AJP - Heart and Circulatory Physiology, Vol 255, Issue 4 980-H984, Copyright © 1988 by American Physiological Society
A new oil-gate concentration jump technique applied to inside-out patch-clamp recording
D. Y. Qin and A. Noma
Department of Physiology, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
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.
