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Fig 3. Single channel and whole-cell data reveal incomplete activation of GIRK1/2 by agonist compared to Gβγ.(A) Activity of GIRK1/2 in a cell-attached patch of an oocyte expressing the channel, m2R and Gβγ, without an agonist in the pipette. Right panel shows a 2 minutes segment of record, with zoom (below) on a shorter segment. The amplitude distribution histogram of the same 2 min-segment is shown on the right. Red line shows a two-component Gaussian fit. Isingle was determined as the difference between the fitted midpoints (μ) of the GIRK current peak on the right (μ2) and the left peak which corresponds to noise (μ1). (B) Activity of GIRK1/2 channels in a cell-attached patch of an oocyte expressing the channel and m2R and activated by 2 μM ACh present in the patch pipette. (Asterisks denote artifacts produced by capacity discharges of patch clamp headstage). The corresponding amplitude histogram of the 2 min-segment of the record is shown on the right. In A and B, GIRK1/2 was expressed at low densities (GIRK1, 10–50 pg RNA; GIRK2, 7–17 pg RNA) whereas RNAs of m2R (1–2 ng/oocyte) and Gβγ (5:1 ng/oocyte) were chosen to produce saturating concentrations of these proteins. Inward K+ currents are shown as upward deflections from zero level. In the traces shown, acquisition was at 20 KHz with 5 KHz analog filter. Very similar values of Isingle were obtained with 2 KHz filtering (not shown). (C) Single channel currents (left plot) are identical with either ACh or Gβγ. (D) Po is lower with ACh than with Gβγ (p = 0.029). Bars in C and D show mean±SEM, number of patches is shown above the bars. (E) Summary of whole-cell GIRK1/2 currents at three expression levels (densities). See Table 1 for details. (F) Left panel shows the Itotal/Iβγ ratios at three channel densities, calculated from data of Table 1. The right panel shows the fractional open probabilities of channels occupied by 0–4 Gβγ, same as in Fig 2C but in a simple graphic form. The red dotted lines are drawn to allow direct comparison of the experimental data from the left panel with the estimates of fractional Po from the right panel. Image published in: Yakubovich D et al. (2015) Image reproduced on Xenbase with permission of the publisher and the copyright holder. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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