Giant excised cardiac sarcolemmal membrane patches: sodium and sodium-calcium exchange currents

A tightly-sealed cardiac sarcolemmal patch preparation of large diameter (10-16 microns with 5-25 G delta seals) has been developed to study regulation of selected electrogenic mechanisms. Formation of a large readily accessible membrane surface area is achieved by incubating freshly isolated myocytes in a high KCl/zero calcium solution, which promotes separation of sarcolemma from myofilaments. The formation of large-diameter, high resistance seals is facilitated by depositing a neutral hydrocarbon film on electrode tips. Sodium currents in excised patches are stable for patch life-times of 15-40 min with peak current densities of 120 to 350 microA/cm2. Outward sodium-calcium exchange current is identified by its specific dependence on external calcium and internal sodium, inhibition by cobalt and dichlorobenzamil, and activation by less than 0.1 microM internal free calcium. Maximum exchange current density is greater than 20 microA/cm2.