This chapter describes appropriate methods to investigate mammalian cardiac channels properties at the single channel level. Cell isolation is performed from new born or adult heart by enzymatic digestion on minced tissue or using the Langendorff apparatus. Isolation proceeding is suitable for rabbit, rat, and mouse hearts. In addition, isolation of human atrial cardiomyocytes is described. Such freshly isolated cells or cells maintained in primary culture are suitable for patch-clamp studies. Here we describe the single channel variants of the patch-clamp technique (cell-attached, inside-out, outside-out) used to investigate channel properties. Proceedings for the evaluation of biophysical properties such as conductance, ionic selectivity, regulations by extracellular and intracellular mechanisms are described. To illustrate the study, we provide an example by the characterization of a calcium-activated non-selective cation channel (TRPM4).
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The author is in indebted to Pr. Patrick Bois and Pr. Jacques Teulon for helpful comments on this manuscript and teaching in both tissue preparation and patch-clamp proceedings.
Langendorff O (1895) Untersuchungen am uberlebenden Saugethierherzen. Pflügers Arch 61:291–332CrossRefGoogle Scholar
Bouron A, Potreau D, Besse C, Raymond G (1990) An efficient isolation procedure of Ca-tolerant ventricular myocytes from ferret heart for applications in electrophysiological studies. Biol Cell 70(3):121–127PubMedCrossRefGoogle Scholar
Guinamard R, Sallé L, Simard C (2011) The non-selective monovalent cation channels TRPM4 and TRPM5. Adv Exp Med Biol 704:147–171PubMedCrossRefGoogle Scholar
Bell RM, Mocanu MM, Tellon DM (2011) Retrograde heart perfusion: the Langendorff technique of isolated heart perfusion. J Mol Cell Cardiol 50:940–950PubMedCrossRefGoogle Scholar
Guinamard R, Chatelier A, Demion M et al (2004) Functional characterization of a Ca2+-activated non-selective cation channel in human atrial cardiomyocytes. J Physiol 558:75–83PubMedCentralPubMedCrossRefGoogle Scholar
Demion M, Bois P, Launay P, Guinamard R (2007) TRPM4, a Ca2+-activated nonselective cation channel in mouse sino-atrial node cells. Cardiovasc Res 73:531–538PubMedCrossRefGoogle Scholar
Isenberg G, Klockner U (1982) Calcium tolerant ventricular myocytes prepared by pre-incubation in a “KB medium“. Pflugers Arch 395:6–18PubMedCrossRefGoogle Scholar
Jacobson SL, Piper HM (1986) Cell cultures of adult cardiomyocytes as models of the myocardium. J Mol Cell Cardiol 18:661–678PubMedCrossRefGoogle Scholar
Volz A, Piper HM, Siegmund B, Schwartz P (1991) Longevity of adult ventricular rat heart muscle cells in serum-free primary culture. J Mol Cell Cardiol 23:161–173PubMedCrossRefGoogle Scholar
Fares N, Gomez JP, Potreau D (1996) T-type calcium current is expressed in dedifferentiated adult rat ventricular cells in primary culture. C R Acad Sci III 319:569–576PubMedGoogle Scholar
Brette F, Blandin E, Simard C et al (2013) Epac activator critically regulates action potential duration by decreasing potassium current in rat adult ventricle. J Mol Cell Cardiol 57:96–105PubMedCrossRefGoogle Scholar
Simard C, Hof T, Keddache Z et al (2013) The TRPM4 non-selective cation channel contributes to the mammalian atrial action potential. J Mol Cell Cardiol 59:11–19PubMedCrossRefGoogle Scholar
Guinamard R, Rahmati M, Lenfant J, Bois P (2002) Characterization of a Ca2+-activated nonselective cation channel during dedifferentiation of cultured rat ventricular cardiomyocytes. J Membr Biol 188:127–135PubMedCrossRefGoogle Scholar
Hamill OP, Marty A, Neher E et al (1981) Improved patch-clamp techniques for high-resolution current recording from cell-free membrane patches. Pflugers Arch 391:85–100PubMedCrossRefGoogle Scholar
Guinamard R, Chatelier A, Lenfant J, Bois P (2004) Activation of the Ca2+-activated nonselective cation channel by diacylglycerol analogues in rat cardiomyocytes. J Cardiovasc Electrophysiol 15:342–348PubMedCrossRefGoogle Scholar
Hille B (1984) Selective permeabilities. Ionic channels of excitable membranes. Sinauer Associated Inc Publishers, Sunderland, MAGoogle Scholar
Nastuk WI, Hodkin AL (1950) The electrical activity of single muscles fibers. J Cell Comp Physiol 35:39–73CrossRefGoogle Scholar
Barry PH (1994) JPCalc, a software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurements. J Neurosci Methods 51:107–116PubMedCrossRefGoogle Scholar
Ng B, Barry PH (1995) The measurement of ionic conductivities and mobilities of certain less common organic ions needed for junction potential corrections in electrophysiology. J Neurosci Methods 56:37–41PubMedCrossRefGoogle Scholar
Benardeau A, Hatem SN, Rucker-Martin C et al (1996) Contribution of Na+/Ca2+ exchange to action potential of human atrial myocytes. Am J Physiol 271:H1151–H1161PubMedGoogle Scholar