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Am J Physiol Cell Physiol 271: C524-C532, 1996;
0363-6143/96 $5.00
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AJP - Cell Physiology, Vol 271, Issue 2 C524-C532, Copyright © 1996 by American Physiological Society

ARTICLES

Perforated patch-clamp recording in cardiac myocytes using cation-selective ionophore gramicidin

Y. Tajima, K. Ono and N. Akaike
Department of Physiology, Kyushu University Faculty of Medicine, Fukuoka, Japan.

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.


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