Research paper
Perforated-patch recording with gramicidin avoids artifactual changes in intracellular chloride concentration

https://doi.org/10.1016/0165-0270(94)00116-XGet rights and content

Abstract

The antibiotic gramicidin, when incorporated into lipid membranes, forms pores that are exclusively permeable to monovalent cations and small uncharged molecules. We report the use of gramicidin for perforated patch-clamp recordings in the whole-cell mode. Recordings were performed in cultured rat spinal cord dorsal horn neurons. Cells had stable resting potentials and series resistances for times routinely exceeding 60 min. To test if intracellular chloride concentration ([Cl]i) remains stable with this technique, we measured responses to agonists of glycine and GABAA receptors, both of which gate chloride conductances. The driving force for these responses remained stable at values that differed significantly from values that would be expected if [Cl]i were biased towards pipette [Cl]. We conclude that gramicidin perforated-patch recording, in addition to other properties of the perforated-patch recording technique, has the advantage of not altering [Cl]i. It is, therefore, an electrophysiological method particularly suitable for studies of anionic channels when [Cl]i is a variable of interest, as well as for studies of homeostatic [Cl]i regulation.

References (25)

  • R. Horn et al.

    Muscarinic activation of ionic currents measured by a new whole-cell recording method

    J. Gen. Physiol.

    (1988)
  • M.E. Kleinberg et al.

    Single-length and double length channels formed by nystatin in lipid bilayer membranes

    J. Membr. Biol.

    (1984)
  • Cited by (283)

    • Analysis of neuronal Ca<sup>2+</sup> handling properties by combining perforated patch clamp recordings and the added buffer approach

      2021, Cell Calcium
      Citation Excerpt :

      The original and most commonly used perforating agents have been the antibiotic polyenes nystatin and amphotericin B, and the antibiotic polypeptide gramicidin [2,18,19,24–27]. While polyenes and the peptide exhibit differences in their pore-forming mechanisms and ion selectivities [28–31], their pores share key common properties: they are permeable to small molecules with a molecular weight up to ∼200 Da, including monovalent ions [27,32,33], but they are neither permeable to divalent ions like Ca2+ nor to Ca2+ indicators like fura-2 (whose molecular weight is >600 Da). While the combination of perforated patch clamp recordings and the added buffer approach would be ideal, it cannot be implemented with the previous standard small pore-forming perforating agents.

    View all citing articles on Scopus
    1

    Present address: Department of Anatomy, Box 0452A, University of California, San Francisco, San Francisco, CA 94143, USA.

    View full text