A new oil-gate concentration jump technique applied to inside-out patch-clamp recording

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Abstract
A new method was developed to instantaneously replace the solution on the inner side of an inside-out membrane patch in order to measure time courses with which active substances acted on single ionic channels. Inside-out membrane patches were isolated from single ventricular cells of the guinea pig heart. The recording bath consisted of two chambers separated by a partition having a narrow slit. Mixing of two test solutions through this slit was prevented by filling it with paraffin oil. The pipette tip with a tightly sealed inside-out membrane patch was moved through the oil from one solution to the other so that the pipette tip was instantaneously exposed to a new solution. When the pipette tip was jumped between different K+ concentrations, the leak current through the membrane patch increased or decreased with a half time of 6.3 +/- 3.0 ms (n = 15). The amplitude of single K+ channel currents changed to a new steady level within approximately 20 ms. These time courses were well explained by diffusion of K+ in the dead space between the pipette tip opening and the membrane patch. An application of this method to the ATP-regulated K+ channel revealed a latent period of 1-2 s before the channel started its activity after the instantaneous removal of ATP, whereas no obvious latency was observed in the rapid suppression of the channel, which was completed in 100-300 ms after reapplying ATP.