Skip to main content
PMC home page
The Journal of General Physiology logoLink to The Journal of General Physiology
. 1991 Jul 1;98(1):131–161. doi: 10.1085/jgp.98.1.131

Whole-cell and single channel K+ and Cl- currents in epithelial cells of frog skin

PMCID: PMC2229038  PMID: 1719124

Abstract

Whole-cell and single channel currents were studied in cells from frog (R. pipiens and R. catesbiana) skin epithelium, isolated by collagenase and trypsin treatment, and kept in primary cultures up to three days. Whole-cell currents did not exhibit any significant time-dependent kinetics under any ionic conditions used. With an external K gluconate Ringer solution the currents showed slight inward rectification with a reversal potential near zero and an average conductance of 5 nS at reversal. Ionic substitution of the external medium showed that most of the cell conductance was due to K and that very little, if any, Na conductance was present. This confirmed that most cells originate from inner epithelial layers and contain membranes with basolateral properties. At voltages more positive than 20 mV outward currents were larger with K in the medium than with Na or N-methyl-D-glucamine. Such behavior is indicative of a multi-ion transport mechanism. Whole-cell K current was inhibited by external Ba and quinidine. Blockade by Ba was strongly voltage dependent, while that by quinidine was not. In the presence of high external Cl, a component of outward current that was inhibited by the anion channel blocker diphenylamine-2-carboxylate (DPC) appeared in 70% of the cells. This component was strongly outwardly rectifying and reversed at a potential expected for a Cl current. At the single channel level the event most frequently observed in the cell-attached configuration was a K channel with the following characteristics: inward-rectifying I-V relation with a conductance (with 112.5 mM K in the pipette) of 44 pS at the reversal potential, one open and at least two closed states, and open probability that increased with depolarization. Quinidine blocked by binding in the open state and decreasing mean open time. Several observations suggest that this channel is responsible for most of the whole-cell current observed in high external K, and for the K conductance of the basolateral membrane of the intact epithelium. On a few occasions a Cl channel was observed that activated upon excision and brief strong depolarization. The I-V relation exhibited strong outward rectification with a single channel conductance of 48 pS at 0 mV in symmetrical 112 mM Cl solutions. Kinetic analysis showed the presence of two open and at least two closed states. Open time constants and open probability increased markedly with depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Full Text

The Full Text of this article is available as a PDF (2.2 MB).

Selected References

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

  1. ADRIAN R. H. Movement of inorganic ions across the membrane of striated muscle. Circulation. 1962 Nov;26:1214–1223. doi: 10.1161/01.cir.26.5.1214. [DOI] [PubMed] [Google Scholar]
  2. Adrian R. H. Rectification in muscle membrane. Prog Biophys Mol Biol. 1969;19(2):339–369. [PubMed] [Google Scholar]
  3. Bleich M., Schlatter E., Greger R. The luminal K+ channel of the thick ascending limb of Henle's loop. Pflugers Arch. 1990 Jan;415(4):449–460. doi: 10.1007/BF00373623. [DOI] [PubMed] [Google Scholar]
  4. Bokvist K., Rorsman P., Smith P. A. Block of ATP-regulated and Ca2(+)-activated K+ channels in mouse pancreatic beta-cells by external tetraethylammonium and quinine. J Physiol. 1990 Apr;423:327–342. doi: 10.1113/jphysiol.1990.sp018025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bokvist K., Rorsman P., Smith P. A. Effects of external tetraethylammonium ions and quinine on delayed rectifying K+ channels in mouse pancreatic beta-cells. J Physiol. 1990 Apr;423:311–325. doi: 10.1113/jphysiol.1990.sp018024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang D., Dawson D. C. Digitonin-permeabilized colonic cell layers. Demonstration of calcium-activated basolateral K+ and Cl- conductances. J Gen Physiol. 1988 Sep;92(3):281–306. doi: 10.1085/jgp.92.3.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dörge A., Beck F. X., Wienecke P., Rick R. Cl transport across the basolateral membrane of principal cells in frog skin. Miner Electrolyte Metab. 1989;15(3):155–162. [PubMed] [Google Scholar]
  8. Eaton D. C., Brodwick M. S. Effects of barium on the potassium conductance of squid axon. J Gen Physiol. 1980 Jun;75(6):727–750. doi: 10.1085/jgp.75.6.727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Farquhar M. G., Palade G. E. Adenosine triphosphatase localization in amphibian epidermis. J Cell Biol. 1966 Aug;30(2):359–379. doi: 10.1083/jcb.30.2.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Farquhar M. G., Palade G. E. Cell junctions in amphibian skin. J Cell Biol. 1965 Jul;26(1):263–291. doi: 10.1083/jcb.26.1.263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fisher R. S., Erlij D., Helman S. I. Intracellular voltage of isolated epithelia of frog skin: apical and basolateral cell punctures. J Gen Physiol. 1980 Oct;76(4):447–453. doi: 10.1085/jgp.76.4.447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Foskett J. K., Ussing H. H. Localization of chloride conductance to mitochondria-rich cells in frog skin epithelium. J Membr Biol. 1986;91(3):251–258. doi: 10.1007/BF01868818. [DOI] [PubMed] [Google Scholar]
  13. Friedrich F., Paulmichl M., Kolb H. A., Lang F. Inward rectifier K channels in renal epithelioid cells (MDCK) activated by serotonin. J Membr Biol. 1988 Dec;106(2):149–155. doi: 10.1007/BF01871397. [DOI] [PubMed] [Google Scholar]
  14. Frizzell R. A., Rechkemmer G., Shoemaker R. L. Altered regulation of airway epithelial cell chloride channels in cystic fibrosis. Science. 1986 Aug 1;233(4763):558–560. doi: 10.1126/science.2425436. [DOI] [PubMed] [Google Scholar]
  15. Garcia-Diaz J. F., Essig A. Capacitative transients in voltage-clamped epithelia. Biophys J. 1985 Sep;48(3):519–523. doi: 10.1016/S0006-3495(85)83807-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. García-Díaz J. F., Baxendale L. M., Klemperer G., Essig A. Cell K activity in frog skin in the presence and absence of cell current. J Membr Biol. 1985;85(2):143–158. doi: 10.1007/BF01871267. [DOI] [PubMed] [Google Scholar]
  17. Giebisch G., Hunter M., Kawahara K. Apical potassium channels in Amphiuma diluting segment: effect of barium. J Physiol. 1990 Jan;420:313–323. doi: 10.1113/jphysiol.1990.sp017914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Giraldez F., Ferreira K. T. Intracellular chloride activity and membrane potential in stripped frog skin (Rana temporaria). Biochim Biophys Acta. 1984 Feb 15;769(3):625–628. doi: 10.1016/0005-2736(84)90062-2. [DOI] [PubMed] [Google Scholar]
  19. Glavinović M. I., Trifaró J. M. Quinine blockade of currents through Ca2+-activated K+ channels in bovine chromaffin cells. J Physiol. 1988 May;399:139–152. doi: 10.1113/jphysiol.1988.sp017072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Greger R., Bleich M., Schlatter E. Ion channels in the thick ascending limb of Henle's loop. Ren Physiol Biochem. 1990 Jan-Apr;13(1-2):37–50. doi: 10.1159/000173346. [DOI] [PubMed] [Google Scholar]
  21. Greger R., Gögelein H., Schlatter E. Potassium channels in the basolateral membrane of the rectal gland of the dogfish (Squalus acanthias). Pflugers Arch. 1987 Jun;409(1-2):100–106. doi: 10.1007/BF00584755. [DOI] [PubMed] [Google Scholar]
  22. Greger R., Schlatter E., Gögelein H. Chloride channels in the luminal membrane of the rectal gland of the dogfish (Squalus acanthias). Properties of the "larger" conductance channel. Pflugers Arch. 1987 Jun;409(1-2):114–121. doi: 10.1007/BF00584757. [DOI] [PubMed] [Google Scholar]
  23. Gögelein H., Greger R. Properties of single K+ channels in the basolateral membrane of rabbit proximal straight tubules. Pflugers Arch. 1987 Oct;410(3):288–295. doi: 10.1007/BF00580279. [DOI] [PubMed] [Google Scholar]
  24. Gögelein H., Greger R., Schlatter E. Potassium channels in the basolateral membrane of the rectal gland of Squalus acanthias. Regulation and inhibitors. Pflugers Arch. 1987 Jun;409(1-2):107–113. doi: 10.1007/BF00584756. [DOI] [PubMed] [Google Scholar]
  25. Halm D. R., Rechkemmer G. R., Schoumacher R. A., Frizzell R. A. Apical membrane chloride channels in a colonic cell line activated by secretory agonists. Am J Physiol. 1988 Apr;254(4 Pt 1):C505–C511. doi: 10.1152/ajpcell.1988.254.4.C505. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Helman S. I., Fisher R. S. Microelectrode studies of the active Na transport pathway of frog skin. J Gen Physiol. 1977 May;69(5):571–604. doi: 10.1085/jgp.69.5.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hermann A., Gorman A. L. Action of quinidine on ionic currents of molluscan pacemaker neurons. J Gen Physiol. 1984 Jun;83(6):919–940. doi: 10.1085/jgp.83.6.919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hille B., Schwarz W. Potassium channels as multi-ion single-file pores. J Gen Physiol. 1978 Oct;72(4):409–442. doi: 10.1085/jgp.72.4.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Horn R., Marty A. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol. 1988 Aug;92(2):145–159. doi: 10.1085/jgp.92.2.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Horowicz P., Gage P. W., Eisenberg R. S. The role of the electrochemical gradient in determining potassium fluxes in frog striated muscle. J Gen Physiol. 1968 May;51(5 Suppl):193S+–193S+. [PubMed] [Google Scholar]
  33. Hunter M., Kawahara K., Giebisch G. Potassium channels along the nephron. Fed Proc. 1986 Nov;45(12):2723–2726. [PubMed] [Google Scholar]
  34. Hunter M., Oberleithner H., Henderson R. M., Giebisch G. Whole-cell potassium currents in single early distal tubule cells. Am J Physiol. 1988 Oct;255(4 Pt 2):F699–F703. doi: 10.1152/ajprenal.1988.255.4.F699. [DOI] [PubMed] [Google Scholar]
  35. KOEFOED-JOHNSEN V., USSING H. H. The nature of the frog skin potential. Acta Physiol Scand. 1958 Jun 2;42(3-4):298–308. doi: 10.1111/j.1748-1716.1958.tb01563.x. [DOI] [PubMed] [Google Scholar]
  36. Kawahara K., Hunter M., Giebisch G. Potassium channels in Necturus proximal tubule. Am J Physiol. 1987 Sep;253(3 Pt 2):F488–F494. doi: 10.1152/ajprenal.1987.253.3.F488. [DOI] [PubMed] [Google Scholar]
  37. Kunzelmann K., Pavenstädt H., Beck C., Unal O., Emmrich P., Arndt H. J., Greger R. Characterization of potassium channels in respiratory cells. I. General properties. Pflugers Arch. 1989 Jul;414(3):291–296. doi: 10.1007/BF00584629. [DOI] [PubMed] [Google Scholar]
  38. Kunzelmann K., Pavenstädt H., Greger R. Properties and regulation of chloride channels in cystic fibrosis and normal airway cells. Pflugers Arch. 1989 Nov;415(2):172–182. doi: 10.1007/BF00370589. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Mancilla E., Rojas E. Quinine blocks the high conductance, calcium-activated potassium channel in rat pancreatic beta-cells. FEBS Lett. 1990 Jan 15;260(1):105–108. doi: 10.1016/0014-5793(90)80078-w. [DOI] [PubMed] [Google Scholar]
  41. Martinez-Palomo A., Erlij D., Bracho H. Localization of permeability barriers in the frog skin epithelium. J Cell Biol. 1971 Aug;50(2):277–287. doi: 10.1083/jcb.50.2.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. McCann J. D., Li M., Welsh M. J. Identification and regulation of whole-cell chloride currents in airway epithelium. J Gen Physiol. 1989 Dec;94(6):1015–1036. doi: 10.1085/jgp.94.6.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. McCann J. D., Welsh M. J. Regulation of Cl- and K+ channels in airway epithelium. Annu Rev Physiol. 1990;52:115–135. doi: 10.1146/annurev.ph.52.030190.000555. [DOI] [PubMed] [Google Scholar]
  44. Miller C., Latorre R., Reisin I. Coupling of voltage-dependent gating and Ba++ block in the high-conductance, Ca++-activated K+ channel. J Gen Physiol. 1987 Sep;90(3):427–449. doi: 10.1085/jgp.90.3.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Mills J. W., Ernst S. A., DiBona D. R. Localization of Na+-pump sites in frog skin. J Cell Biol. 1977 Apr;73(1):88–110. doi: 10.1083/jcb.73.1.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Nagel W. Chloride conductance of frog skin: localization to the tight junctions? Miner Electrolyte Metab. 1989;15(3):163–170. [PubMed] [Google Scholar]
  47. Nagel W., Dörge A. Analysis of anion conductance in frog skin. Pflugers Arch. 1990 Apr;416(1-2):53–61. doi: 10.1007/BF00370221. [DOI] [PubMed] [Google Scholar]
  48. Nagel W., Garcia-Diaz J. F., Armstrong W. M. Intracellular ionic activities in frog skin. J Membr Biol. 1981;61(2):127–134. doi: 10.1007/BF02007639. [DOI] [PubMed] [Google Scholar]
  49. Nagel W., García-Díaz J. F., Essig A. Voltage dependence of cellular current and conductances in frog skin. J Membr Biol. 1988 Nov;106(1):13–28. doi: 10.1007/BF01871763. [DOI] [PubMed] [Google Scholar]
  50. Nagel W. Inhibition of potassium conductance by barium in frog skin epithelium. Biochim Biophys Acta. 1979 Apr 4;552(2):346–357. doi: 10.1016/0005-2736(79)90289-x. [DOI] [PubMed] [Google Scholar]
  51. Nagel W. The dependence of the electrical potentials across the membranes of the frog skin upon the concentration of sodium in the mucosal solution. J Physiol. 1977 Aug;269(3):777–796. doi: 10.1113/jphysiol.1977.sp011929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Noble D., Tsien R. W. The kinetics and rectifier properties of the slow potassium current in cardiac Purkinje fibres. J Physiol. 1968 Mar;195(1):185–214. doi: 10.1113/jphysiol.1968.sp008454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Parent L., Cardinal J., Sauvé R. Single-channel analysis of a K channel at basolateral membrane of rabbit proximal convoluted tubule. Am J Physiol. 1988 Jan;254(1 Pt 2):F105–F113. doi: 10.1152/ajprenal.1988.254.1.F105. [DOI] [PubMed] [Google Scholar]
  54. Rae J. L., Dewey J., Cooper K., Gates P. A non-selective cation channel in rabbit corneal endothelium activated by internal calcium and inhibited by internal ATP. Exp Eye Res. 1990 Apr;50(4):373–384. doi: 10.1016/0014-4835(90)90138-k. [DOI] [PubMed] [Google Scholar]
  55. Rae J. L., Dewey J., Cooper K., Gates P. Potassium channel in rabbit corneal endothelium activated by external anions. J Membr Biol. 1990 Mar;114(1):29–36. doi: 10.1007/BF01869382. [DOI] [PubMed] [Google Scholar]
  56. Richards N. W., Dawson D. C. Single potassium channels blocked by lidocaine and quinidine in isolated turtle colon epithelial cells. Am J Physiol. 1986 Jul;251(1 Pt 1):C85–C89. doi: 10.1152/ajpcell.1986.251.1.C85. [DOI] [PubMed] [Google Scholar]
  57. Rick R., Dörge A., von Arnim E., Thurau K. Electron microprobe analysis of frog skin epithelium: evidence for a syncytial sodium transport compartment. J Membr Biol. 1978 Mar 20;39(4):313–331. doi: 10.1007/BF01869897. [DOI] [PubMed] [Google Scholar]
  58. Sackin H., Palmer L. G. Basolateral potassium channels in renal proximal tubule. Am J Physiol. 1987 Sep;253(3 Pt 2):F476–F487. doi: 10.1152/ajprenal.1987.253.3.F476. [DOI] [PubMed] [Google Scholar]
  59. Sansom S. C., La B. Q., Carosi S. L. Double-barreled chloride channels of collecting duct basolateral membrane. Am J Physiol. 1990 Jul;259(1 Pt 2):F46–F52. doi: 10.1152/ajprenal.1990.259.1.F46. [DOI] [PubMed] [Google Scholar]
  60. Schoen H. F., Erlij D. Current-voltage relations of the apical and basolateral membranes of the frog skin. J Gen Physiol. 1985 Aug;86(2):257–287. doi: 10.1085/jgp.86.2.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Schoumacher R. A., Ram J., Iannuzzi M. C., Bradbury N. A., Wallace R. W., Hon C. T., Kelly D. R., Schmid S. M., Gelder F. B., Rado T. A. A cystic fibrosis pancreatic adenocarcinoma cell line. Proc Natl Acad Sci U S A. 1990 May;87(10):4012–4016. doi: 10.1073/pnas.87.10.4012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Segal Y., Reuss L. Ba2+, TEA+, and quinine effects on apical membrane K+ conductance and maxi K+ channels in gallbladder epithelium. Am J Physiol. 1990 Jul;259(1 Pt 1):C56–C68. doi: 10.1152/ajpcell.1990.259.1.C56. [DOI] [PubMed] [Google Scholar]
  63. Sheppard D. N., Giraldez F., Sepúlveda F. V. Kinetics of voltage- and Ca2+ activation and Ba2+ blockade of a large-conductance K+ channel from Necturus enterocytes. J Membr Biol. 1988 Oct;105(1):65–75. doi: 10.1007/BF01871107. [DOI] [PubMed] [Google Scholar]
  64. Smith P. G. The low-frequency electrical impedance of the isolated frog skin. Acta Physiol Scand. 1971 Mar;81(3):355–366. doi: 10.1111/j.1748-1716.1971.tb04910.x. [DOI] [PubMed] [Google Scholar]
  65. Spalding B. C., Senyk O., Swift J. G., Horowicz P. Unidirectional flux ratio for potassium ions in depolarized frog skeletal muscle. Am J Physiol. 1981 Jul;241(1):C68–C75. doi: 10.1152/ajpcell.1981.241.1.C68. [DOI] [PubMed] [Google Scholar]

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

RESOURCES