Skip to main content
SpringerLink
Log in

Whole-cell and single-channel α1 β1 γ2S GABAA receptor currents elicited by a “multipuffer” drug application device

  • Instruments and Techniques
  • Molecular and Cellular Physiology
  • Published:
Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

Abstract

Pharmacological characterization of ion channels and receptors in cultured neurons or transfected cell lines requires microapplication of multiple drug solutions during electrophysiological recording. An ideal device could apply a large number of solutions to a limited area with rapid arrival and removal of drug solutions. We describe a novel “multipuffer” rapid application device, based on a modified T-tube with a nozzle made from a glass micropipette tip. Drug solutions are drawn via suction from open reservoirs mounted above the recording chamber through the device into a waste trap. Closure of a solenoid valve between the device and the waste trap causes flow of drug solution though the T-tube nozzle. Any number of drug solutions can be applied with rapid onset (50–100 ms) after a brief fixed delay (100–200 ms). Recombinant α1β1γ2S GABAA receptors (GABARs) transfected into L929 fibroblasts were recorded using whole-cell and single-channel configurations. Application of GABA resulted in chloride currents with an EC50 of 12.2 μM and a Hill slope of 1.27, suggesting more than one binding site for GABA. GABAR currents were enhanced by diazepam and pentobarbital and inhibited by bicuculline and picrotoxin. Single-channel recordings revealed a main conductance state of 26–28 pS. This device is particularly suitable for rapid, spatially controlled drug applications onto neurons or other cells recorded in the whole-cell configuration, but is also appropriate for isolated single-channel or multichannel membrane patch recordings.

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

References

  1. Angelotti TP, Macdonald RL (1993) Assembly of GABAA receptor subunits: α1β1 and α1β1γ2S subunits produce unique ion channels with dissimilar single-channel properties. J Neurosci 13: 1429–1440

    CAS  PubMed  Google Scholar 

  2. Angelotti TP, Uhler MD, Macdonald RL (1993) Assembly of GABAA receptor subunits: analysis of transient single-cell expression utilizing a fluorescent substrate/marker gene technique. J Neurosci 13: 1418–1428

    CAS  PubMed  Google Scholar 

  3. Betz H (1990) Ligand-gated ion channels in the brain: the amino acid receptor superfamily. Neuron 5: 383–392

    Article  CAS  PubMed  Google Scholar 

  4. Burgard EC, Tietz EI, Neelands TR, Macdonald RL (1996) Properties of recombinant GABAA receptor isoforms containing the α5 subunit subtype. Mol Pharmacol 50: 119–127

    CAS  PubMed  Google Scholar 

  5. Celentano JJ, Wong RKS (1994) Multiphasic desensitization of the GABA(A) receptor in outside-out patches. Biophys J 66: 1039–1050

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Dekoninck Y, Mody I (1994) Noise analysis of miniature IPSCs in adult rat brain slices — properties and modulation of synaptic GABA(A) receptor channels. J Neurophysiol 71: 1318–1335

    CAS  Google Scholar 

  7. Frosch MP, Lipton SA, Dichter MA (1992) Desensitization of GABA-activated currents and channels in cultured cortical neurons. J Neurosci 12: 3042–3053

    CAS  PubMed  Google Scholar 

  8. Gingrich KJ, Roberts WA, Kass RS (1995) Dependence of the GABAA receptor gating kinetics on the α-subunit isoform: implications for structure-function relations and synaptic transmission. J Physiol (Lond) 489: 529–543

    Article  CAS  Google Scholar 

  9. Greenfield LJ, Uhler MD, Macdonald RL (1994) Cyclic GMP-dependent protein kinase II regulates recombinant GABAA receptor currents in L929 cells (abstract). Soc Neurosci Abstr 20: 503

    Google Scholar 

  10. Hamill OP, 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. Pflugers Arch 391: 85–100

    Article  CAS  PubMed  Google Scholar 

  11. Hille B (1992): Ionic channels of excitable membranes, 2nd edn. Sinauer, Sunderland, Mass., pp 146–158

    Google Scholar 

  12. Jones MV, Westbrook GL (1995) Desensitized states prolong GABAA channel responses to brief agonist pulses. Neuron 15: 181–191

    Article  CAS  PubMed  Google Scholar 

  13. Kapur J, Macdonald RL (1996) Pharmacological properties of GABAA receptors from acutely dissociated rat dentate granule cells. Mol Pharmacol (in press)

  14. Kume A, Greenfield LJJ, Macdonald RL, Albin RL (1996) Felbamate inhibits [3H]t-butylbicycloorthobenzoate binding and enhances Cl− current at the gamma-aminobutyric acidA receptor. J Pharmacol Exp Ther 277: 1784–1792

    CAS  PubMed  Google Scholar 

  15. Lin Y, Angelotti TP, Dudek EM, Browning MD, Macdonald RL (1996) Enhancement of recombinant α1β1γ2L GABAA receptor whole cell currents by protein kinase C is mediated by phosphorylation of both β1 and γ2L subunits. Mol Pharmacol 50: 185–195

    CAS  PubMed  Google Scholar 

  16. Lin Y, Browning MD, Dudek EM, Macdonald RL (1994) Protein kinase C enhances recombinant bovine α1β1γ2L GABAA receptor whole-cell currents expressed in L929 fibroblasts. Neuron 13: 1421–1431

    Article  CAS  PubMed  Google Scholar 

  17. Macdonald RL, Olsen RW (1994) GABAA receptor channels. Annu Rev Neurosci 17: 569–602

    Article  CAS  PubMed  Google Scholar 

  18. Macdonald RL, Weddle MG, Gross RA (1986) Benzodiazepine, beta-carboline, and barbiturate actions on GABA responses. Adv Biochem Psychopharmacol 41: 67–78

    CAS  PubMed  Google Scholar 

  19. Macdonald RL, Rogers CJ, Twyman RE (1989) Kinetic properties of the GABAA receptor main conductance state of mouse spinal cord neurones in culture. J Physiol (Lond) 410: 479–499

    Article  CAS  Google Scholar 

  20. Maconochie DJ, Knight DE (1994) A method for making solution changes in the sub-millisecond range at the tip of a patch pipette. Pflügers Arch 414: 589–596

    Article  Google Scholar 

  21. Maconochie DJ, Zempel JM, Steinbach JH (1994) How quickly can GABA(A) receptors open? Neuron 12: 61–71

    Article  CAS  PubMed  Google Scholar 

  22. Murase K, Ryu PD, Randic M (1989) Excitatory and inhibitory amino acids and peptide-induced responses in acutely isolated rat spinal dorsal horn neurons. Neurosci Lett 103: 56–63

    Article  CAS  PubMed  Google Scholar 

  23. Newland CF, Cull-Candy SG (1992) On the mechanism of action of picrotoxin on GABA receptor channels in dissociated sympathetic neurones of the rat. J Physiol (Lond) 447: 191–213

    Article  CAS  Google Scholar 

  24. Porter NM, Angelotti TP, Twyman RE, Macdonald RL (1992) Kinetic properties of alpha 1 beta 1 gamma-aminobutyric acidA receptor channels expressed in Chinese Hamster Ovary cells: regulation by pentobarbital and picrotoxin. Mol Pharmacol 42: 872–881

    CAS  PubMed  Google Scholar 

  25. Puia G, Costa E, Vicini S (1994) Functional diversity of GABA-activated Cl− currents in purkinje versus granule neurons in rat cerebellar slices. Neuron 12: 117–126

    Article  CAS  PubMed  Google Scholar 

  26. Rogers CJ, Twyman RE, Macdonald RL (1994) Benzodiazepine and beta-carboline regulation of single GABA(A) receptor channels of mouse spinal neurones in culture. J Physiol (Lond) 475: 69–82

    Article  CAS  Google Scholar 

  27. Saxena NC, Macdonald RL (1994) Assembly of GABAA receptor subunits: role of the delta subunit. J Neurosci 14: 7077–7086

    CAS  PubMed  Google Scholar 

  28. Schofield PR, Darlison MG, Fujita N, Burt DR, Stephenson FA, Rodriguez H, Rhee LM, Ramachandran J, Reale V, Glencorse TA (1987) Sequence and functional expression of the GABA-A receptor shows a ligand-gated receptor super-family. Nature 328: 221–227

    Article  CAS  PubMed  Google Scholar 

  29. Twyman RE (1994) GABA-A receptor channel opening rates in excised patches of mouse cortical neurons. Jpn J Physiol 44: S87-S90

    CAS  PubMed  Google Scholar 

  30. Twyman RE, Rogers CJ, Macdonald RL (1989) Pentobarbital and picrotoxin have reciprocal actions on single GABAA receptor channels. Neurosci Lett 96: 89–95

    Article  CAS  PubMed  Google Scholar 

  31. Twyman RE, Rogers CJ, Macdonald RL (1990) Intraburst kinetic properties of the GABAA receptor main conductance state of mouse spinal cord neurones in culture. J Physiol (Lond) 423: 193–220

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Departments of Neurology, Neuroscience Laboratory Building. 1103 E. Huron, 48904-1687, Ann Arbor, MI, USA

    L. John Greenfield

  2. Department of Neurology and Physiology, University of Michigan Medical Center, Michigan, USA

    Robert L. Macdonald

Authors
  1. L. John Greenfield
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert L. Macdonald
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Greenfield, L.J., Macdonald, R.L. Whole-cell and single-channel α1 β1 γ2S GABAA receptor currents elicited by a “multipuffer” drug application device. Pflügers Arch — Eur J Physiol 432, 1080–1090 (1996). https://doi.org/10.1007/s004240050238

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s004240050238

Key words

Search

Navigation