Developmental regulation of G protein-gated inwardly-rectifying K+ (GIRK/Kir3) channel subunits in the brain.
- Authors
- Fernández-Alacid, Laura; Watanabe, Masahiko; Molnár, Elek; Wickman, Kevin; Luján, Rafael
- Year
- 2011
- Journal
- The European journal of neuroscience
- PMID
- 22098295
- DOI
- 10.1111/j.1460-9568.2011.07886.x
- PMCID
- PMC3936682
G protein-gated inwardly-rectifying K(+) (GIRK/family 3 of inwardly-rectifying K(+) ) channels are coupled to neurotransmitter action and can play important roles in modulating neuronal excitability. We investigated the temporal and spatial expression of GIRK1, GIRK2 and GIRK3 subunits in the developing and adult brain of mice and rats using biochemical, immunohistochemical and immunoelectron microscopic techniques. At all ages analysed, the overall distribution patterns of GIRK1-3 were very similar, with high expression levels in the neocortex, cerebellum, hippocampus and thalamus. Focusing on the hippocampus, histoblotting and immunohistochemistry showed that GIRK1-3 protein levels increased with age, and this was accompanied by a shift in the subcellular localization of the subunits. Early in development (postnatal day 5), GIRK subunits were predominantly localized to the endoplasmic reticulum in the pyramidal cells, but by postnatal day 60 they were mostly found along the plasma membrane. During development, GIRK1 and GIRK2 were found primarily at postsynaptic sites, whereas GIRK3 was predominantly detected at presynaptic sites. In addition, GIRK1 and GIRK2 expression on the spine plasma membrane showed identical proximal-to-distal gradients that differed from GIRK3 distribution. Furthermore, although GIRK1 was never found within the postsynaptic density (PSD), the level of GIRK2 in the PSD progressively increased and GIRK3 did not change in the PSD during development. Together, these findings shed new light on the developmental regulation and subcellular diversity of neuronal GIRK channels, and support the contention that distinct subpopulations of GIRK channels exert separable influences on neuronal excitability. The ability to selectively target specific subpopulations of GIRK channels may prove effective in the treatment of disorders of excitability.
Developmental and regional distribution of GIRK1 and GIRK2 subunits in the mouse brain. (A) GIRK protein distribution was visualized on histoblots of brain horizontal sections at various stages of postnatal development using affinity-purified anti-GIRK1 and anti-GIRK2 antibodies. The two GIRK channel subunits exhibited broad and overlapping distributions in the developing and adult brain. In particular, strong immunoreactivity for GIRK1 and GIRK2 was detected in the neocortex, cerebellum, hippocampus and thalamus, with the lowest intensity in the caudate putamen. (B and C) The histoblots were scanned and densitometric measurements from five independent experiments were averaged to compare the protein densities for each developmental time point. Error bars indicate SEM; *p < 0.001 compared with P60. Scale bar, 0.4 cm.
Developmental and regional distribution of the GIRK3 subunit in the rat brain. (A) GIRK protein distribution was visualized on histoblots of brain horizontal sections at various stages of postnatal development using affinity-purified anti-GIRK3 antibodies. GIRK3 exhibited a broad distribution pattern in the developing and adult brain. Strong immunoreactivity for GIRK3 was detected in the neocortex, hippocampus, thalamus and cerebellum, with the lowest intensity in the caudate putamen. (B and C) The developed histoblots were scanned and densitometric measurements from five independent experiments were averaged together to compare the protein densities for each age. In all brain regions analysed, as well as in all layers or subfields from each region, the GIRK3 protein increased from its lowest expression at P5 to a peak at P60. Error bars indicate SEM; *p < 0.001 compared with P60. Scale bar, 0.4 cm.
Regulation of GIRK1 expression in the brain of GIRK2 null mice. Protein distribution was visualized on histoblots of brain horizontal sections at P60 using affinity-purified anti-GIRK1 antibodies. The developed histoblots were scanned and densitometric measurements from five independent experiments were averaged together to compare the protein densities for each age. (A, C and E) The pattern of expression of GIRK1 observed in the wild-type (A) was consistently reduced in the brain of GIRK2 null mice (C). In all brain regions analysed, as well as in all layers or subfields from each region, the expression of GIRK1 was significantly reduced (E). (B, D and F) GIRK2 immunoreactivity was completely absent in the brain of GIRK2 null mice (D and F), demonstrating the specificity of the antibody. Error bars indicate SEM; *p < 0.001 compared with P60 in all cases. Scale bar, 0.5 cm.
Immunoreactivity for GIRK1, GIRK2 and GIRK3 in the hippocampus during postnatal development using a pre-embedding immunoperoxidase method. sp, stratum pyramidale; gc, granule cell layer; h, hilus; ml, molecular layer. Scale bar: A-C, 0.5 cm.
Developmental shift in the subcellular localization of GIRK1 and GIRK2. Electron micrographs of the CA1 region of the hippocampus showing immunogold particles for GIRK1 and GIRK2 during postnatal development, as detected using a pre-embedding method. GIRK1 and GIRK2 showed similar distribution patterns throughout development. At P5, immunoparticles for GIRK1 (A) and GIRK2 (E) were mainly associated with the endoplasmic reticulum (ER) in the cytoplasm (crossed arrows) of pyramidal cells. In the stratum radiatum, immunoparticles for GIRK1 (B) and GIRK2 (not shown) were mainly detected at intracellular sites (crossed arrows) in the dendritic shafts (Den) of pyramidal cells, and only a few immunoparticles were found along the plasma membrane (arrows). At P15, immunoparticles for GIRK1 (C) and GIRK2 (F) were detected along the plasma membrane (arrows) of dendritic spines (s) shafts (Den) and at intracellular sites (crossed arrows). A few immunoparticles for GIRK and GIRK2 were also found in axon terminals (at) establishing asymmetrical synapses with dendritic spines (s). At P60, immunoparticles for GIRK1 (D) and GIRK2 (G) were mainly detected at postsynaptic sites along the plasma membrane (arrows) of dendritic spines (s) shafts (Den, arrows) and at intracellular sites (crossed arrows) in associated with membranes in spines (s) and dendrites (Den). In less proportion, immunoparticles for GIRK and GIRK2 were also found in axon terminals (at) establishing asymmetrical synapses with dendritic spines (s). Quantitative analysis showing the percentage of immunoparticles for GIRK1 (H) and GIRK2 (I) at intracellular sites vs. plasma membrane (left histogram) and along the plasma membrane of postsynaptic compartments, such as dendrites, and spines vs. presynaptic compartments, such as axon terminals (right histogram), during postnatal development. Scale bars: A and F, 0.5 μm; B-D, F and G, 0.2 μm.
Electron micrographs of the CA1 region of the hippocampus showing immunogold particles for GIRK3 during postnatal development, as detected using a pre-embedding method. (A and B) At P5, immunoparticles for GIRK3 were mainly associated with the endoplasmic reticulum (ER) in the cytoplasm (crossed arrows) of pyramidal cells. In the stratum radiatum (B), immunoparticles for GIRK3 were mainly detected at presynaptic sites (arrowheads) in axon terminals (at) establishing asymmetrical synapses, as well as at intracellular sites in the dendritic shafts (Den) and spines (s) of pyramidal cells, and only a few immunoparticles were found along the plasma membrane (arrows). (C and D) At P15, immunoparticles for GIRK3 were detected along the plasma membrane (arrows) and intracellular sites (crossed arrows) of dendritic shafts (Den) and spines (s). GIRK3 immunoparticles were also observed in axon terminals (at) (arrowheads) establishing asymmetrical synapses with dendritic spines (s). (E-G) At P60, immunoparticles for GIRK3 were localized along the plasma membrane and intracellular sites of dendritic spines (s) and shafts (Den) (arrows), and along the plasma membrane of axon terminals (at) (arrowheads), in the stratum radiatum. In the stratum lucidum of the CA3 region, (G), GIRK3 immunoparticles were also observed in mossy fibres (at) (arrowheads) establishing asymmetrical synapses with dendritic spines (s) of pyramidal cells. (H) Quantitative analysis showing the percentage of immunoparticles for GIRK3 at intracellular sites vs. plasma membrane (top histogram) and along the plasma membrane of postsynaptic compartments, such as dendrites, and spines vs. presynaptic compartments, such as axon terminals (bottom histogram), during postnatal development. Scale bars: A, 0.5 μm; B-G, 0.2 μm.
Electron micrographs of the CA1 region of the hippocampus showing immunoparticles for GIRK1, GIRK2 and GIRK3 in the stratum radiatum, as detected using a postembedding immunogold method at P5, P15 and P60. (A-C) GIRK1 was never detected along the PSD of dendritic spines (s) of CA1 pyramidal cells establishing asymmetrical synapses with axon terminals (at), probably Schaffer collaterals, at any developmental age. Immunoparticles for GIRK1 were only found at extrasynaptic or perisynaptic sites (crossed arrows). (D-F) Immunoreactivity for GIRK2 at the PSD (arrows) was low at P5, increased at P15 and was high at P60. Immunoparticles for GIRK2 were also detected at extrasynaptic or perisynaptic sites (crossed arrows). (G-I) Immunoreactivity for GIRK3 at the PSD (arrows) was similar throughout postnatal development. Scale bar: 0.2 μm.
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