Perforated patch-clamp recording in cardiac myocytes using cation-selective ionophore gramicidin.
Whole cell currents were recorded in single myocytes dissociated from guinea pig ventricles by the patch-clamp technique. The addition of 0.1 mg/ml gramicidin D, a cation-selective ionophore, into the pipette solution induced a gradual spontaneous perforation of the patch membrane under a conventional cell-attached configuration. The access resistance, measured at approximately 12 min after formation of a gigaohm seal, was 9.2 +/- 1.5 M omega (n = 12). The perforated patch membrane exhibited ionic selectivity for various monovalent cations, with a relative order of Cs+ (1.11) > K+ (1.0) > Na+ (0.65) >> tris(hydroxymethyl)aminomethane+ (approximately 0) but was not permeable for Cl-. Under the gramicidin-perforated patch recording configuration, the cells showed the typical electrophysiological properties for ventricular cells reported previously. The intracellular Cl- concentration, estimated from the reversal potential of the catecholamine-induced Cl- current, was 36.3 +/- 2.9 mM (n = 17). We thus conclude that the gramicidin-perforated patch recording mode provides a useful tool for recording the ionic currents while maintaining the intracellular Cl- concentration.
Tajima
Y
Y
Department of Physiology, Kyushu University Faculty of Medicine, Fukuoka, Japan.
Ono
K
K
Akaike
N
N
D000200:Action Potentials; D000818:Animals; D002412:Cations; D002712:Chlorides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004553:Electric Conductivity; D006096:Gramicidin / Q000494:pharmacology*; D006168:Guinea Pigs; D006321:Heart / Q000187:drug effects* / Q000502:physiology*; D007476:Ionophores / Q000494:pharmacology; D008566:Membranes / Q000502:physiology; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D009994:Osmolar Concentration; D018408:Patch-Clamp Techniques
D002412:Cations; D002712:Chlorides; D007476:Ionophores; D006096:Gramicidin