uKATP-1) in rat brain

Molecular Brain Research 74 Ž1999. 15–25 www.elsevier.comrlocaterbres

Research report

Localization of the ATP-sensitive potassium channel subunit žKir6.1ruK ATP-1 / in rat brain Ming Zhou a,) , Osamu Tanaka a , Masaki Sekiguchi a , Kou Sakabe a , Masahiko Anzai b, Ibuki Izumida b, Tomoko Inoue a , Katsumasa Kawahara b, Hiroshi Abe a a

Department of Morphology, Tokai UniÕersity School of Medicine, Bohseidai, Isehara, Kanagawa, 259-1193, Japan b Department of Physiology, Kitasato UniÕersity School of Medicine, Sagamihara, 228-0829, Japan Accepted 20 July 1999

Abstract The Kir6.1ruK ATP-1, subunit of ATP-sensitive Kq channels ŽK ATP ., was localized in adult rat brain by in situ hybridization and immunohistochemistry. The mRNA of this subunit was ubiquitously expressed in various neurons and nuclei of the adult rat brain. Interestingly, Kir6.1ruK ATP-1 mRNA was also expressed in glial cells. Kir6.1ruK ATP-1 protein staining gave a dispersed array of fine dots throughout all neurons and glial cells examined. Under electron microscope, the immunoreactive products were specifically restricted to the mitochondria. The present study indicates that this K ATP subunit is localized in the mitochondria and may play a fundamental role in vital brain function. q 1999 Elsevier Science B.V. All rights reserved. Keywords: ATP-sensitive potassium channel; Neuron; Glia; In situ hybridization; Immunohistochemistry; Electron microscopy

1. Introduction The ATP-sensitive Kq-channel ŽK ATP ., discovered originally by Noma w25x in the cardiac muscle, is regulated by intracellular ATP ŽATPi.. The current–voltage relation measured for the single K ATP shows inward rectification when the channel is exposed to roughly symmetrical Kqconcentrations with outward currents being significantly smaller than inward currents w5,25x. Later other cells such as smooth muscles w17x, neurons w22x, and even mitochondria w16x were found to have functional Kq channels. The Kq influx into mitochondria is mediated by the mitochondrial K ATP channel, which is regulated by the same biochemical and pharmacological mechanism as those in plasma membrane, although heteromultimer composition of mitochondrial K ATP channel remains to be elucidated w3,6x. Recently, a new K ATP subunit called Kir6.1ruK ATP-1 was cloned by Inagaki et al. w14,15x. This subunit is a 424-amino acid residue protein ŽMr s 47 960., and belongs to the inwardly rectifying Kq channel family, which is ) Corresponding author. Present address: Dept. of Anatomy, Akita University School of Medicine, Honndou 1-1-1, Akita, 010-8543, Japan. Fax: q81-018-884-6440; E-mail: [email protected]

characterized by two putative membrane spanning regions and one pore-forming region w15,21x. RNA blot analysis showed that this subunit was expressed ubiquitously in all tissues examined including brain w15x. To gain further insight into the function of Kir6.1ruK ATP-1, especially in brain, we performed in situ hybridization histochemistry and immunohistochemistry to analyze its cellular and subcellular localization in more detail. Kir6.1ruK ATP-1 mRNA and protein were widely expressed in most neuronal populations of adult rat brain, and interestingly, also in glial cells in the corpus callosum and cerebellar white matter. Electron microscopy showed that this channel subunit was restricted to the mitochondria, suggesting this subunit is a candidate of mitochondrial K ATP which has important roles in neuronal and glial functions. 2. Materials and methods 2.1. Animals and section preparation Ten male Wistar rats Ž6–10 weeks. of 180–240 g in weight were used. They were divided into two groups Ž5 in each., one for in situ hybridization histochemistry and another for immunohistochemistry. Under deep anesthesia

0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 2 3 2 - 6

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M. Zhou et al.r Molecular Brain Research 74 (1999) 15–25

by inhaling ether, those for immunohistochemistry were perfused through the left ventricle with 4% paraformaldehyde in 0.1 M phosphate buffered saline ŽPBS. at pH 7.3. The excised brains were placed in the same fixative for 4 h at 48C and subsequently transferred into 30% sucrose in PBS. For in situ hybridization histochemistry, brains were quickly removed and immediately frozen on powdered dry ice. Cryosections for both groups were cut at 10 to 20 mm thick and thaw-mounted on silane-coated glass slides, and stored at y808C until use. 2.2. Preparation of probes Antisense 45-mer oligonucleotides and cRNA probes specific to Kir6.1ruK ATP-1 were used for in situ hybridization. The antisense oligonucleotide, which was complementary to the nucleotide 1514–1558 of the 3X non-coding sequences of rat Kir6.1ruK ATP-1 cDNA w15x, was 3X end-labeled with w a-35 Sx dATP ŽNew England Nuclear. by terminal deoxynucleotidyl transferase ŽBethesda Research Labs, USA.. A sense oligonucleotide was also prepared for control study. Digoxigenin ŽDIG. labeled single strand riboprobes were prepared using a DIG RNA Labeling Kit ŽBoehringer Mannheim. according to the manufacturer’s instructions. In brief, template for synthesis of DIG-labeled RNA probes was generated by using PCR amplification from an adult rat brain cDNA library. Ž5X-GGCGACAGGTCCG ATACTTCGATCACCAGAACTCGACAAACTGTC-3X . and Ž3X-TCCATCTTGATTCAGACCCTCCAAAAGAGTAACTCGCACCAG5X . which were designed to amplify nucleotides 1266–1559 of Kir6.1ruK ATP-1 ŽGenbank accession number D42145. were used as 5X and 3X primer, respectively. PCR was with Taq polymerase ŽTakara. for 34 cycles of 948C = 30 s denaturation, 428C = 30 s annealing, and 728C = 1 min

extension each, with a final extension of 7 min at 728C. The amplified products were isolated and subcloned into pCRII cloning vector ŽInvitrogen. for generation of rat Kir6.1ruK ATP-1 riboprobes. This vector was either linearized with BamHI ŽBiolabs. and transcribed with T7 RNA polymerase ŽBoehringer Mannheim. to get antisense probe or linearized with EcoRI ŽNippon Gene. and transcribed with Sp6 RNA polymerase ŽBoehringer Mannheim. to get sense probe, in the presence of 350 mM DIG-linked UTP in a 20 ml reaction mixture. 2.3. In situ hybridization by oligonucleotide probe In situ hybridization with isotope-labeled oligonucleotide probes was carried out as described previously w32x. In brief, fresh frozen sections were fixed in 4% paraformaldehyde in sodium phosphate buffer ŽpH 7.2. for 15 min and rinsed in PBS containing 2 mgrml glycine for 20 min, then they were acetylated in 0.25% acetic anhydrider0.1 M triethanolamine ŽpH 8.0. for 10 min. Following dehydration through graded ethanol and air-drying, prehybridization was performed for 1 h at room temperature in the prehybridization buffer containing 50% deionized formamide, 4 = Standard Saline Citrate ŽSSC., 1 = Denhardt’s solution, 2% sodium N-lauroyl sarcosinate ŽSalkosyl., 0.1 M sodium phosphate buffer ŽpH 7.2., and 250 mgrml heat-denatured salmon sperm DNA. Hybridization was performed at 428C overnight under a Parafilm coverslip in a moisture chamber, in prehybridization buffer with 10% dextran sulfate, 0.1 mM dithiothreitol, and 35 S-labeled oligonucleotide probe Ž10 = 10 5 cpmrslide.. After hybridization, the sections were washed once in 2 = SSCr0.1% Sarkosyl at room temperature for 15 min and three times in 0.1 = SSCr0.1% Sarkosyl at 428C for 30 min. The sections were exposed to Hyperfilm b-max

Fig. 1. A sagittal section of adult rat brain in dark field showing that Kir6.1ruK AT P -1 mRNA was widely expressed in various neuronal populations by antisense oligonucleotide probe labeled with 35 S-dATP. Cb, cerebellar cortex; CPu, caudate putamen; Cx, neocortex; DG, dentate gyrus; H, hippocampus; Mi, mitral cell layer of olfactory bulb; Pn, pontine nucleus; Th, thalamus. Bar s 2 mm.

M. Zhou et al.r Molecular Brain Research 74 (1999) 15–25

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Fig. 2. Kir6.1ruK AT P -1 mRNA in olfactory bulb in dark field Ža. and bright field Žb.. Mitral cells Žarrows. and Tufted cells Žarrowheads. have strong expression. Bar s 50 mm.

ŽAmersham. for three weeks and were then dipped in NTB2 nuclear track emulsion ŽKodak.. After exposure for 6–10 weeks, the emulsion-covered sections were developed in D19 developer ŽKodak.. For identification and confirmation of brain structures with bright- and dark-field optics, the sections were counter-stained with hematoxylin. Brain structures were identified and confirmed according to Paxinos and Watson w26x. In order to verify the specificity of antisense oligonucleotide probe, brain sections were hybridized with a 100-fold excess of unlabeled oligonucleotide probes added to individual 35 S-labeled probe, or with the 35 S-labeled sense oligonucleotide. 2.4. In situ hybridization by cRNA probe In situ hybridization by using DIG-labeled riboprobe was carried out as described previously w11x with some modifications. Briefly, fresh frozen sections of adult rat brains were fixed with 4% paraformaldehyde for 15 min, and then digested with 10 mgrml proteinase K at room

temperature for 30 min. They were refixed in 4% paraformaldehyde for 10 min, then treated with 0.2 N HCl for 10 min for inactivation of internal alkaline phosphatase, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine pH 8.0 for 10 min. After dehydration with an ethanol series and air drying, hybridization was performed at 508C overnight Ž16 h. under a Parafilm coverslip, in a moisture chamber with the hybridization buffer, containing 50% deionized formamide, 10% dextran sulfate, 10 mM Tris–HCl pH 7.6, 200 mgrml salmon sperm DNA, 1 = Denhardt’s solution, 600 mM NaCl, 0.25% SDS, 1 mM EDTA, and approximately 0.1–0.5 mgrml cRNA probe. After rinsing and treatment with 50 mgrml RNase at 378C for 30 min, hybridized DIG-labeled probe was detected by Nucleic Acid Detection Kit ŽBoehringer Mannheim. according to the manufacturer’s instructions. After color reaction, sections were rinsed by 10 mM Tris–HCl pH 7.6, 1.0 mM EDTA, postfixed with 4% paraformaldehyde, rinsed with distilled water and mounted without counter staining. As a control, the same procedure was employed using sense cRNA probe.

Fig. 3. Kir6.1ruK AT P -1 mRNA is moderately expressed in hippocampus and intensely in dentate gyrus. Dark field Ža. and bright field Žb.. Bar s 50 mm.

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Fig. 4. Kir6.1ruK AT P -1 mRNA localization in the corpus callosum and in the CA1 of Ammon’s horn ŽCA1. in dark field Ža. and bright field Žb.. Note the small cells Žarrows. in the corpus callosum have weak expression, indicating that glial cells express Kir6.1ruK AT P -1. Bar s 50 mm.

2.5. Immunohistochemistry for light microscopy Cryosections were kept in PBS contained 0.3% Polyoxyethylene Ž20. sorbitan monolaurate Žequal to Tween 20. for 30 min. After preincubation with 5% normal goat serum for 1 h, the sections were incubated with rabbit anti-Kir6.1ruK ATP-1 antibody at a dilution of 1:100 ; 1:300 overnight Žabout 12 h. at room temperature. Detailed procedures for the preparation and specificity of the antibody were described elsewhere w30x. The sections were then treated with Vectastain ABC kit ŽVector Lab.. according to the manufacturer’s instructions. Reactivity was visualized by DAB Ž3,3X-diaminobenzidine tetrahydrochloride. reaction, and counterstained with methyl green. 2.6. Immunohistochemistry for electron microscopy The perfused brains were cut into 40 mm thick parasagittal sections with a Microslicer ŽDSK.. They were rinsed several times with PBS and treated with 0.03%

Polyoxyethylene sorbitan monolaurate in PBS for 1 h and preincubated in 5% normal goat serum for 1 h at room temperature. The sections were then incubated with antiKir6.1ruK ATP-1 antibody Ž1:100. overnight Žabout 12 h. at room temperature. After thorough rinsing in PBS, they were treated with Vectastain ABC kit ŽVector Lab.., and DAB was used as chromogen. The sections were post fixed with 1% OsO4 in PBS for 30 min, dehydrated in ethanol series, and embedded in Quetol-812.

3. Results 3.1. Localization of Kir6.1 r uK AT P-1 mRNA Kir6.1ruK ATP-1 mRNA, observed as silver grains, was found in somata of all neurons examined ŽFig. 1.. In addition, a faint level of grain accumulation was observed in small cells in the cerebellar white matter and corpus callosum.

Fig. 5. Kir6.1ruK AT P -1 mRNA in the cerebellum in dark field Ža. and bright field Žb.. The Purkinje cell layer ŽPCL. and granule cell layer ŽGCL. have moderate expression. Some neurons Žarrowheads. in the molecular layer ŽML. have weak expression. Weak expression could also be observed in the small cells in white matter Žarrows.. Bar s 25 mm.

M. Zhou et al.r Molecular Brain Research 74 (1999) 15–25 Table 1 Localization of mRNA and protein for uK AT P -1 in rat brain ", faint; q, weak; qq, moderate; qqq, intense.

Olfactory bulb External plexiform layer Internal granular layer Glomerular layer Mitral cell layer Tufted cell Cerebrum Anterior olfactory nucleus Olfactory tubercle Piriform cortex Amygdaloid nucleus Hippocampus CA1 CA2 CA3 Dentate gyrus Caudate-putamen Accumbens nucleus Globus pallidus Neocortex Corpus callosum Diencephalon Hypothalamus Paraventricular nucleus Supraoptic nucleus Mammillary nucleus Habenula medial nucleus lateral nucleus Pineal body Zona incerta Thalamus Mesencephalon Substantia nigra Red nucleus Central gray Superior colliculus Inferior colliculus Locus ceruleus Dorsal raphe nucleus Pons and medulla oblongata Pontine nucleus Pontine reticular nucleus Motor trigeminal nucleus Spinal trigeminal nucleus Spinal vestibular nucleus Lateral vestibular nucleus Locus ceruleus Medial vestibular nucleus Mesencephalic trigeminal nucleus Facial nucleus Vestibular nucleus Hypoglossal nucleus Gracile nucleus cuneate nucleus Inferior olivary nucleus Raphe magnus nucleus

mRNA

Protein

q " ;q q qqq qq

q " q qq qq

q qq q q

qq ;qqq qq q q

qq qq qq qqq q q " q ;qq "

q qq qq q q q " q ;qq q

q q " q

q q q q

q " ;q qq " ;q q

q q qq q q

q ;qq q ;qq q q q q qqq

q qq qq q q q qqq

qq ;qqq qq qq qq qqq qqq qqq qqq qqq

qqq qqq qqq qqq qqq qq qq qqq qqq

qq qq qq qq qq q ;qq q

qqq qqq qq qq qq qqq q

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Table 1 Žcontinued.

Cerebellum Purkinje cell Granule cell Basket cell Stellate cell Deep cerebellar nuclei White matter Artery Žsmooth muscle. Choroid Plexus Ependymal cell

mRNA

Protein

qq qq q q q ;qq " q q q

qq q q q qqq " qqq qq q

In the olfactory bulb, Kir6.1ruK ATP-1 mRNA was weakly expressed in external plexiform, internal granular, and glomerular layers, while moderate to intense level of expression were observed in Tufted and mitral cells ŽFig. 2a, b.. In the forebrain, weak expression was detected in the anterior olfactory nucleus, amygdaloid nucleus, caudate-putamen, and accumbens nucleus. Cells in the neocortex had weak to moderate expression, and olfactory tubercle had moderate mRNA expression. In the hippocampal formation, moderate expression was detected in the CA1– CA3 regions of the pyramidal cell layers of the hippocampus and intense expression was detected in the granule cell layer of the dentate gyrus ŽFig. 3a, b.. In diencephalon, expression was weak in the thalamus and hypothalamus, habenular nucleus, zona incerta, and was moderate in the pineal body. In addition, moderate expression was detected in the choroid plexus of the lateral and third ventricle. In the corpus callosum, faint expression was detected in small cells ŽFig. 4a, b.. In the midbrain and hindbrain, moderate to intense expression of Kir6.1ruK ATP-1 mRNA was noted in the pontine nucleus. There was moderate expression in the pontine reticular nucleus, motor and spinal trigeminal nuclei, facial nucleus, vestibular nuclei, hypoglossal nucleus, and gracile and cuneate nuclei. The inferior olivary nucleus, red nucleus and substantia nigra had weak to moderate expression, whereas most other nuclei had only weak expression. In the cerebellum, the Purkinje and granule cell layers moderately expressed Kir6.1ruK ATP-1 mRNA ŽFig. 5a, b.. In addition small neurons in the molecular layer had weak expression. Weak to moderate expression was noted in the deep cerebellar nuclei. In white matter of the cerebellum, faint grain accumulation was detected in the arbor vitae. No specific signals above the background levels were detected on control sections. The relative intensities for uK ATP-1 mRNA expression in different nuclei and regions of the brain are shown in Table 1. To verify the localization of Kir6.1ruK ATP-1 mRNA detected by isotope labeled oligonucleotide probe, DIGlabeled cRNA probe was used. The overall pattern of expression in the adult rat brain was similar to the results

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M. Zhou et al.r Molecular Brain Research 74 (1999) 15–25

observed with 35 S-labeled oligonucleotide probe, although there were some differences, such as in the granular cells of the olfactory bulb and cerebellum which had a weaker signal with the cRNA probe than with oligonucliotide probe. Purple staining was observed in virtually all neurons examined ŽFigs. 6–8. and in the corpus callosum ŽFig. 7.. Sense probe was used as a control, and gave no staining above background levels ŽFig. 9.. 3.2. Localization of Kir6.1 r uK AT P-1 protein Immunohistochemistry using anti-Kir6.1ruK ATP-1 antibody demonstrated that this channel subunit was widely expressed in neurons in whole brain, as well as small cells in the corpus callosum and cerebellar white matter. Overall distribution of the Kir6.1ruK ATP-1 immunoreactive cells was similar to that of Kir6.1ruK ATP-1 mRNA observed by in situ hybridization with the antisense oligonucleotide and cRNA probe. In the olfactory bulb, moderate immunoreactivity for Kir6.1ruK ATP-1 was detected in mitral cells and Tufted cells. Cells immunoreactive for Kir6.1ruK ATP-1 were also distributed in external and internal plexiform layers and the glomerular layer. The olfactory glomeruli were slightly immunopositive ŽFig. 10.. In the forebrain, weak to moderate immunoreactive cells were widely distributed in olfactory tubercle, piriform cortex, amygdaloid nucleus, caudate-putamen, accumbens nucleus, hippocampal formation, neocortex, hypothalamus,

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thalamus, and pineal body. In the hippocampal formation, pyramidal cells in CA2 and CA3 exhibited more intense immunoreactivity than those in CA1, with granular immunoreaction products being dispersed in their somata and thick apical dendrites ŽFig. 11.. In the dentate gyrus, the outer part of the granule cell layer was more intensely immunoreactive than the inner part ŽFig. 11.. In addition, small cells in the corpus callosum exhibited weak immunoreactivity ŽFig. 12.. Punctate immunoreaction products were observed in these small cells. In the midbrain and hindbrain, intense Kir6.1ruK ATP-1 immunoreactivity was detected in many neurons in the pontine nucleus, pontine reticular nucleus, motor and spinal trigeminal nuclei, mesencephalic trigeminal nucleus, facial nucleus, and inferior olivary nucleus. In the vestibular nuclear complex, neurons in the spinal and medial vestibular nuclei had intense immunoreactivity, and those in lateral vestibular nucleus had moderate immunoreactivity. Weak to moderate immunoreactive neurons were distributed in the substantia nigra, superior and inferior colliculi, locus ceruleus, dorsal motor nucleus of vagus, hypoglossal nucleus, gracile and cuneate nuclei, and raphe magnus nucleus. In the cerebellum, moderate immunoreactivity was observed in Purkinje cells ŽFig. 13. and neurons of the deep cerebellar nuclei, while granule cells, basket cells and stellate cells exhibited weak immunoreactivity. The neuronal somata of the deep cerebellar nuclei and Purkinje cells had punctate immunoreactivity. Moreover, in the

Fig. 6. Kir6.1ruK AT P -1 mRNA is detected in olfactory bulb by antisense cRNA probe. Moderate to intense expression can be seen in the mitral cell layer ŽMi.; and Tufted cells ŽTuf.. Bar s 50 mm. Fig. 7. A sagittal section through rat hippocampal formation showing Kir6.1ruK AT P -1 mRNA expression detected by antisense cRNA probe. Note moderate to intense expression in the hippocampal pyramidal cells and granule cells of the dentate gyrus ŽDG.. Weaker expression could be observed in the corpus callosum Žcc., indicating glial cells also contain this gene as shown in Fig. 4. Moderate to intense expression also can be seen in the Choroid Plexus Žarrow.. CA1–3, fields of CA1–3 of Ammon’s horn; H; hilus of dentate gyrus. Bar s 100 mm. Fig. 8. Kir6.1ruK AT P -1 mRNA expression in cerebellar cortex detected by antisense cRNA probe. Note the Purkinje cell layer ŽPCL. and Golgi cells Žarrows. express Kir6.1ruK AT P -1 moderately to intensely, while the granule cell layer ŽGCL. has only low expression Žcompare to oligonucleotide probe shown in Fig. 5.. Bar s 50 mm. Fig. 9. A sagittal section through cerebellum probed with sense cRNA probe as a control of Figs. 6–8. No significant levels of expression were detected. Bar s 100 mm. Fig. 10. Distribution of Kir6.1ruK AT P -1 immunoreactivity in the olfactory bulb of adult rat brain. Moderate immunoreactivity can be observed in the Tufted cells Žarrows., while glomerular layer of olfactory bulb ŽGl. shows faint reactivity. Bar s 50 mm. Fig. 11. A sagittal section of the hippocampal formation of adult rat. Note pyramidal cells in CA2 and CA3 exhibit more intense Kir6.1ruK AT P -1 immunoreactivity than those in CA1. In the dentate gyrus ŽDG., the outer part of the granule cell layer is more intensely immunoreactive than the inner part of the granule cell layer. Blood vessels Žarrowhead. show intense Kir6.1ruK AT P -1 immunoreactivity. CA1-3, fields of CA1-3 of Ammon’s horn; cc, corpus callosum; H, hilus of dentate gyrus. Bar s 100 mm. Fig. 12. Small cells Žarrows. in the corpus callosum exhibit weak immunoreactivity for Kir6.1ruK AT P -1, corresponding to the expression of mRNA shown in Figs. 4 and 7. Bar s 25 mm. Fig. 13. A part of cerebellar hemisphere showing distribution of Kir6.1ruK AT P -1 immunoreactivity. Note punctate immunoreaction products can be observed in the cytoplasm of the Purkinje cells ŽPCL., evidence for specific intracellular localization of Kir6.1ruK AT P -1. In the molecular layer ŽML., some stellate cells Žarrowheads. show weak immunoreactivity. In the granule cell layer ŽGCL. faint to weak immunoreactivity can be seen. Bar s 25 mm.

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Fig. 14. Electron micrograph of the cerebellar Purkinje cell immunoreactive for Kir6.1ruK AT P -1 in the adult rat brain. Immunoreaction products were located in the mitochondria ŽM.. No other cell organelles were positive immunoreaction to anti-Kir6.1ruK AT P -1, including Golgi complex ŽG. and endoplasmic reticulum Žarrows.. N, nucleus; Nu, nucleolus. Bar s 2 mm.

cerebellar white matter and inferior cerebellar peduncle, some small cells showed weak immunoreactivity for Kir6.1ruK ATP-1. Cells of the choroid plexuses had moderate immunoreactivity. and vascular smooth muscle cells of the pial surface of the brain had intense Kir6.1ruK ATP-1 immunoreactivity. Granular, punctate immunoreaction products were localized in virtually all immunopositive cells throughout the rat brain. The relative intensities of distribution of Kir6.1ruK ATP-1 protein are summarized in Table 1. To determine where this subunit is localized at the subcellular level, immunoelectron microscopy was performed. The specific labeling of Kir6.1ruK ATP-1 protein was localized in the mitochondria of the cerebellar Purkinje cells ŽFig. 14., granule cells ŽFig. 15., and neurons in the deep cerebellar nucleus Ždata not shown.. The mitochondria which were positive for anti-Kir6.1ruK ATP-1 antibody were dispersed in the neurons in the same manner as the punctate reaction products in neurons observed under light microscopy. Some mitochondria were stained intensely, some moderately, and some were negative ŽFig. 14.. Closer observation of these mitochondria which were immunoreactive to anti-Kir6.1ruK ATP-1 antibody, showed that the immunoreaction products were restricted inside the

mitochondria, suggesting that the Kir6.1ruK ATP-1 subunit is localized in the inner membrane of mitochondria, although the mitochondrial matrix was also stained, because of diffusion of the immunoreaction products near to cristae. No specific labeling was observed on the plasma and nuclear membranes, endoplasmic reticulum, or Golgi apparatus.

4. Discussion The present study demonstrated that the Kir6.1ruK ATP1 is widely and differentially expressed in various neuronal populations in adult rat brain. We also observed expression of this subunit in small cells in the corpus callosum and in the cerebellar white matter, showing for the first time that Kir6.1ruK ATP-1 subfamily proteins are also localized in glial cells, although Kir6.2 has been shown to be localized in glial cells by PCR analysis w19x. Such wide localization of Kir6.1ruK ATP-1 is in contrast to the relatively limited expression of other Kq-channels such as GIRK1 ŽG-protein gated inward rectifier Kq-channel. which is restricted to olfactory bulb, neocortex, hippocampus, and cerebellum w28x, IRK1 which is restricted in certain subsets of neurons, IRK2 which is limited in some brain regions, IRK3

M. Zhou et al.r Molecular Brain Research 74 (1999) 15–25

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Fig. 15. Electron micrograph of the granule cells of cerebellum. Positive reaction to anti-Kir6.1ruK AT P -1 antibody could be observed in the mitochondria ŽM.. Note the cristae Žarrow. clearly contain positive reaction products. N, nucleus of the granule cells. Bar s 500 nm.

which is restricted in the forebrain w12x and the ATP-dependent Kq-channel ŽK AB -2. expressed only in the glial cells w31x, suggesting that Kir6.1ruK ATP-1 may play important roles in neuronal and glial cell function. Karschin et al., w20x described that two mRNA transcripts ŽKir6.2 and SUR1. were extensively overlapped in the rodent brain and may form K ATP , and they suggested that Kir6.1 mRNA localized to small cells. In the present study, however, we found expressions for Kir6.1ruK ATP-1 mRNA in both small and large neurons, with large neurons showing more intense reactivity by in situ hybridization. This result was further confirmed by in situ hybridization with DIG-labeled cRNA probe and immunohistochemistry with anti-Kir6.1ruK ATP-1 antibody. Recently, hippocampal pyramidal neurons and interneurons in CA1 were also shown to contain Kir6.1 by a single cell RT-PCR technique w35x. Electron microscopy showed that the immunoreaction products for Kir6.1ruK ATP-1 were deposited in the mitochondria, strongly suggesting that this protein is a component of the mitochondrial K ATP ŽmitoK ATP .. This subunit may be located on the inner membrane of mitochondria, since the immunoreaction products were restricted inside the mitochondria. Suzuki et al. w30x reported that Kir6.1 was stained as a dispersed array of fine dots in the rat skeletal muscle and liver, and showed specific labeling of Kir6.1 in the mitochondrial inner membrane.

Inoue et al. w16x reported that mitochondria contain a Kq-channel which is blocked by ATP and glibenclamide by using the patch clamp technique on fused giant mitoplasts isolated from rat liver mitochondria. Beavis et al. w3x also showed that the Kq-channel sensitive to ATP and ATP-analogues is localized in the inner membrane of mitochondria. In the present study, we did not find any positive reaction for anti-Kir6.1ruK ATP-1 antibody in the plasma membrane, because the plasma membrane may not contain detectable concentrations of this protein or antigenic sites may be blocked w30x. In addition, we noticed that large neurons such as mitral cells and Tufted cells in the olfactory bulb, pyramidal cells in CA2 and CA3, neurons in the cerebellar nuclei or brain stem nuclei and Purkinje cells in the cerebellum tend to show the intense immunoreactivity for Kir6.1ruK ATP-1 than relatively small neurons such as granule cells in the olfactory bulb, dentate gyrus and cerebellum. This may reflect the number of mitochondria in these neuronal somata. The mitoK ATP may be responsible for maintaining and regulating the matrix volume of mitochondria during energy metabolism. Mitochondria generate a Kq conductance when the ATP level on the matrix side becomes deficient, and mitochondria in living cells swell, then contract by changing their water content w16x. This matrix expansion plays an important role in cell-signaling pathways after the mitoK ATP opening, resulting in activation of

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electron transport and stimulation of fatty acid oxidation w18x. These functions seem to be different from Kir6.2, despite the 70% homology between Kir6.1 and Kir6.2 w1,13x, since Kir6.2 is predominantly localized in the plasma membrane of in vitro COSm6 cells w23x, pancreatic islet cells w29x, neurons, and glial cells w35x, and is involved in insulin secretion w24,27,33x and transmitter release w2x. It is believed that K ATP channel may play an important role in cerebral ischemic tolerance w9,10x and in cardioprotection w7,8,18x. In hippocampus pyramidal cells, K ATP channel opening will protect the neurons from degeneration during cerebral ischemia w9x. Our present study showed that the hippocampal pyramidal cells in CA2 and CA3 exhibit more intense immunoreactivity for Kir6.1ruK ATP-1 than those in CA1 ŽFig. 11., which is more susceptible to ischemic injury than CA3 w4x. The main cause of damage to neuronal tissue following ischemia are ATP depletion w9x. Thus, Kir6.1ruK ATP-1 may be related to susceptibility to ischemic injury in the hippocampus. In cardiac muscle the mitoK ATP can be the receptor for Kq channel openers ŽKCO. which have cardioprotective effects w7,8,18x. It is important to determine whether Kir6.1ruK ATP-1 is the exact subunit which functions in these pathological conditions. Kir 6.1 coexpressed with SUR1 forms an K ATP channel current w22x, while Kir6.1 coexpressed with SUR2B forms a sulfonylurea sensitive but ATP-insensitive Kq channel current w34x. In the present study, the distribution of Kir6.1 mRNA in medium- and large-sized neurons was similar to that of SUR1 reported by Karschin et al. w20x. We suggest that Kir6.1ruK ATP-1 may be coexpressed with SUR1 in various neurons. Further study is needed to analyze the co-localization and diversity of SURs with the Kir6.1ruK ATP-1 to give functional channel activity, not only in physiological but in pathological conditions in the mitochondrial inner membrane in neurons and glial cells.

Acknowledgements The authors wish to thank Prof. S. Seino, Chiba University School of Medicine, for kindly supplying antiKir6.1ruK ATP-1 antibody. We also thank Prof. H. Kondo, Tohoku University School of Medicine, for reviewing this manuscript. This work was supported by Grants in Aid from the Ministry of Education, Science and Culture of Japan, No. 09670031 and No. 11670022 to H. A., and by CREST of Japan Science and Technology Corporation ŽJST. to K.K.

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