Cell-attached patch clamp measurement of macroscopic rapid inward sodium current in cultured heart cell reaggregates

A megaohm seal, cell-attached patch clamp technique utilizingplastic suction pipettes for measuring macroscopic rapid inward sodium current ( I Na ) was applied to cell membrane patches 30 to 100 μm 2 in area at the periphery of reaggregates of myocardial cells from newborn rats cultured for 3 to 7 days. The reaggregates were composed of about 20 to several thousand of electrically well-coupled cells, the latter forming spheroidal reaggregates with a diameter of maximally 300 μm. This system was shown to guarantee satisfactory voltage-clamp control during ionic current measurements. The time and voltage dependence of the I Na recorded during periods of up to 90 min was similar to that observed in single cardiac cell preparations from adult rats. I Na inactivation was described by two time constant ( tau h1 , and tau h2 ). For maximal I Na tau h1 and tau h2 had values of 1.5±0.25 ms and 8.7±3.9 ms ( n =4), respectively. The time course for recovery of I Na from inactivation at the reaggregate resting potential exhibited also two time constants ( tau re1 =9.8±3 ms, tau re2 =193±50 ms, n =5). Estimated current density was about 10 pA/μm 2 . The concentration of tetrodotoxin needed to reduce maximal I Na by 75% was 85 times lower in the reaggregates than it was in freshly isolated heart muscle cells from adult rats. The present system offers a combination of features that should make it well-suited for the study of both short- and long-term effects on the sodium channels in neonatal heart cells: Easy handling of the object, great electric stability, high time and amplitude resolution during ionic current measurements, and viability for at least one week.