Summary
Enterrocytes from the intestinal epithelium of the winter flounder were isolated by collagenase digestion and incubated in flounder Ringer solution. Conventional whole-cell and amphotericin-perforated whole-cell recording techniques were used to characterize the properties of a voltage-activated K current present in dissociated cells. Resting membrane potentials and series resistances were significantly lower (from −23 to − 39 mV and 29 to 13 MΩ, respectively) when amphotericin was used to achieve the whole-cell configuration. When cells were placed in flounder Ringer solution, held at −80 mV and subsequently stepped to a series of depolarizing voltages (from−70 to 0 mV), an outward current was observed that exhibited inactivation at voltages above −20 mV. This current was sensitive to holding potential and was not activated when the cells were held at −40 mV or above. When cells were bathed in symmetric K Ringer solution and the same voltage protocol was applied to the cell, inward currents were observed in response to the negative intracellular potentials. Reversal potentials at two different extracellular K concentrations were consistent with K as the currentcarrying ion. BaCl2 (2 mM) and CsCl (0.5 mM) both produced voltage-dependent blockade of the current when added to the bathing solution. Charybdotoxin (300 nM extracellular concentration) completely blocked the current. The IC50 for charybdotoxin was 50 nM. Cyclic. GMP inhibited the voltage-activated current in flounder Ringer and in symmetric K Ringer solution. The cyclic GMP analog, 8-Br cGMP, lowered the threshold for voltage activation and potentiated inactivation of the current at voltages above−40 mV. Previous studies with intact flounder epithelium showed that K recycling and net K secretion were inhibited by Ba2+ and by cGMP. We suggest that the channel responsible for the whole-cell current described in this study may be important in K recycling and secretion.
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Castle, N.A., Haylett, D.G., Jenkinson, D.G., Jenkinson, D.H. 1989. Toxins in the characterization of potassium channels.Trends Neurosci. 12:59–65
Dawson, D.C., Richards, N.W. 1990. Basolateral K conductance: Role in regulation of NaCl absorption and secretion.Am. J. Physiol. 259:C181–195
Duffey, M.E., Thompson, S.M., Frizzell, R.A., Schultz, S.G. 1979. Intracellular chloride activities and active chloride absorption in the intestinal epithelium of the winter flounder.J. Membrane Biol. 50:331–341
Field, M., Karnaky, K.J., Jr., Smith, P.L., Bolton, J.E., Kinter, W. B. 1978. Ion transport across the isolated intestinal mucosa of the winter flounder,Pseudopleuronectes americanus: I. Functional and structural properties of cellular and paracellular pathways for Na and Cl.J. Membrane Biol. 41:265–293
Hagiwara, S., Miyasaki, S., Rosenthal, N.P. 1976. Potassium current and the effects of cesium on this current during anomalous rectification of the egg cell membrane of the starfish.J. Gen. Physiol. 67:621–638
Halm, D.R., Krasny, E.J., Frizzell, R.A. (1985). Electrophysiology of the flounder intestinal mucosa. l. Conductance properties of the cellular and paracellular pathways.J. Gen. Physiol. 85:843–864
Hunter, M., Geibisch, G. 1988. Calcium-activated K channel ofAmphiuma early distal tubule: Inhibition by ATP.Pfluegers Arch. 412:331–333
Lewis, R.S., Cahalan, M.D. 1990.Ion channels and signal transsduction in lymphocytes.Annu. Rev. Physiol. 52:415–430
Light, D.B., Corbin, J.D., Stanton B.A. 1990. Dual ion channel regulation by cGMP and cGMP-dependent protein kinase.Nature 344:336–339
Light, D.B., McCann, F.V. Keller, T.M., Stanton, B.A. 1988. Amiloride sensitive cation duct.Am. J. Physiol. 255:F278-F286
McCann, J.D., Welsh, M.J. 1990. Regulation of Cl and K channels in airway epithelium.Annu. Rev. Physiol. 52:115–135
Miller, C., Moczydlowski, E., Latorre, R., Phillips M. 1985. Charybdotoxin, a protein inhibitor of single Ca-activated K channels from mammalian skeletal muscle.Nature 313:316–318
Musch, M.W., Orellana, S.A., Kimberg, L.S., Filed, M., Halm, D.R., Krasny, E.J., Frizzell, R.A. 1982. Na-K-Cl cotransport in the intestine of a marine teleost.Nature 300:351–353
O'Grady, S.M., DeJonge, H.R., Vaandrager, A.B., Field, M. 1988. Cyclic nucleotide-dependent protein kinase inhibition by H-8: Effects on ion transport.Am. J. Physiol. 254:C115-C121
O'Grady, S.M., Palfrey, C.H., Field, M. 1987. Characteristics and functions of Na-K-Cl cotransport in epithelial tissues.Am. J. Physiol. 253:C177–192
Rae, J., Cooper, K., Gates, P., Watsky, M. 1991. Low access resistance perforated patch recordings using amphotericin B.J. Neurosci. Methods 37:15–26
Rao, M.C., Nash, N.T., Field, M. 1984. Differing effects of cGMP and cAMP on ion transport across flounder intestine.Am. J. Physiol. 246:C167-C171
Rudy, B. 1988. Diversity and ubiquity of K channels.Neuroscience 25:729–749
Sullivan, S.K., Swamy, K., Greenspan, N.R., Field, M. 1990. Epithelial K channel expressed inXenopus oocytes is inactivated by protein kinase C.Proc. Natl. Acad. Sci. USA 87:4553–4556
Wang, W., White, S., Geibel, J., Giebisch,. 1990. A potassium channel in the apical membrane of the rabbit thick ascending limb of Henle's loop.Am. J. Physiol. 258:F244-F253
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O'Grady, S.M., Cooper, K.E. & Rae, J.L. Cyclic GMP regulation of a voltage-activated K channel in dissociated Enterocytes. J. Membrain Biol. 124, 159–167 (1991). https://doi.org/10.1007/BF01870460
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DOI: https://doi.org/10.1007/BF01870460