Skip to main content
PMC home page
The Journal of General Physiology logoLink to The Journal of General Physiology
. 1988 Feb 1;91(2):193–222. doi: 10.1085/jgp.91.2.193

Single-channel recordings from cultured human retinal pigment epithelial cells

PMCID: PMC2216133  PMID: 2453602

Abstract

We have applied patch-clamp techniques to on-cell and excised-membrane patches from human retinal pigment epithelial cells in tissue culture. Single-channel currents from at least four ion channel types were observed: three or more potassium-selective channels with single- channel slope conductances near 100, 45, and 25 pS as measured in on- cell patches with physiological saline in the pipette, and a relatively nonselective channel with subconductance states, which has a main-state conductance of approximately 300 pS at physiological ion concentrations. The permeability ratios, PK/PNa, measured in excised patches were 21 for the 100-pS channels, 3 for the 25-pS channels, and 0.8 for the 300-pS nonselective channel. The 45-pS channels appeared to be of at least two types, with PK/PNa's of approximately 41 for one type and 3 for the other. The potassium-selective channels were spontaneously active at all potentials examined. The average open time for these channels ranged from a few milliseconds to many tens of milliseconds. No consistent trend relating potassium-selective channel kinetics to membrane potential was apparent, which suggests that channel activity was not regulated by the membrane potential. In contrast to the potassium-selective channels, the activity of the nonselective channel was voltage dependent: the open probability of this channel declined to low values at large positive or negative membrane potentials and was maximal near zero. Single-channel conductances observed at several symmetrical KCl concentrations have been fitted with Michaelis-Menten curves in order to estimate maximum channel conductances and ion-binding constants for the different channel types. The channels we have recorded are probably responsible for the previously observed potassium permeability of the retinal pigment epithelium apical membrane.

Full Text

The Full Text of this article is available as a PDF (2.0 MB).

Selected References

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

  1. Benham C. D., Bolton T. B. Patch-clamp studies of slow potential-sensitive potassium channels in longitudinal smooth muscle cells of rabbit jejunum. J Physiol. 1983 Jul;340:469–486. doi: 10.1113/jphysiol.1983.sp014774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blatz A. L., Magleby K. L. Single voltage-dependent chloride-selective channels of large conductance in cultured rat muscle. Biophys J. 1983 Aug;43(2):237–241. doi: 10.1016/S0006-3495(83)84344-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Conti F., Neher E. Single channel recordings of K+ currents in squid axons. Nature. 1980 May 15;285(5761):140–143. doi: 10.1038/285140a0. [DOI] [PubMed] [Google Scholar]
  4. Cooper K. E., Tang J. M., Rae J. L., Eisenberg R. S. A cation channel in frog lens epithelia responsive to pressure and calcium. J Membr Biol. 1986;93(3):259–269. doi: 10.1007/BF01871180. [DOI] [PubMed] [Google Scholar]
  5. Crawford B. J. Some factors controlling cell polarity in chick retinal pigment epithelial cells in clonal culture. Tissue Cell. 1983;15(6):993–1005. doi: 10.1016/0040-8166(83)90064-2. [DOI] [PubMed] [Google Scholar]
  6. Fenwick E. M., Marty A., Neher E. Sodium and calcium channels in bovine chromaffin cells. J Physiol. 1982 Oct;331:599–635. doi: 10.1113/jphysiol.1982.sp014394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Fox J. A. An improved method for illuminating pipet tips for fire-polishing. J Neurosci Methods. 1985 Nov-Dec;15(3):239–241. doi: 10.1016/0165-0270(85)90104-9. [DOI] [PubMed] [Google Scholar]
  9. Fukushima Y. Single channel potassium currents of the anomalous rectifier. Nature. 1981 Nov 26;294(5839):368–371. doi: 10.1038/294368a0. [DOI] [PubMed] [Google Scholar]
  10. García-Díaz J. F., Nagel W., Essig A. Voltage-dependent K conductance at the apical membrane of Necturus gallbladder. Biophys J. 1983 Sep;43(3):269–278. doi: 10.1016/S0006-3495(83)84350-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gray P. T., Bevan S., Ritchie J. M. High conductance anion-selective channels in rat cultured Schwann cells. Proc R Soc Lond B Biol Sci. 1984 Jun 22;221(1225):395–409. doi: 10.1098/rspb.1984.0041. [DOI] [PubMed] [Google Scholar]
  12. Griff E. R., Steinberg R. H. Origin of the light peak: in vitro study of Gekko gekko. J Physiol. 1982 Oct;331:637–652. doi: 10.1113/jphysiol.1982.sp014395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Hanrahan J. W., Alles W. P., Lewis S. A. Single anion-selective channels in basolateral membrane of a mammalian tight epithelium. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7791–7795. doi: 10.1073/pnas.82.22.7791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hess P., Lansman J. B., Tsien R. W. Different modes of Ca channel gating behaviour favoured by dihydropyridine Ca agonists and antagonists. Nature. 1984 Oct 11;311(5986):538–544. doi: 10.1038/311538a0. [DOI] [PubMed] [Google Scholar]
  16. Hille B. Ionic selectivity of Na and K channels of nerve membranes. Membranes. 1975;3:255–323. [PubMed] [Google Scholar]
  17. Horn R., Patlak J. Single channel currents from excised patches of muscle membrane. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6930–6934. doi: 10.1073/pnas.77.11.6930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Horn R., Vandenberg C. A., Lange K. Statistical analysis of single sodium channels. Effects of N-bromoacetamide. Biophys J. 1984 Jan;45(1):323–335. doi: 10.1016/S0006-3495(84)84158-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Immel J., Steinberg R. H. Spatial buffering of K+ by the retinal pigment epithelium in frog. J Neurosci. 1986 Nov;6(11):3197–3204. doi: 10.1523/JNEUROSCI.06-11-03197.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jacob T. J., Bangham J. A., Duncan G. Characterization of a cation channel on the apical surface of the frog lens epithelium. Q J Exp Physiol. 1985 Jul;70(3):403–421. doi: 10.1113/expphysiol.1985.sp002925. [DOI] [PubMed] [Google Scholar]
  21. Kolb H. A., Brown C. D., Murer H. Identification of a voltage-dependent anion channel in the apical membrane of a Cl(-)-secretory epithelium (MDCK). Pflugers Arch. 1985 Mar;403(3):262–265. doi: 10.1007/BF00583597. [DOI] [PubMed] [Google Scholar]
  22. Krouse M. E., Schneider G. T., Gage P. W. A large anion-selective channel has seven conductance levels. Nature. 1986 Jan 2;319(6048):58–60. doi: 10.1038/319058a0. [DOI] [PubMed] [Google Scholar]
  23. Labarca P., Lindstrom J., Montal M. Acetylcholine receptor in planar lipid bilayers. Characterization of the channel properties of the purified nicotinic acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayers. J Gen Physiol. 1984 Apr;83(4):473–496. doi: 10.1085/jgp.83.4.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lasansky A., De Fisch F. W. Potential, current, and ionic fluxes across the isolated retinal pigment epithelium and choriod. J Gen Physiol. 1966 May;49(5):913–924. doi: 10.1085/jgp.49.5.913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Latorre R., Miller C. Conduction and selectivity in potassium channels. J Membr Biol. 1983;71(1-2):11–30. doi: 10.1007/BF01870671. [DOI] [PubMed] [Google Scholar]
  26. Lindemann B. Fluctuation analysis of sodium channels in epithelia. Annu Rev Physiol. 1984;46:497–515. doi: 10.1146/annurev.ph.46.030184.002433. [DOI] [PubMed] [Google Scholar]
  27. Linsenmeier R. A., Steinberg R. H. A light-evoked interaction of apical and basal membranes of retinal pigment epithelium: c-wave and light peak. J Neurophysiol. 1983 Jul;50(1):136–147. doi: 10.1152/jn.1983.50.1.136. [DOI] [PubMed] [Google Scholar]
  28. Linsenmeier R. A., Steinberg R. H. Origin and sensitivity of the light peak in the intact cat eye. J Physiol. 1982 Oct;331:653–673. doi: 10.1113/jphysiol.1982.sp014396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Läuger P. Ion transport through pores: a rate-theory analysis. Biochim Biophys Acta. 1973 Jul 6;311(3):423–441. doi: 10.1016/0005-2736(73)90323-4. [DOI] [PubMed] [Google Scholar]
  30. Maruyama Y., Gallacher D. V., Petersen O. H. Voltage and Ca2+-activated K+ channel in baso-lateral acinar cell membranes of mammalian salivary glands. Nature. 1983 Apr 28;302(5911):827–829. doi: 10.1038/302827a0. [DOI] [PubMed] [Google Scholar]
  31. Miller S. S., Steinberg R. H. Passive ionic properties of frog retinal pigment epithelium. J Membr Biol. 1977 Sep 15;36(4):337–372. doi: 10.1007/BF01868158. [DOI] [PubMed] [Google Scholar]
  32. Miller S. S., Steinberg R. H. Potassium transport across the frog retinal pigment epithelium. J Membr Biol. 1982;67(3):199–209. doi: 10.1007/BF01868661. [DOI] [PubMed] [Google Scholar]
  33. Moczydlowski E., Garber S. S., Miller C. Batrachotoxin-activated Na+ channels in planar lipid bilayers. Competition of tetrodotoxin block by Na+. J Gen Physiol. 1984 Nov;84(5):665–686. doi: 10.1085/jgp.84.5.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Morris C. E., Wong B. S., Jackson M. B., Lecar H. Single-channel currents activated by curare in cultured embryonic rat muscle. J Neurosci. 1983 Dec;3(12):2525–2531. doi: 10.1523/JNEUROSCI.03-12-02525.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nelson D. J., Tang J. M., Palmer L. G. Single-channel recordings of apical membrane chloride conductance in A6 epithelial cells. J Membr Biol. 1984;80(1):81–89. doi: 10.1007/BF01868692. [DOI] [PubMed] [Google Scholar]
  36. Oakley B., 2nd Effects of maintained illumination upon [K+]0 in the subretinal space of the isolated retina of the toad. Vision Res. 1983;23(11):1325–1337. doi: 10.1016/0042-6989(83)90108-6. [DOI] [PubMed] [Google Scholar]
  37. Oakley B., 2nd, Miller S. S., Steinberg R. H. Effect of intracellular potassium upon the electrogenic pump of frog retinal pigment epithelium. J Membr Biol. 1978 Dec 29;44(3-4):281–307. doi: 10.1007/BF01944225. [DOI] [PubMed] [Google Scholar]
  38. Oakley B., 2nd Potassium and the photoreceptor-dependent pigment epithelial hyperpolarization. J Gen Physiol. 1977 Oct;70(4):405–425. doi: 10.1085/jgp.70.4.405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ohmori H., Yoshida S., Hagiwara S. Single K+ channel currents of anomalous rectification in cultured rat myotubes. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4960–4964. doi: 10.1073/pnas.78.8.4960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Patlak J., Horn R. Effect of N-bromoacetamide on single sodium channel currents in excised membrane patches. J Gen Physiol. 1982 Mar;79(3):333–351. doi: 10.1085/jgp.79.3.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Pfeffer B. A., Clark V. M., Flannery J. G., Bok D. Membrane receptors for retinol-binding protein in cultured human retinal pigment epithelium. Invest Ophthalmol Vis Sci. 1986 Jul;27(7):1031–1040. [PubMed] [Google Scholar]
  43. Rae J. L., Levis R. A. Patch Clamp Recordings from the Epithelium of the Lens Obtained using Glasses Selected for Low Noise and Improved Sealing Properties. Biophys J. 1984 Jan;45(1):144–146. doi: 10.1016/S0006-3495(84)84142-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rae J. L. The application of patch clamp methods to ocular epithelia. Curr Eye Res. 1985 Apr;4(4):409–420. doi: 10.3109/02713688509025155. [DOI] [PubMed] [Google Scholar]
  45. Sakmann B., Trube G. Conductance properties of single inwardly rectifying potassium channels in ventricular cells from guinea-pig heart. J Physiol. 1984 Feb;347:641–657. doi: 10.1113/jphysiol.1984.sp015088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sather W. A., Bodoia R. D., Detwiler P. B. Does the plasma membrane of the rod outer segment contain more than one type of ion channel? Neurosci Res Suppl. 1985;2:S89–S99. doi: 10.1016/0921-8696(85)90009-x. [DOI] [PubMed] [Google Scholar]
  47. Schein S. J., Colombini M., Finkelstein A. Reconstitution in planar lipid bilayers of a voltage-dependent anion-selective channel obtained from paramecium mitochondria. J Membr Biol. 1976 Dec 28;30(2):99–120. doi: 10.1007/BF01869662. [DOI] [PubMed] [Google Scholar]
  48. Schmidt R., Steinberg R. H. Rod-dependent intracellular responses to light recorded from the pigment epithelium of the cat retina. J Physiol. 1971 Aug;217(1):71–91. doi: 10.1113/jphysiol.1971.sp009560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Schneider G. T., Cook D. I., Gage P. W., Young J. A. Voltage sensitive, high-conductance chloride channels in the luminal membrane of cultured pulmonary alveolar (type II) cells. Pflugers Arch. 1985 Aug;404(4):354–357. doi: 10.1007/BF00585348. [DOI] [PubMed] [Google Scholar]
  50. Schwarze W., Kolb H. A. Voltage-dependent kinetics of an anionic channel of large unit conductance in macrophages and myotube membranes. Pflugers Arch. 1984 Nov;402(3):281–291. doi: 10.1007/BF00585511. [DOI] [PubMed] [Google Scholar]
  51. Simons K., Fuller S. D. Cell surface polarity in epithelia. Annu Rev Cell Biol. 1985;1:243–288. doi: 10.1146/annurev.cb.01.110185.001331. [DOI] [PubMed] [Google Scholar]
  52. Steinberg R. H., Linsenmeier R. A., Griff E. R. Three light-evoked responses of the retinal pigment epithelium. Vision Res. 1983;23(11):1315–1323. doi: 10.1016/0042-6989(83)90107-4. [DOI] [PubMed] [Google Scholar]
  53. Steinberg R. H., Oakley B., 2nd, Niemeyer G. Light-evoked changes in [K+]0 in retina of intact cat eye. J Neurophysiol. 1980 Nov;44(5):897–921. doi: 10.1152/jn.1980.44.5.897. [DOI] [PubMed] [Google Scholar]
  54. Trautmann A., Marty A. Activation of Ca-dependent K channels by carbamoylcholine in rat lacrimal glands. Proc Natl Acad Sci U S A. 1984 Jan;81(2):611–615. doi: 10.1073/pnas.81.2.611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Tsuboi S., Manabe R., Iizuka S. Aspects of electrolyte transport across isolated dog retinal pigment epithelium. Am J Physiol. 1986 May;250(5 Pt 2):F781–F784. doi: 10.1152/ajprenal.1986.250.5.F781. [DOI] [PubMed] [Google Scholar]
  56. Van Driessche W., Zeiske W. Ionic channels in epithelial cell membranes. Physiol Rev. 1985 Oct;65(4):833–903. doi: 10.1152/physrev.1985.65.4.833. [DOI] [PubMed] [Google Scholar]
  57. Welsh M. J., McCann J. D. Intracellular calcium regulates basolateral potassium channels in a chloride-secreting epithelium. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8823–8826. doi: 10.1073/pnas.82.24.8823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zampighi G. A., Hall J. E., Kreman M. Purified lens junctional protein forms channels in planar lipid films. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8468–8472. doi: 10.1073/pnas.82.24.8468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. la Cour M., Lund-Andersen H., Zeuthen T. Potassium transport of the frog retinal pigment epithelium: autoregulation of potassium activity in the subretinal space. J Physiol. 1986 Jun;375:461–479. doi: 10.1113/jphysiol.1986.sp016128. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES