA could be of great use in characterizing components of neuronal IK.Biochemical characterization of K+ channel proteins has lagged behind similar studies of voltage-sensitive Na+ and Ca2+ channels. This fact is primarily due to the lack of naturally occurring high-affinity toxins for most K+ channel classes; this situation is especially true for neuronal IK. The generation of antibodies to immunogens derived from cloned K+ channel cDNAs provides an approach to characterize biochemical properties of the corresponding proteins in the absence of purified material. This report describes the use of such immunochemical reagents to begin to characterize the drkl polypeptide in adult rat brain. MATERIALS AND METHODSMaterials. '25I-labeled protein A was from ICN, and Na'251 was from NEN. All other reagents were molecular biology grade from Sigma or Schwarz/Mann.Production of Fusion Proteins and Antibodies. An 85-basepair (bp) insert of a drkl cDNA (pMK4; J.S.T., unpublished work) was ligated into the vector pGEX-2 (25) to create pGEXdrkl, containing amino acids 516-533 of the drkl polypeptide, which was used to immunize rabbits for production of antisera.A synthetic peptide, KC, corresponding to the C terminus (amino acids 837-853) of the drkl polypeptide, was conjugated to keyhole limpet hemocyanin and injected into rabbits for production of antisera. A recombinant pGEX fusion protein corresponding to the C terminus of drkl polypeptide was also constructed as follows. The PCR was used to amplify a genomic fragment of drkl-coding region corresponding to nucleotides 1537-2590. A 126-bp HincII fragment (nucleotides 2465-2590, polylinker HincII site) was ligated into pGEX-2 to create pGEXKC.A...

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Section: Resultsmentioning

confidence: 99%

Immunological identification and characterization of a delayed rectifier K+ channel polypeptide in rat brain.

Trimmer

1991

Proc. Natl. Acad. Sci. U.S.A.

251

266

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“…Microsequencing, by one group, of the N-terminus of the Dtx-purified a-subunit (Scott et al, 1990), together with gene cloning (Reid et al, 1992) has shown that this protein is homologous with a rat brain cloned K+ channel, rKvl.2 (also known as RCK5), which is sensitive to a-Dtx when expressed in Xenopus oocytes (Stiihmer et al, 1989). Other microsequencing studies together with the use of specific antibodies suggest that Kvl.1 (also known as MK1, RCK1 and RBK1) channels also contribute to purified brain Dtxbinding protein (Rehm et al, 1989). Kvl.1 cRNA + cDNA also gives rise to 'delayed-rectifier-like', Dtx-sensitive currents when expressed in Xenopus oocytes (Stiihmer et al, 1989) and mammalian cells (Owen et al, 1992;Robertson & Owen, 1993;Bosma et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

Blockade by dendrotoxin homologues of voltage‐dependent K⁺ currents in cultured sensory neurones from neonatal rats

Hall

Stow

Sorensen

et al. 1994

British J Pharmacology

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1Homologues of dendrotoxin (Dtx) were isolated from the crude venom of Green and Black Mamba snakes and examined for K+ channel blocking activity in neonatal rat dorsal root ganglion cells (DRGs) by whole-cell patch clamp recording. 2 Outward potassium current activated by depolarization was composed of two major components: a slowly inactivating current (SIC, tdecay ; 50 ms, 200 ms and 2 s), and a non-inactivating current (NIC, tdecay> 2 min). Tail current analysis revealed two time constants of deactivation of total outward current, 3-12 ms and 50-150 ms (at -80 mV) which corresponded to SIC and NIC, respectively.3 All the homologues (a-, P-, 'y-and 6-Dtx and toxins I and K) blocked outward current activated by depolarization in a dose-dependent manner. The most potent in blocking total outward current was 6-Dtx (ECm of 0.5 ± 0.2 nM), although there were no statistically significant differences in potency between any of the homologues. 4 Qualitative differences in the nature of the block were noted between homologues. In particular, the block by 6-Dtx was time-dependent, whereas that by a-Dtx was not. 5 a-Dtx was a much better blocker of SIC (EC50 = 1.0 ± 0.4 nM) than was 6-Dtx (EC50 = 17.6 ± 5.8 nM). Furthermore, 6-Dtx was selective for NIC (EC5 ± 0.24 ± 0.03 nM) over SIC and reduced the slow component of tail currents (NIC), preferentially. On the other hand, a-Dtx did not significantly distinguish between SIC and NIC although tail current analysis showed that a-Dtx preferentially reduced the fast component of tail currents (SIC). 6 The results confirm, using direct electrophysiological methods, that homologues of dendrotoxins from Mamba snake venom block K+ channels in rat sensory neurones. Furthermore, a-Dtx and 6-Dtx distinguish between sub-types of K+ channels in these cells and may thus be useful pharmacological tools in other neuronal K+ channel studies.

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“…The Kv1.1 channel has only one extracellular N-glycosylation site yet the glycoprotein has about 24 kDa of carbohydrate/monomer, of which 12-18 kDa is sialic acid (20). It is unlikely that 24 kDa of carbohydrate is attached to this one site, and the channel may have O-linked carbohydrates.…”

Section: Fig 4 Effects Of Varying Extracellular Camentioning

confidence: 99%

“…Kv1.1 is a delayed rectifier K ϩ channel that was originally cloned from rodent brain cDNA libraries (27)(28)(29)(30) and is a heavily sialidated protein (20). We investigated the possible role of sialic acids by comparing the functional properties of Kv1.1 in control and glycosylation-deficient CHO 1 cell lines (Lec mutants).…”

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confidence: 99%

Expression of Kv1.1 Delayed Rectifier Potassium Channels in Lec Mutant Chinese Hamster Ovary Cell Lines Reveals a Role for Sialidation in Channel Function

Thornhill

Jiang

et al. 1996

Journal of Biological Chemistry

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Immunological evidence for a relationship between the dendrotoxin‐binding protein and the mammalian homologue of the Drosophila Shaker K⁺ channel

Cited by 41 publications

References 35 publications

Immunological identification and characterization of a delayed rectifier K+ channel polypeptide in rat brain.

Immunological identification and characterization of a delayed rectifier K+ channel polypeptide in rat brain.

Blockade by dendrotoxin homologues of voltage‐dependent K⁺ currents in cultured sensory neurones from neonatal rats

Expression of Kv1.1 Delayed Rectifier Potassium Channels in Lec Mutant Chinese Hamster Ovary Cell Lines Reveals a Role for Sialidation in Channel Function

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Immunological evidence for a relationship between the dendrotoxin‐binding protein and the mammalian homologue of the Drosophila Shaker K+ channel

Cited by 41 publications

References 35 publications

Immunological identification and characterization of a delayed rectifier K+ channel polypeptide in rat brain.

Immunological identification and characterization of a delayed rectifier K+ channel polypeptide in rat brain.

Blockade by dendrotoxin homologues of voltage‐dependent K+ currents in cultured sensory neurones from neonatal rats

Expression of Kv1.1 Delayed Rectifier Potassium Channels in Lec Mutant Chinese Hamster Ovary Cell Lines Reveals a Role for Sialidation in Channel Function

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Immunological evidence for a relationship between the dendrotoxin‐binding protein and the mammalian homologue of the Drosophila Shaker K⁺ channel

Blockade by dendrotoxin homologues of voltage‐dependent K⁺ currents in cultured sensory neurones from neonatal rats