Skip to main content
SpringerLink
Log in

Expression of a genomic clone encoding a brain potassium channel in mammalian cells using lipofection

  • Published:
European Biophysics Journal Aims and scope Submit manuscript

Abstract

A genomic clone encoding a mouse brain K+ channel (MBK1) was isolated, characterized and expressed in COS cells using the lipofection technique. Transfected COS cells expressed voltage-dependent K+ currents that activated within 20 ms at 0 mV and showed less than 10% inactivation during 250 ms depolarizing pulses at 60 mV. Expressed K+ currents were reversibly blocked by 4-aminopyridine and tetraethylammonium, and were moderately sensitive to dendrotoxin, but insensitive to charybdotoxin. Thus MBK1, expressed transiently in a mammalian cell line, exhibits features characteristic of non-inactivating K+ channels with a conspicuous insensitivity to charybdotoxin. Lipofection is, therefore, a valuable strategy for expression of channel proteins in mammalian cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

4-AP:

4 aminopyridine

TEA:

tetraethylammonium

CTX:

charybdotoxin

DTX:

dendrotoxin

V:

applied voltage

Vrev :

reversal potential

I:

current

G:

conductance

MBK1:

mouse brain potassium channel 1

TES:

N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid

References

  • Armstrong C, Bezanilla F (1977) Inactivation of sodium channel 11. Gating current experiments. J Gen Physiol 70:567–590

    Google Scholar 

  • Baumann A, Grupe A, Ackermann A, Pongs O (1988) Structure of the voltage-dependent potassium channel is highly conserved from Drosophila to vertebrate central nervous systems. EMBO J 7:2457–2463

    Google Scholar 

  • Carbone E, Lux HD (1987) Kinetics and selectivity of a low-voltageactivated calcium current in chick and rat sensory neurons. J Physiol 386:547–570

    Google Scholar 

  • Chandy KG, Williams CB, Spencer RH, Aguilar BA, Ghanshani S, Tempel BL, Gutman GA (1990) A family of three mouse potassium channel genes with intronless coding regions. Science 247:973–975

    Google Scholar 

  • Christie MJ, Adelman JP, Douglass J, North RA (1989) Expression of a cloned rat brain potassium channel in Xenopus oocytes. Science 244:221–224

    Google Scholar 

  • Felgner PL, Gadek TR, Holm M, Roman R, Chan HW Wenz M, Northrop JP, Ringold GM, Danielsen M (1987) Lipofection: A highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA 84:7413–7417

    Google Scholar 

  • Ferroni S, Planells-Cases R, Ahmed CMI, Montal M (1992) Expression of channel clones in mammalian cells by lipofection. Biophys J 61:A250

    Google Scholar 

  • Graham FL, Van der Eb, AJ (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456–461

    Google Scholar 

  • Hamill PO, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391:85–100

    CAS  Google Scholar 

  • Hille B (1991) Ionic channels of excitable membranes (2nd ed) Sinnauer Associates, Sunderland, MA

    Google Scholar 

  • Hodgkin AL, Huxley AF (1952) A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol 117:500–544

    CAS  PubMed  Google Scholar 

  • Hoger JH, Walter AE, Vance D, Yu L, Lester HA, Davidson N (1991) Modulation of a cloned mouse brain potassium channel. Neuron 6:227–236

    Google Scholar 

  • Hoshi T, Zagotta WN, Aldrich RW (1990) Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science 250:533–538

    CAS  PubMed  Google Scholar 

  • Jan LY, Jan YN (1990) How might the diversity of potassium channels be generated? Trends Neurosci 13:415–419

    Google Scholar 

  • Kamb A, Tseng-Crank J, Tanouye MA (1988) Multiple products of the Drosophila Shaker gene may contribute to potassium channel diversity. Neuron 1:421–430

    Google Scholar 

  • Klaiber K, Williams N, Roberts TM, Papazian DM, Jan LY, Miller C (1990) Functional expression of Shaker K+ channels in a baculovirus-infected insect cell line. Neuron 5:221–226

    Google Scholar 

  • Koren G, Liman ER, Logothetis, DE, Nadal-Ginard B, Hess P (1990) Gating mechanism of a cloned potassium channel expressed in frog oocytes and mammalian cells. Neuron 4:39–51

    Google Scholar 

  • Leonard RJ, Karschin A, Jayashree-Aiyar S, Davidson N, Tanouye MA, Thomas L, Thomas G, Lester HA (1989) Expression of Drosophila Shaker potassium channels in mammalian cells infected with recombinant vaccinia virus. Proc Natl Acad Sci USA 86:7629–7633

    Google Scholar 

  • Lester HA (1988) Heterologous expression of excitability proteins: Route to more specific drugs? Science 241:1057–1063

    Google Scholar 

  • MacKinnon R (1991) Determination of subunit stoichiometry of a voltage-activated potassium channel. Nature 350:232–235

    Article  CAS  PubMed  Google Scholar 

  • MacKinnon R, Miller C (1989) Mutant potassium channels with altered binding of charybdotoxin, a pore-blocking peptide inhibitor. Science 245:1382–1385

    Google Scholar 

  • Moczydlowski E, Lucchesi K, Ravindran A (1988) An emerging pharmacology of peptide toxins targeted against potassium channels. J Membr Biol 105:95–111

    Google Scholar 

  • Mongkolsuk S (1988) Novel eukaryotic expression vectors which permit single-stranded replication in Escherichia coli and in vitro translational analysis of cloned genes. Gene 70:313–319

    Google Scholar 

  • Neher E (1971) Two fast transient current components during voltage clamp on snail neurons. J Gen Physiol 58:36–53

    Google Scholar 

  • Oliva C, Folander K, Smith JS (1991) Charybdotoxin is not a high affinity blocker of Shaker K+ channels expressed in Xenopus oocytes. Biophys J 59:450A

    Google Scholar 

  • Pongs O, Kecskemethy N, Muller R, Krah-Jentgens I, Baumann A, Kiltz HH, Canal I, Llamazares S, Ferrus A (1988) Shaker encodes a family of putative potassium channels proteins in the nervous system of Drosophila. EMBO J 7:1087–1096

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual.Cold Spring Laboratory Press, New York

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467

    CAS  PubMed  Google Scholar 

  • Schwarz TL, Tempel BL, Papazian DM, Jan YN, Jan LY (1988) Multiple potassium-channel components are produced by alternative splicing at the Shaker locus in Drosophila. Nature 331:137–142

    Google Scholar 

  • Stühmer W, Ruppersberg JP, Schroter KH, Sakmann B, Stoker 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 11:3235–3244

    Google Scholar 

  • Stühmer W, Stocker M, Sakmann B, Seeburg P, Baumann A, Grupe A, Pongs O (1988) Potassium channels expressed from rat brain cDNA have delayed rectifier properties. FEBS Lett 242:199–206

    Google Scholar 

  • Tempel BL, Jan YN, Jan LY (1988) Cloning of a probable potassium channel gene from mouse brain Nature 332:837–839

    Google Scholar 

  • Thompson SH (1977) Three pharmacologically distinct potassium channels in molluscan neurones. J Physiol 265:465–488

    Google Scholar 

  • Zagotta WN, Germeraad S, Garber SS, Hoshi T Aldrich RW (1989) Properties of ShB A-type potassium channels expressed in Shaker mutant Drosophila by germline transformation. Neuron 3:773–782

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Departments of Biology and Physics, University of California San Diego, 92093-0319, La Jolla, CA, USA

    Stefano Ferroni, Rosa Planells-Cases, C. M. Iqbal Ahmed & Mauricio Montal

Authors
  1. Stefano Ferroni
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rosa Planells-Cases
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C. M. Iqbal Ahmed
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mauricio Montal
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Correspondence to: M. Montal.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ferroni, S., Planells-Cases, R., Ahmed, C.M.I. et al. Expression of a genomic clone encoding a brain potassium channel in mammalian cells using lipofection. Eur Biophys J 21, 185–191 (1992). https://doi.org/10.1007/BF00196762

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00196762

Key words

Search

Navigation