Skip to main content
PMC home page
The Journal of General Physiology logoLink to The Journal of General Physiology
. 1987 Jun 1;89(6):985–997. doi: 10.1085/jgp.89.6.985

A comparison of calcium-activated potassium channel currents in cell- attached and excised patches

PMCID: PMC2215963  PMID: 2440980

Abstract

Single channel currents from Ca-activated K channels were recorded from cell-attached patches, which were then excised from 1321N1 human astrocytoma cells. Cells were depolarized with K (110 mM) so that the membrane potential was known in both patch configurations, and the Ca ionophore A23187 or ionomycin (20-100 microM) was used to equilibrate intracellular and extracellular [Ca] (0.3 or 1 microM). Measurements of intracellular [Ca] with the fluorescent Ca indicator quin2 verified that [Ca] equilibration apparently occurred in our experiments. Under these conditions, where both membrane potential and intracellular [Ca] were known, we found that the dependence of the channel percent open time on membrane potential and [Ca] was similar in both the cell- attached and excised patch configuration for several minutes after excision. Current-voltage relations were also similar, and autocorrelation functions constructed from the single channel currents revealed no obvious change in channel gating upon patch excision. These findings suggest that the results of studies that use excised membrane patches can be extrapolated to the K-depolarized cell-attached configuration, and that the relation between [Ca] and channel activity can be used to obtain a quantitative measure of [Ca] near the membrane intracellular surface.

Full Text

The Full Text of this article is available as a PDF (778.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Albano J. D., Brown B. L., Ekins R. P., Tait S. A., Tait J. F. The effects of potassium, 5-hydrocytryptamine, adrenocorticotrophin and angiotensin II on the concentration of adenosine 3':5'-cyclic monophosphate in suspensions of dispersed rat adrenal zona glomerulosa and zona fasciculata cells. Biochem J. 1974 Aug;142(2):391–400. doi: 10.1042/bj1420391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baker P. F., Knight D. E., Umbach J. A. Calcium clamp of the intracellular environment. Cell Calcium. 1985 Apr;6(1-2):5–14. doi: 10.1016/0143-4160(85)90030-2. [DOI] [PubMed] [Google Scholar]
  3. Barrett J. N., Magleby K. L., Pallotta B. S. Properties of single calcium-activated potassium channels in cultured rat muscle. J Physiol. 1982 Oct;331:211–230. doi: 10.1113/jphysiol.1982.sp014370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cachelin A. B., De Peyer J. E., Kokubun S., Reuter H. Sodium channels in cultured cardiac cells. J Physiol. 1983 Jul;340:389–401. doi: 10.1113/jphysiol.1983.sp014768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Colquhoun D., Hawkes A. G. Relaxation and fluctuations of membrane currents that flow through drug-operated channels. Proc R Soc Lond B Biol Sci. 1977 Nov 14;199(1135):231–262. doi: 10.1098/rspb.1977.0137. [DOI] [PubMed] [Google Scholar]
  6. Cook D. L., Ikeuchi M., Fujimoto W. Y. Lowering of pHi inhibits Ca2+-activated K+ channels in pancreatic B-cells. Nature. 1984 Sep 20;311(5983):269–271. doi: 10.1038/311269a0. [DOI] [PubMed] [Google Scholar]
  7. Ewald D. A., Williams A., Levitan I. B. Modulation of single Ca2+-dependent K+-channel activity by protein phosphorylation. Nature. 1985 Jun 6;315(6019):503–506. doi: 10.1038/315503a0. [DOI] [PubMed] [Google Scholar]
  8. Fernandez J. M., Fox A. P., Krasne S. Membrane patches and whole-cell membranes: a comparison of electrical properties in rat clonal pituitary (GH3) cells. J Physiol. 1984 Nov;356:565–585. doi: 10.1113/jphysiol.1984.sp015483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gallin E. K. Calcium- and voltage-activated potassium channels in human macrophages. Biophys J. 1984 Dec;46(6):821–825. doi: 10.1016/S0006-3495(84)84080-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  11. Kojima I., Kojima K., Rasmussen H. Intracellular calcium and adenosine 3',5'-cyclic monophosphate as mediators of potassium-induced aldosterone secretion. Biochem J. 1985 May 15;228(1):69–76. doi: 10.1042/bj2280069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kostyuk P. G. Metabolic control of ionic channels in the neuronal membrane. Neuroscience. 1984 Dec;13(4):983–989. doi: 10.1016/0306-4522(84)90282-3. [DOI] [PubMed] [Google Scholar]
  13. Kunze D. L., Lacerda A. E., Wilson D. L., Brown A. M. Cardiac Na currents and the inactivating, reopening, and waiting properties of single cardiac Na channels. J Gen Physiol. 1985 Nov;86(5):691–719. doi: 10.1085/jgp.86.5.691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Latorre R., Alvarez O., Cecchi X., Vergara C. Properties of reconstituted ion channels. Annu Rev Biophys Biophys Chem. 1985;14:79–111. doi: 10.1146/annurev.bb.14.060185.000455. [DOI] [PubMed] [Google Scholar]
  15. Lew V. L., Garcia-Sancho J. Use of the ionophore A23187 to measure and control cytoplasmic Ca2+ levels in intact red cells. Cell Calcium. 1985 Apr;6(1-2):15–23. doi: 10.1016/0143-4160(85)90031-4. [DOI] [PubMed] [Google Scholar]
  16. Liebovitch L. S., Fischbarg J. Determining the kinetics of membrane pores from patch clamp data without measuring the open and closed times. Biochim Biophys Acta. 1985 Feb 28;813(1):132–136. doi: 10.1016/0005-2736(85)90353-0. [DOI] [PubMed] [Google Scholar]
  17. Meeker R. B., Harden T. K. Muscarinic cholinergic receptor-mediated activation of phosphodiesterase. Mol Pharmacol. 1982 Sep;22(2):310–319. [PubMed] [Google Scholar]
  18. Methfessel C., Boheim G. The gating of single calcium-dependent potassium channels is described by an activation/blockade mechanism. Biophys Struct Mech. 1982;9(1):35–60. doi: 10.1007/BF00536014. [DOI] [PubMed] [Google Scholar]
  19. Moczydlowski E., Alvarez O., Vergara C., Latorre R. Effect of phospholipid surface charge on the conductance and gating of a Ca2+-activated K+ channel in planar lipid bilayers. J Membr Biol. 1985;83(3):273–282. doi: 10.1007/BF01868701. [DOI] [PubMed] [Google Scholar]
  20. Moczydlowski E., Latorre R. Gating kinetics of Ca2+-activated K+ channels from rat muscle incorporated into planar lipid bilayers. Evidence for two voltage-dependent Ca2+ binding reactions. J Gen Physiol. 1983 Oct;82(4):511–542. doi: 10.1085/jgp.82.4.511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Neher E., Sakmann B. Noise analysis of drug induced voltage clamp currents in denervated frog muscle fibres. J Physiol. 1976 Jul;258(3):705–729. doi: 10.1113/jphysiol.1976.sp011442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Neher E., Stevens C. F. Conductance fluctuations and ionic pores in membranes. Annu Rev Biophys Bioeng. 1977;6:345–381. doi: 10.1146/annurev.bb.06.060177.002021. [DOI] [PubMed] [Google Scholar]
  23. Noma A. ATP-regulated K+ channels in cardiac muscle. Nature. 1983 Sep 8;305(5930):147–148. doi: 10.1038/305147a0. [DOI] [PubMed] [Google Scholar]
  24. Owen J. D. The determination of the stability constant for calcium-EGTA. Biochim Biophys Acta. 1976 Nov 18;451(1):321–325. doi: 10.1016/0304-4165(76)90282-8. [DOI] [PubMed] [Google Scholar]
  25. Petersen O. H., Maruyama Y. Calcium-activated potassium channels and their role in secretion. Nature. 1984 Feb 23;307(5953):693–696. doi: 10.1038/307693a0. [DOI] [PubMed] [Google Scholar]
  26. Pontén J., Macintyre E. H. Long term culture of normal and neoplastic human glia. Acta Pathol Microbiol Scand. 1968;74(4):465–486. doi: 10.1111/j.1699-0463.1968.tb03502.x. [DOI] [PubMed] [Google Scholar]
  27. Sakmann B., Neher E. Patch clamp techniques for studying ionic channels in excitable membranes. Annu Rev Physiol. 1984;46:455–472. doi: 10.1146/annurev.ph.46.030184.002323. [DOI] [PubMed] [Google Scholar]
  28. Siegelbaum S. A., Camardo J. S., Kandel E. R. Serotonin and cyclic AMP close single K+ channels in Aplysia sensory neurones. Nature. 1982 Sep 30;299(5882):413–417. doi: 10.1038/299413a0. [DOI] [PubMed] [Google Scholar]
  29. Trube G., Hescheler J. Inward-rectifying channels in isolated patches of the heart cell membrane: ATP-dependence and comparison with cell-attached patches. Pflugers Arch. 1984 Jun;401(2):178–184. doi: 10.1007/BF00583879. [DOI] [PubMed] [Google Scholar]
  30. Tsien R. Y., Pozzan T., Rink T. J. Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J Cell Biol. 1982 Aug;94(2):325–334. doi: 10.1083/jcb.94.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wong B. S., Lecar H., Adler M. Single calcium-dependent potassium channels in clonal anterior pituitary cells. Biophys J. 1982 Sep;39(3):313–317. doi: 10.1016/S0006-3495(82)84522-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of General Physiology are provided here courtesy of The Rockefeller University Press

RESOURCES