IRK(1-3) and GIRK(1-4) inwardly rectifying K+ channel mRNAs are differentially expressed in the adult rat brain

Abstract

Molecular cloning together with functional characterization has shown that the newly identified family of inwardly rectifying K+ channels consists of several closely related members encoded by separate genes. In this report we demonstrate the differential mRNA expression and detailed cellular localization in the adult rat brain of seven members of the IRK and GIRK subfamilies. Using both radiolabeled cRNA riboprobes and specific oligonucleotide probes directed to nonconserved regions of both known and newly isolated rat brain cDNAs, in situ hybridization revealed wide distribution with partly overlapping expression of the mRNAs of IRK1-3 and GIRK1-4. Except for the low levels of GIRK4 transcripts observed, the overall distribution patterns of the other GIRK subunits were rather similar, with high levels of expression in the olfactory bulb, hippocampus, cortex, thalamus, and cerebellum. Marked differences in expression levels existed only in some thalamic, brainstem, and midbrain nuclei, e.g., the substantial nigra, superior colliculus, or inferior olive. In contrast, IRK subunits were expressed more differentially: all mRNAs were abundant in dentate gyrus, olfactory bulb, caudate putamen, and piriform cortex. IRK1 and IRK3 were restricted to these regions, but they were absent from most parts of the thalamus, cerebellum, and brainstem, where IRK2 was expressed predominantly. Because channel subunits may assemble as heteromultimers, additional functional characterization based on overlapping expression patterns may help to decipher the native K+ channels in neurons and glial cells.

Fig. 1.

X-ray film images of sagittal rat brain sections show distribution of mRNAs as detected by in situhybridization with oligonucleotide probes specific for IRK1 (A), IRK2 (B), IRK3 (C), GIRK1 (E), GIRK2 (F), GIRK3 (G), and GIRK4 (H). D, Control section hybridized with sense probe. Scale bar (shown in H): 5 mm. Exposure times are 8–21 d.

Fig. 2.

Dark-field photomicrographs of adjacent sagittal sections through the rat hippocampal region hybridized with oligonucleotides specific for IRK1 (A), IRK2 (B), IRK3 (C), GIRK1 (D), GIRK2 (E), and GIRK3 (F). CA1, CA3, Pyramidal cell layer of the CA1 and CA3 fields of the Ammon’s horn; DG, granule cell layer of the dentate gyrus; PoDG, polymorphic layer of the dentate gyrus. Scale bar (shown in F): 300 μm.

Fig. 3.

Dark-field photomicrographs of adjacent coronal sections through the parietal cortex hybridized with oligonucleotides specific for GIRK1 (A), GIRK3 (B), and IRK1 (C). D, Adjacent Nissl-stained section showing distribution of cells in cortical layers I–V. Scale bar (shown inD): 150 μm.

Fig. 4.

(A, C) Dark-field photomicrographs of sections through the caudate putamen hybridized with oligonucleotides specific for IRK1 (A) and IRK2 (C). Bright-field high-power photomicrographs in B and Ddemonstrate expression of IRK1 in most cells except the large neurons (B), whereas IRK2 mRNA is expressed predominantly by this population of large cells (D). Scale bars: A, C, 150 μm; B, D, 15 μm.

Fig. 5.

Dark-field photomicrographs of adjacent sections through the region of the dorsal lateral geniculate of the rat thalamus hybridized to (A) IRK1-, (B) IRK2-, (C) GIRK1-, and (D) GIRK3-specific oligonucleotides. Scale bar (shown in D): 150 μm.

Fig. 6.

Dark-field photomicrographs of adjacent sagittal sections through the rat SN and VTA hybridized to oligonucleotides specific for GIRK2 (A), GIRK3 (B), and IRK3 (C). Note the intense labeling of putatively dopaminergic neurons with GIRK2 mRNA. VTA, Ventral tegmental area;SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata. Scale bar (shown in C): 150 μm.

Fig. 7.

Dark-field photomicrographs of adjacent sections through the rat upper brainstem region hybridized to oligonucleotides specific for IRK2 (A), IRK3 (B), and GIRK3 (C). For orientation, part of a cerebellar lobe (Cb) is shown in the upper left corner. IRK2 mRNA expression is restricted to the motor trigeminal nucleus (Mo5), GIRK3 mRNA is abundantly expressed throughout the brainstem region, and IRK3 is completely absent from this area. Scale bar (shown in C): 300 μm.

Fig. 8.

Dark-field photomicrographs of coronal sections through the lower brainstem. Note that neurons in the inferior olive are intensely labeled with IRK2 (A), moderately express GIRK3 mRNA (B), and do not express GIRK1 mRNA (C). Scale bar (shown in C): 300 μm.