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

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We have used the nystatin perforated patch technique to study ionic currents in rat pancreatic β-cells. The access resistance (R a) between the pipette and the cell cytoplasm, measured by analyzing capacitive currents, decreased with a slow exponential time course (τ=5.4±2.7 min) after seal formation. AsR a decreased, the magnitude of voltage-dependent K and Ca currents increased with a similar time course, and their activation kinetics became faster. AfterR a stabilized, the macroscopic currents remained stable for up to an hour or more. When the finalR a 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 (K v ) channels. Whole-cell recordings of KATP currents in the absence of ATP in the pipette showed that the maximum KATP conductance of the β-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 β-cell. In classical whole-cell recordings. K v 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, K v activation kinetics were nearly identical to the steady-state whole cell measurements.

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Sala, S., Parsey, R.V., Cohen, A.S. et al. Analysis and use of the perforated patch technique for recording ionic currents in pancreaticβ-cells. J. Membrain Biol. 122, 177–187 (1991). https://doi.org/10.1007/BF01872640

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Key Words

  • perforated-patch
  • nystatin
  • patch clamp
  • β-cell
  • K channel
  • Ca channel