Bard J et al. (2000), Single channel studies of inward rectifier pota...

XB-ART-10062

J Gen Physiol 2000 Nov 01;1165:645-52.

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Single channel studies of inward rectifier potassium channel regulation by muscarinic acetylcholine receptors.

Bard J , Kunkel MT , Peralta EG .

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Negative regulation of the heartbeat rate involves the activation of an inwardly rectifying potassium current (I(KACh)) by G protein-coupled receptors such as the m2 muscarinic acetylcholine receptor. Recent studies have shown that this process involves the direct binding of G(betagamma) subunits to the NH(2)- and COOH-terminal cytoplasmic domains of the proteins termed GIRK1 and GIRK4 (Kir3.1 and Kir3.4/CIR), which mediate I(KACh). Because of the very low basal activity of native I(KACh), it has been difficult to determine the single channel effect of G(betagamma) subunit binding on I(KACh) activity. Through analysis of a novel G protein-activated chimeric inward rectifier channel that displays increased basal activity relative to I(KACh), we find that single channel activation can be explained by a G protein-dependent shift in the equilibrium of open channel transitions in favor of a bursting state of channel activity over a long-lived closed state.

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Species referenced: Xenopus
Genes referenced: kcnj12 kcnj3 kcnj5

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	Figure 1. Schematic representation of inward rectifier channel topology. Sequence from GIRK1 is in light gray. Sequence from RB-IRK2 is in black. Chimera GR7.1 contains amino acids 1–84 and 290–501 of GIRK1 fused to amino acids 85–289 of RB-IRK2.
	Figure 3. Single channel recordings of GR7.1. (A) Excerpt from a recording of a single GR7.1 channel before addition of carbachol to the pipet. (B) Excerpt from a recording of the same patch after addition of carbachol to the pipet. Bars to the left of traces indicate the current level when the channel is closed. Insets show the indicated region of the trace with a magnified time scale.
	Figure 2. GIRK1/GIRK4 and GR7.1 currents are activated via similar membrane-delimited pathways. Cell-attached patches containing multiple channels from oocytes expressing GIRK1/GIRK4 (A), RB-IRK2 (B), or chimera GR7.1 (C), were held at −60 mV for 800 ms, and then depolarized to +60 mV for 800 ms every 5 s for the duration of the experiment. Carbachol was added to the bath at the indicated time by hand pipetting. Carbachol was perfused into the pipet by suction (see Materials and Methods) over the indicated time period. Each point on the larger plot represents the integrated current over the portion of the trace at one holding potential for that time point. The insets show representative traces from before the addition of carbachol to the bath (left), after addition to the bath (center), and after addition to the pipet (right). The transient increase in outward current during the perfusion reflects activation of the oocyte's endogenous stretch-activated channels by the suction applied to the pipet. D shows the average percent change in integrated current after carbachol addition to the bath and to the pipet for GR7.1 (filled bars, n = 5) and RB-IRK2 (open bars, n = 5); error bars represent SEM. Because there is virtually no current before addition of carbachol to the pipet for GIRK1/GIRK4, the percent change is undefined.
	Figure 4. Analysis of GR7.1 single channel parameters. (A–H) Dwell-time histograms from the channel shown in Fig. 3 before (left) and after (right) perfusion of the pipet with carbachol. Maximum likelihood fits to exponential functions are shown for each. All except the closed time histograms were fit to single exponentials. The closed time histograms were fit to sums of three exponentials. The fits of the Before Carbachol data are shown as a dotted line in the After Carbachol histograms for comparison. (I) Mean change in single channel properties after addition of carbachol to the pipet for 5 GR7.1 channels (filled bars) and 4 RB-IRK2 channels (open bars). Stars above bars indicate the significance level of the difference between the properties of each channel before and after carbachol treatment based on a paired t test. Stars above brackets indicate the significance of the difference between the percent changes for each of the two types of channel based on a Kolmogorov-Smirnov test. (*) P > 99%; () P > 95%; (*) P > 90%. Error bars represent SEM.
	Figure 5. Single channel behavior of GIRK1/GIRK4 with and without carbachol in the pipet. (A) A portion of a recording of a patch containing an unknown number of channels at −60 mV without carbachol in the pipet. Bars to the left of traces indicate current level where no channels are open. (B) A portion of a recording from a different patch containing a single GIRK1/GIRK4 channel at −60 mV with 10 μM carbachol in the pipet. (C and E) Closed and open dwell-time histograms from the experiment in A. (D and F) Closed and open dwell-time histograms from the recording in B. The histogram in E is fit to a single exponential function with τ = 2.1 ms. The histogram in F is fit to the sum of two exponential functions with τ1 = 0.7 ms and τ2 = 5.9 ms. The closed dwell-time histograms are shown to illustrate the increased presence of short, intraburst closings. Because the number of channels in each patch is unknown, the durations of longer closings (shown at the right of each histogram) do not have any significance.

References [+] :

Breitwieser, Uncoupling of cardiac muscarinic and beta-adrenergic receptors from ion channels by a guanine nucleotide analogue. , Pubmed