(A) Targeted disruption of the mouse GIRK2 gene in ES cells and mice. The genomic structure and restriction map of the mouse GIRK2 gene locus and targeting vectors pPNT-76 used to disrupt the GIRK2 gene are shown. Shaded boxes represent coding sequences of the exons (boxes). The probe 3′ of the deletion is used for Southern blot analysis and shown as an open bar. B, BglII; A, AccI; E, EcoRI; X, XbaI. (B) Southern analysis of transfected ES cells. Lanes: 1, targeted clone containing the targeted allele, as assessed by the presence of an additional 1.9-kb BglII fragment; 2: parental clone containing the normal 1.6-kb fragment; 3–9, genotypes from tail biopsies of GIRK2 +/+, +/−, and −/− mice. (C) Reverse transcriptase (RT)-PCR analysis from brain mRNA of GIRK2 +/+, +/−, and −/− mice. (Upper) GIRK2 wild-type and mutant alleles are shown with the position of oligonucleotides used as PCR primers. D, deleted region into which the pgk-neomycin resistance cassette was inserted. No primer-pair amplified products in the absence of reverse transcriptase. Each sample started with an equal amount of cDNAs. A 5′-truncated mRNA terminating in pgk-neomycin can be detected by PCR at reduced levels in +/− and −/− animals. The sequences of the oligonucleotide used are available upon request.

(A) Western blots of membrane prepared from GIRK2 +/+, +/−, and −/− mouse brains show that both GIRK2 and GIRK1 protein levels are reduced in the GIRK2 knockout mice. (B) RT-PCR showing equal mRNA expression of GIRK1 in +/+, +/−, and −/− mouse brain. No product is amplified by any of the four pairs of primers in the absence of reverse transcriptase (−/− −RT; lane 4), confirming that all products were amplified from cDNA rather than contaminating genomic DNA. Hypoxanthine phosphoribosyltransferase (HPRT) primers amplify a comparable level of product in all samples, indicating that the same amount of template is present. Amplified products by GIRK1, GIRK4, IRK1, and HPRT primers are of expected sizes.

(A) Coronal sections of brain from GIRK2 +/+ and −/− mouse brains stained with antibodies against the N terminus of GIRK2, the N terminus of GIRK1, and the C terminus of IRK1 show that there is no detectable GIRK2 and dramatically reduced GIRK1 immunoreactivity in the GIRK2 −/− mice. The IRK1 staining patterns are the same for these mice. CTX, cerebral cortex; HP, hippocampus; Th, thalamus. (Bar = 10 mm.) (B) Sagittal views of cerebella from GIRK2 +/+ and −/− mice stained with the antibodies described in A. Although there is no detectable GIRK2 protein in the −/− mice, there is still significant GIRK1 staining, whereas the level of IRK1 expression appears to be the same. g, granule cell layer; m, molecular layer. (Bar = 20 mm.) (C) Coronal sections of ventral midbrain from +/+, −/−, wv/−, and wv/wv mice are stained with antibodies against TH and the N terminus of GIRK2. The TH staining of +/+, −/−, and wv/− midbrain appears similar, whereas there are fewer TH-positive neurons in the substantia nigra pars compacta (SNc) of the wv/wv midbrain. GIRK2 immunoreactivity is absent in the −/− midbrain but can still be found in the wv/− and wv/wv mice, although the dendritic staining is dramatically reduced. SNr, substantia nigra pars reticulata; VTA, ventral tegmental area. (Bar = 1 mm.) (D) High magnification views of parasagittal cerebellar sections from +/+, −/−, wv/−, and wv/wv mice. The sections are counterstained with toluidine blue or stained with antibody against the C terminus of IRK1, which stains the cell body and dendrites of the Purkinje cells as well as the dendrites in the granule cell layer. The wv/− cerebellum is mostly wild-type in appearance except for regions with a Purkinje cell layer more disorganized and broader than that in GIRK2 +/+ and −/− mice. Some Purkinje cells and their dendrites can be found in the granule cell layer of wv/− mice. In the wv/wv cerebellum, there is no granule cell layer, and the Purkinje cells with disorganized dendrites are scattered throughout the cerebellum. m, molecular layer; p, Purkinje cell layer; g, granule cell layer; p/g, cell layer where both Purkinje cells and granule cells are found; m/p, cell layer where molecular layer and Purkinje cell layers collapse into one in the absence of granule cell layer; wm, white matter. (Bar = 0.2 mm.)

Susceptibility of GIRK2-deficient mice to PTZ-induced seizures. (A) Response of mice receiving one injection of 50 mg/kg PTZ i.p. (0, no response; 1, isolated twitches; 2, tonic-clonic convulsions; 3, tonic extension and/or death). GIRK2 −/− mice (n = 16) tend to progress to more severe stages than +/+ or +/− mice (n = 13 and 12, respectively; P < 0.004, Mann–Whitney U–Wilcoxon rank sum test). No statistically significant difference was observed between +/+ and +/− animals. (B) Seizure latency. The PTZ seizure latency was defined as the time elapsed from PTZ injection to the first obvious sign of tonic-clonic convulsion or tonic extension. The latency to seizures was shorter for the −/− mice (∗, P < 0.002, unpaired test).