Analysis and use of the perforated patch technique for recording ionic currents in pancreatic beta-cells

Abstract

We have used the nystatin perforated patch technique to study ionic currents in rat pancreatic beta-cells. The access resistance (Ra) between the pipette and the cell cytoplasm, measured by analyzing capacitive currents, decreased with a slow exponential time course (tau = 5.4 +/- 2.7 min) after seal formation. As Ra decreased, the magnitude of voltage-dependent K and Ca currents increased with a similar time course, and their activation kinetics became faster. After Ra stabilized, the macroscopic currents remained stable for up to an hour or more. When the final Ra was sufficiently low, Ca tail currents could be resolved which had properties similar to those recorded with the classical whole-cell technique. Two types of K channels could be characterized with perforated patch recordings of macroscopic K currents: (i) ATP-blockable K (KATP) channels which generate a time and voltage independent current that is blocked by glyburide and enhanced by pinacidil and (ii) voltage-dependent K (Kv) channels. Whole-cell recordings of KATP currents in the absence of ATP in the pipette showed that the maximum KATP conductance of the beta-cell was 83.8 +/- 40 nS. Perforated patch recordings show that the resting KATP conductance is 3.57 +/- 2.09 nS, which corresponds to about 4% of the channels being open in the intact beta-cell. In classical whole-cell recordings. Kv activation kinetics become faster during the first 10-15 min of recording, probably due to a dissipating Donnan potential. In perforated patch recordings where the Donnan potential is very small, Kv activation kinetics were nearly identical to the steady-state whole cell measurements.