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Analysis and use of the perforated patch technique for recording ionic currents in pancreaticβ-cells

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Summary

We have used the nystatin perforated patch technique to study ionic currents in rat pancreatic β-cells. The access resistance (R a) between the pipette and the cell cytoplasm, measured by analyzing capacitive currents, decreased with a slow exponential time course (τ=5.4±2.7 min) after seal formation. AsR a decreased, the magnitude of voltage-dependent K and Ca currents increased with a similar time course, and their activation kinetics became faster. AfterR a stabilized, the macroscopic currents remained stable for up to an hour or more. When the finalR a was sufficiently low, Ca tail currents could be resolved which had properties similar to those recorded with the classical whole-cell technique. Two types of K channels could be characterized with perforated patch recordings of macroscopic K currents: (i) ATP-blockable K (KATP) channels which generate a time and voltage independent current that is blocked by glyburide and enhanced by pinacidil and (ii) voltage-dependent K (K v ) channels. Whole-cell recordings of KATP currents in the absence of ATP in the pipette showed that the maximum KATP conductance of the β-cell was 83.8±40 nS. Perforated patch recordings show that the resting KATP conductance is 3.57±2.09 nS, which corresponds to about 4% of the channels being open in the intact β-cell. In classical whole-cell recordings. K v activation kinetics become faster during the first 10–15 min of recording, probably due to a dissipating Donnan potential. In perforated patch recordings where the Donnan potential is very small, K v activation kinetics were nearly identical to the steady-state whole cell measurements.

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References

  1. Arena, J.P., Kass, R.S. 1989. Enhancement of potassium-sensitive current in heart cells by pinacidil.Circ. Res. 65:436–445

  2. Armstrong, C.M., Bezanilla, F. 1974. Charge movement associated with the opening and closing of the activation gates of the Na channel.J. Gen. Physiol. 63:533–552

  3. Armstrong, C.M., Matteson, D.R. 1985. Two distinct populations of calcium channels in a clonal line of pituitary cells.Science 227:65–67

  4. Ashcroft, F.M. 1988. Adenosine 5′-triphosphate-sensitive potassium channels.Annu. Rev. Neurosci. 11:97–118

  5. Ashcroft, F.M., Harrison, D.E., Ashcroft, S.J.H. 1984. Glucose induces closure of single potassium channels in isolated rat pancreatic B-cells.Nature 312:446–448

  6. Ashcroft, F.M., Kakei, M., Kelly, R.P. 1989. Rubidium and sodium permeability of the ATP-sensitive K+ channel in single rat pancreatic beta-cells.J. Physiol. 408:413–429

  7. Ashcroft, F.M., Kelley, R.P., Smith, P.A. 1990. Two types of Ca channel in rat pancreatic B-cells.Pfluegers Arch. 415:504–506

  8. Cahalan, M.D., Chandy, K.G., DeCoursey, T.E., Gupta, S. 1985. A voltage-gated potassium channel in human T-lymphocytes.J. Physiol. 358:197–237

  9. Carbone, E., Lux, H.D. 1984. A low voltage activated, fully inactivating Ca channel in vertebrate sensory neurones.Nature 310:501–511

  10. Carbone, E., Lux, H.D. 1987. Kinetics and selectivity of a lowvoltage-activated calcium current in chick and rat sensory neurones.J. Physiol. 386:547–570

  11. Chen, C., Hess, P. 1990. Mechanisms of gating of T-type calcium channels.J. Gen. Physiol. 96:603–630

  12. Cohen, A.S., Matteson, D.R., Parsey, R.V., Sala, S. 1990. Ionic currents in rat pancreatic beta cells recorded with the perforated patch technique.Biophys. J. 57:509a

  13. Cohen, C.J., McCarthy, R.T., Barrett, P.Q., Rasmussen, H. 1988. Ca channels in adrenal glomerulosa cells: K+ and angiotensin II increase T-type Ca channel current.Proc. Natl. Acad. Sci. USA 85:2412–2416

  14. Cook, D.L., Hales, C.N. 1984. Intracellular ATP directly blocks K+ channels in pancreatic B-cells.Nature 311:271–273

  15. Cook, D.L., Satin, L.S., Ashford, M.L.J., Hales, C.N. 1988. ATP-sensitive K+ channels in pancreatic β-cells.Diabetes 37:495–498

  16. Cota, G. 1986. Calcium channel currents in pars intermedia cells of the rat pituitary gland.J. Gen. Physiol. 88:83–105

  17. Falke, L.C., Gillis, K.D., Pressel, D.M., Misler, S. 1989. ‘Perforated patch recording’ allows long-term monitoring of metabolite-induced electrical activity and voltage-dependent Ca2+ currents in pancreatic islet B cells.FEBS Lett. 251:167–172

  18. Fernandez, J.M., Fox, A.P., Krasne, S. 1984. Membrane patches and whole-cell membranes: A comparison of electrical properties in rat clonal pituitary (GH3) cells.J. Physiol. 356:565–585

  19. Hagiwara, S., Byerly, L. 1983. The calcium channel.Trends Neurosci. 6:189–193

  20. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J. 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.Pfluegers Arch. 391:85–100

  21. Hiriart, M., Matteson, D.R. 1988. Sodium channels and two types of calcium channels in rat pancreatic beta cells identified with the reverse hemolytic plaque assay.J. Gen. Physiol. 91:617–639

  22. Horn, R., Marty, A. 1988. Muscarinic activation of ionic currents measured by a new whole-cell recording method.J. Gen. Physiol. 92:145–159

  23. Kakei, M., Kelly, R.P., Ashcroft, S.J.H., Ashcroft, F.M. 1986. The ATP-sensitivity of K+ channels in rat pancreatic B-cells is modulated by ADP.FEBS Lett. 208:63–66

  24. Korn, S.J., Horn, R. 1989. Influence of sodium-calcium exchange on calcium current rundown and the duration of calcium-dependent chloride currents in pituitary cells, studied with whole cell and perforated patch recording.J. Gen. Physiol. 94:789–812

  25. Kurachi, Y., Asano, Y., Takikawa, R., Sugimoto, T. 1989. Cardiac Ca current does not run down and is very sensitive to isoprenaline in the nystatin method of whole cell recording.Naunyn-Schmiedeberg's Arch.Pharmacol. 340:219–222

  26. Lindau, M., Fernandez, J.M. 1986. IgE-mediated degranulation of mast cells does not require opening of ion channels.Nature 319:150–153

  27. Lucero, M.T., Pappone, P.A. 1990. Membrane responses to NE in cultured brown fat cells.J. Gen. Physiol. 95:523–544

  28. Marty, A., Neher, E. 1983. Tight-seal whole-cell recording.In: Single Channel Recording. B. Sakmann and E. Neher, editors. pp. 107–122. Plenum, New York

  29. Matteson, D.R., Armstrong, C.M. 1986. Properties of two types of calcium channels in clonal pituitary cells.J. Gen. Physiol. 87:161–182

  30. Nowycky, M.C., Fox, A.P., Tsien, R.W. 1985. Three types of neuronal calcium channels with different calcium agonist sensitivity.Nature 316:440–443

  31. Rorsman, P., Trube, G. 1986. Calcium and delayed potassium currents in mouse pancreatic β-cells under voltage clamp conditions.J. Physiol. 374:531–550

  32. Sala, S., Matteson, D.R. 1990. Single-channel recordings of two types of calcium channels in rat pancreatic β-cells.Biophys. J. 58:567–571

  33. Satin, L.S., Cook, D.L. 1988. Evidence for two calcium currents in insulin-secreting cells.Pfluegers Arch. 411:401–409

  34. Sturgess, N.C., Ashford, M.L.J., Cook, D.L., Hales, C.N. 1985. The sulfonylurea receptor may be an ATP-sensitive potassium chennel.Lancet 8453:474–475

  35. Trube, G., Rorsman, P., Ohno-Shosaku, T. 1986. Opposite effects of tolbutamide and diazoxide on the ATP-dependent K+ channel in mouse pancreatic β-cells.Pfluegers Arch. 407:493–499

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Sala, S., Parsey, R.V., Cohen, A.S. et al. Analysis and use of the perforated patch technique for recording ionic currents in pancreaticβ-cells. J. Membrain Biol. 122, 177–187 (1991). https://doi.org/10.1007/BF01872640

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Key Words

  • perforated-patch
  • nystatin
  • patch clamp
  • β-cell
  • K channel
  • Ca channel