G-protein-gated potassium channels containing Kir3.2 and Kir3.3 subunits mediate the acute inhibitory effects of opioids on locus ceruleus neurons

Abstract

Acute opioid administration causes hyperpolarization of locus ceruleus (LC) neurons. A G-protein-gated, inwardly rectifying potassium (GIRK/K(G)) conductance and a cAMP-dependent cation conductance have both been implicated in this effect; the relative contribution of each conductance remains controversial. Here, the contribution of K(G) channels to the inhibitory effects of opioids on LC neurons was examined using mice that lack the K(G) channel subunits Kir3.2 and Kir3.3. Resting membrane potentials of LC neurons in brain slices from Kir3.2 knock-out, Kir3.3 knock-out, and Kir3.2/3.3 double knock-out mice were depolarized by 15-20 mV relative to LC neurons from wild-type mice. [Met](5)enkephalin-induced hyperpolarization and whole-cell current were reduced by 40% in LC neurons from Kir3.2 knock-out mice and by 80% in neurons from Kir3.2/3.3 double knock-out mice. The small opioid-sensitive current observed in LC neurons from Kir3.2/3.3 double knock-out mice was virtually eliminated with the nonselective potassium channel blockers barium and cesium. We conclude that the acute opioid inhibition of LC neurons is mediated primarily by the activation of G-protein-gated potassium channels and that the cAMP-dependent cation conductance does not contribute significantly to this effect.

Fig. 1.

Generation of Kir3.3 knock-out mice.A, Depictions of the mouse Kir3.3 gene, targeting vector, and recombination events. Kir3.3 exons are represented as black rectangles. Vertical arrows indicate the positions of loxP sites.B, BamHI restriction enzyme site;NEO, neomycin resistance gene cassette;DTA, diphtheria toxin gene cassette.B, Predicted membrane topology of the Kir3.3 subunit. The domain of shaded circles represents the coding sequence eliminated in the constitutive null Kir3.3gene. N, N-terminal end of the Kir3.3 subunit;C, C-terminal end of the Kir3.3 subunit. C, Southern blot analysis of ES cell clones derived after transfection of an ES cell line carrying the targeted Kir3.3 allele with the Cre recombinase gene. Cre-transfection promoted the excision of DNA found between two loxP sites. G418 sensitivity was used to screen for the transfected cells that lacked the NEO cassette. Genomic DNA from G418-sensitive clones was digested with BamHI and probed with a radiolabeled fragment corresponding to thestriped rectangle shown in A. Several derivatives harboring the constitutive null [knock-out (KO)] and floxed versions of the Kir3.3gene were isolated; for this study, chimeric animals were generated using cells from lines 60 and 66. WT, Wild-type.

Fig. 2.

Kir3 protein levels in wild-type and knock-out mouse brains. Membrane proteins from wild-type (WT), Kir3.2 knock-out (2 ko), Kir3.3 knock-out (3 ko), and Kir3.2/3.3 double knock-out (2/3 ko) mouse brains were electrophoresed and probed using anti-Kir3 antibodies. Loading consistency was evaluated using an anti-NMDA receptor subunit (NR1) antibody. Kir3.1 appears as a doublet, with the two bands representing differentially glycosylated versions of the core polypeptide (Krapivinsky et al., 1995). Note that the heavily glycosylated form of Kir3.1 is most affected by the ablation of Kir3.2 and/or Kir3.3. Ab, Primary antibody.

Fig. 3.

Average resting membrane potentials of LC neurons from wild-type (−60.2 ± 1.0 mV), Kir3.3 knock-out (3 ko) (−36.9 + 4.6 mV), Kir3.2 heterozygous (2 het) (−45.2 ± 2.7 mV), Kir3.2 heterozygous/Kir3.3 knock-out (2 het/3 ko) (−40.4 ± 2.0 mV), Kir3.2 knock-out (2 ko) (−45.5 ± 3.2 mV), and Kir3.2/3.3 double knock-out (2/3 ko) (−38.8 ± 2.9 mV) mice. The number of independent experiments for each group is shown at thetop of each column; values shown are means ± SEM. *p < 0.001 (unpaired two-tailed ttest).

Fig. 4.

Effect of ME (30 μm) on LC neurons in coronal brain slices from wild-type mice. Representative recordings of the hyperpolarization (A) and outward current (B) induced by a brief application of ME in an LC neuron from a wild-type mouse are shown. C, Whole-cellI–V relationships in LC neurons before (control; open squares) and during the perfusion of ME (filled squares). D,I–V plots of the ME-induced current (I-ME) in 2.5 mm (filled circles) and 6.5 mm (open circles) extracellular potassium. I-ME was calculated by subtracting the current obtained before ME application from the current obtained in the presence of ME. The reversal potential for I-ME was −111 ± 2.1 mV in 2.5 mm bath potassium and −89 ± 4.1 mV in 6.5 mm bath potassium. The reversal potentials were corrected for the liquid junction potential (+3.3 mV). The holding potential was −60 mV. Values shown are means ± SEM.

Fig. 5.

Summary of the ME-induced effects observed in LC neurons from wild-type and Kir3 knock-out mice. A, Individual traces and summary bar graphs representing the hyperpolarization observed in LC neurons from wild-type and Kir3 knock-out mice during application of 30 μm ME. In neurons from Kir3.2 knock-out and Kir3.2/3.3 double knock-out mice, the hyperpolarizing effect of ME was smaller than in wild-type mice (*p < 0.05; **p < 0.01). Statistically significant differences were also observed between the Kir3.2 knock-out and Kir3.2/3.3 double knock-out groups (⁺p < 0.05). B, Individual traces and summary bar graphs representing the amplitudes of the ME-induced (I-ME) current in LC neurons from wild-type and Kir3 knock-out mice. The holding potential was −60 mV. ME-induced currents observed in Kir3.2 heterozygous/Kir3.3 knock-out (2 het/3 ko), Kir3.2 knock-out (2 ko), and Kir3.2/3.3 double knock-out (2/3 ko) neurons were significantly reduced relative to LC neurons from wild-type mice (**p < 0.01). Moreover, ME-induced currents observed in Kir3.2 knock-out and Kir3.2/3.3 double knock-out mice were significantly different (⁺⁺p < 0.01), as were currents observed in Kir3.2 heterozygous/Kir3.3 knock-out (3 ko) and Kir3.2 heterozygous (2 het) groups (⁺p < 0.05). The number of independent experiments for each group in A–C is shown at the top of each column. Values shown are means ± SEM.

Fig. 6.

Reduction of the ME-induced current in LC neurons from Kir3.2/3.3 double knock-out mice. A,I–V relationship of ME-induced current (I-ME) in 2.5 mm (n = 44; open circles) and 6.5 mm(n = 6; filled circles) bath potassium. B, With a cesium-based pipette solution and by adding barium (1 mm) to the bath solution, I-ME in LC neurons from Kir3.2/3.3 double knock-out mice decreased between −80 mV and −50 mV (open squares) compared with currents measured in cesium- and barium-free conditions (filled squares). The I–V plots were obtained from neurons from the same double knock-out mouse. C, Summary of the experiments shown in B displaying I-ME at −60 mV in the absence (n = 6; filled bar) and presence (n = 4; open bar) of barium and cesium. Values shown are means ± SEM. *p < 0.05.

Fig. 7.

The opioid-induced inhibition of voltage-gated calcium channels is normal in LC neurons from Kir3 knock-out mice.A, Example of the inward calcium/barium current elicited by a voltage step from −60 to −20 mV in an LC neuron from a Kir3.2/Kir3.3 double knock-out mouse (control), and its inhibition during ME perfusion. B, Summary of the calcium/barium current inhibition by ME observed in wild-type and Kir3 knock-out mice. Values shown are means ± SEM, and the number of cells tested in each group shown at the top of each column. 3 ko, Kir3.3 knock-out; 2 het, Kir3.2 heterozygous; 2 het/3 ko, Kir3.2 heterozygous/Kir3.3 knock-out; 2 ko, Kir3.2 knock-out;2/3 ko, Kir3.2/3.3 double knock-out.