Pflügers Archiv

, Volume 405, Issue 3, pp 274–284 | Cite as

A comparative electrophysiological study of enzymatically isolated single cells and strips of frog ventricle

  • Leslie Tung
  • Martin Morad
Excitable Tissues and Central Nervous Physiology


  1. 1.

    Single heart cells were obtained from frog ventricle using an enzymatic dispersion technique.

  2. 2.

    The whole cell variation of the patch clamp technique was used to monitor action potential and cell membrane currents. The clamp circuit could be switched electronically between voltage and current clamp modes.

  3. 3.

    The effects of seal leakage currents were to depolarize the cell, reduce the amplitude of the plateau, and lengthen the action potential duration. A scheme to compensate for these currents is presented.

  4. 4.

    The membrane currents obtained from the single cell under voltage clamp conditions were compared to those obtained from multicellular preparations using the single sucrose gap technique.

  5. 5.

    Hyperpolarizing clamps showed time-dependent, depletion-related K+ currents for the multicellular preparation, whereas for the single cell no such currents were observed. The absence of extracellular accumulation or depletion of K+ in the single cell was confirmed by the lack of post-clamp afterpotentials or changes in resting potential following a train of frequently elicited action potentials.

  6. 6.

    The TTX-insensitive inward current was relatively faster in the single cell, compared to that measured in the multicellular preparation.

  7. 7.

    A delayed time-dependent outward current was observed in the positive potential range for both single and multicellular preparations.

  8. 8.

    The isochronal current-voltage (I–V) relations obtained at 400 ms were N-shaped for both preparations, but was more, negative for the single cell at potentials positive to −20 mV.

  9. 9.

    The results indicate a strong similarity between membrane currents obtained in single and multicellular preparations. The differences in the currents in the two preparations are due in large part to accumulation or depletion of K+ in the extracellular space


Key words

(Cardiac) electrophysiology Voltage clamp Single cell Sucrose gap Whole cell clamp Frog heart 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Attwell D, Cohen I (1977) The voltage clamp of multicellular preparations. Prog Biophys Mol Biol 31:201–245Google Scholar
  2. Attwell D, Eisner D, Cohen I (1979) Voltage clamp and tracer flux data: effects of a restricted extracellular space. Q Rev Biophys 12:213–261Google Scholar
  3. Bean BP, Nowycky MC, Tsien RW (1984) β-adrenergic modulation of calcium channels in frog ventricular heart cells. Nature 307:371–375Google Scholar
  4. Beeler GW, McGuigan JAS (1978) Voltage clamping of multicellular preparations: capabilities and limitations of existing methods. Prog Biophys Mol Biol 34:219–254Google Scholar
  5. Bustamonte JO, Watanabe T, McDonald TF (1981) Single cells from adult mammalian heart: isolation procedure and preliminary electrophysiological studies. Can J Physiol Pharmacol 59:907–910Google Scholar
  6. Cleemann L, Morad M (1979a) Extracellular potassium accumulation in voltage-clamped frog ventricular muscle. J Physiol 286:83–111Google Scholar
  7. Cleemann L, Morad M (1979b) Potassium currents in frog ventricular muscle: evidence from voltage clamp currents and extracellular K accumulation. J Physiol 286:113–143Google Scholar
  8. Cleemann L, Suenson M (1984) Reduction of the sucrose-saline interdiffusion in the sucrose gap technique by controlled compression of the extracellular space in myocardial preparations. Acta Phhsiol Scand 120:417–427Google Scholar
  9. Coraboeuf E (1980) In: Zipe DP, Bailey JC, Elharrar V (eds) The slow inward current and cardiac arrhythmias, chapter 3. Martinus Nijhoff, BostonGoogle Scholar
  10. Fischmeister R, Mentrard D, Vassort G (1982) Limitations of voltage clamp studies of slow inward current using the double sucrose gap. Gen Physiol Biophys 1:319–348Google Scholar
  11. Goldman Y, Morad M (1977a) Regenerative repolarization of the frog ventricular action potential: a time and voltage-dependent phenomenon. J Physiol 268:575–611Google Scholar
  12. Goldman Y, Morad M (1977b) Measurement of transmembrane potential and current in cardiac muscle: a new voltage clamp method. J Physiol 268:613–654Google Scholar
  13. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391:85–100Google Scholar
  14. Hewett K, Legato M, Danilo P, Robinson RB (1983) Isolated myocytes from adult canine left ventricle: Ca2+ tolerance, electrophysiology, and ultrastructure. Am J Physiol 245:H830-H839Google Scholar
  15. Hume JR, Giles W (1981) Active and passive electrical properties of single bullfrog atrial cells. J Gen Physiol 78:18–43Google Scholar
  16. Hume JR, Giles W (1983) Ionic currents in single isolated bullfrog atrial cells. J Gen Physiol 81:153–194Google Scholar
  17. Irisawa H, Kokubun S (1983) Modulation by intracellular ATP and cyclic AMP of the slow inward current in isolated single ventricular cells of the guinea pig. J Physiol 338:321–337Google Scholar
  18. Isenberg G, Klockner U (1980) Glycocalyx is not required for slow inward calcium current in isolated rat heart myocytes. Nature 284:358–360Google Scholar
  19. Isenberg G, Klockner U (1982) Calcium tolerant ventricular myocytes prepared by preincubation in a “KB Medium”. Pflügers Arch 395:6–18Google Scholar
  20. Jack JJB, Noble D, Tsien RW (1975) Electrical current flowing in excitable cells. Clarendon Press, OxfordGoogle Scholar
  21. Jirounek P, Jones GJ, Burckhardt CW, Straub RW (1981) The correction factors for sucrose gap measurements and their practical applications. Biophys J 33:107–119Google Scholar
  22. Johnson EA, Lieberman M (1971) Heart: excitation and contraction. Annu Rev Physiol 33:479–529Google Scholar
  23. Kass RS, Siegelbaum SA, Tsien RW (1979) Three-micro-electrode voltage clamp experiments in calf cardiac Purkinje fibres: is slow inward current adequately measured? J Physiol 290:201–225Google Scholar
  24. Kline RP, Morad M (1978) Potassium efflux in heart muscle during activity: extracellular accumulation and its implications. J Physiol 280:537–558Google Scholar
  25. Lammel E (1981) A theoretical study on the sucrose gap technique as applied to multicellular muscle preparations. Biophys J 36:533–553Google Scholar
  26. Lee KS, Tsien RW (1982) Reversal of current through calcium channels in dialysed single heart cells. Nature 297:498–501Google Scholar
  27. Lee KS, Weeks TA, Kao RL, Akaike N, Brown AM (1979) Sodium current in single heart muscle cells. Nature 278:269–271Google Scholar
  28. McGuigan JAS, Tsien RW (1974) Appendix in: Some limitations of the double sucrose gap, and its use in a study of the slow outward current in mammalian ventricular muscle (by JAS McGuigan). J Physiol 240:775–806Google Scholar
  29. Mitra R, Morad M, Tourneur Y (1984) Time-dependent activation of the potassium inward rectifier IKi on isolated guinea-pig cardiac cells. J Physiol 356:68PGoogle Scholar
  30. Morad M, Tung L (1982) Ionic events responsible for the cardiac resting and action potential. Am J Cardiol 49:584–594Google Scholar
  31. New W, Trautwein W (1972) Inward membrane currents in mammalian myocardium. Pflügers Arch 334:1–23Google Scholar
  32. Noble S (1976) Potassium accumulation and depletion in frog atrial muscle. J Physiol 258:579–613Google Scholar
  33. Page SG, Niedergerke R (1972) Structures of physiological interest in the frog heart ventricle. J Cell Sci 11:179–203Google Scholar
  34. Powell T, Twist VW (1976) A rapid technique for the isolation and purification of adult cardiac muscle cells having respiratory control and tolerance to calcium. Biochem Biophys Res Commun 72:327–333Google Scholar
  35. Rais J, Sundberg M, Sundby GV, Donell N, Torling G, Biberfeld P, Jacobson S (1978) A rapid method for the isolation of viable cardiac myocytes from adult rat. Exp Cell Res 115:183–189Google Scholar
  36. Rougier O, Vassort G, Garnier D, Gargouil YM, Coraboeuf E (1969) Existence and role of a slow inward current during the frog atrial action potential. Pflügers Arch 308:91–110Google Scholar
  37. Salama G, Morad M (1983) Diffusion profiles of Na+-fluorescein in frog ventricular muscle. Biophys J 43:225–229Google Scholar
  38. Silver LH, Hemwall EL, Marino TA, Houser SR (1983) Isolation and morphology of calcium tolerant feline myocytes. Am J Physiol 345:H891-H896Google Scholar
  39. Taniguchi J, Kokubun S, Noma A, Irisawa H (1981) Spontaneously active cells isolated from the sino-atrial and atrio-ventricular nodes of the rabbit heart. Jpn J Physiol 31:547–558Google Scholar
  40. Tourneur Y, Mitra R, Morad M (1984) Activation of the inwardly rectifying K+ current in guinea pig isolated atrial and ventricular cells. Biophys J 45:136aGoogle Scholar
  41. Tung L, Morad M (1983) Voltage- and frequency-dependent block of diltiazem on the slow inward current and generation of tension in frog ventricular muscle. Pflügers Arch 398:189–198Google Scholar
  42. Tung L, Morad M (1984) Evaluation of the single suction pipette voltage clamp technique in frog ventricular myocytes. Biophys J 45:279aGoogle Scholar
  43. Undrovinas AI, Yushmanova AV, Hering S, Rosenshtraukh LV (1980) Voltage clamp method on single cardiac cells from adult rat heart. Experientia 36:572–574Google Scholar
  44. Vahouny GV, Wei RW, Tamboli A, Albert EN (1979) Adult canine myocytes: isolation, morphology and biochemical characteristics. J Mol Cell Cardiol 11:339–357Google Scholar
  45. Weidmann S (1956) Shortening of the cardiac action potential due to a brief injection of KCl following the onset of activity. J Physiol 132:157–163Google Scholar

Copyright information

© Springer-Verlag 1985

Authors and Affiliations

  • Leslie Tung
    • 1
  • Martin Morad
    • 1
  1. 1.Department of PhysiologyUniversity of PennsylvaniaPhiladelphiaUSA

Personalised recommendations