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Differential expression of the inwardly-rectifying K-channel ROMK1 in rat brain
MOLECULAR BRAIN RESEARCH ELSEVIER
Molecular Brain Research24 (1994)353-356
Short Communication
Differential expression of the inwardly-rectifying K-channel ROMK1 in rat brain Susan Kenna b, Jochen R6per
a Kevin Ho c, Steven Hebert c, Stephen J.H. Ashcroft b Frances M. Ashcroft a,,
a University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK b Nuffield Departmertt of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK c Harvard Medical School, Department of Medicine, Renal Division, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 21115, USA
(Accepted 29 March 1994)
Abstract
We have investigated the distribution of the inwardly-rectifying K-channel ROMK1 in rat brain using Northern blotting and in situ hybridization. High levels of expression were found in cortex and hippocampus but not in substantia nigra, ventromedial hypothalamus, cerebellum or striatum. PCR primers generated against ROMK1 amplified a 300 bp cDNA fragment from cortex and hippocampus identical in sequence to ROMK1. Key words: ROMK1; ATP-sensitive K-channel; Rat brain
An inwardly-rectifying K-channel, ROMK1, has recently been cloned from the outer medulla of rat kidney [1]. This channel, together with IRK1 [2] and GIRK1 [3] belongs to a new family of inward-rectifier K-channels which have a molecular architecture distinct from that of voltage-gated K-channels. Since ROMK1 possesses a putative ATP-binding site, we have examined whether the distribution of this channel in the brain correlates with that found for ATP-sensitive K-channels [4] (K-ATP channel) or the high-affinity sulphonylurea receptors associated with K-ATP channels [5]. The distribution of ROMK1 was investigated using in situ hybridization and Northern blotting. The 5' end of ROMK1 (nt 0-522) was subcloned into pSPORT1 and used to generate riboprobes for hybridizations. The plasmid was linearised with the restriction enzyme XhoI and in vitro transcribed with the RNA polymerase SP6 for the antisense probe. For the sense
* Corresponding author. Fax: (44) 865-272469. 0169-328X/94/$07.00 © 1994ElsevierScienceB.V. All rights reserved SSDI 0169-328X(94)00071-L
probe, the plasmid was linearised with BamH1 and in vitro transcribed with RNA polymerase T7. In situ hybridization was performed on sagittal and coronal sections of rat brains (15 day postnatal) using the antisense riboprobe. Parallel experiments were carried out using the sense riboprobe to determine the level of background non-specific hybridization. A high density of specific binding was observed only in the hippocampus and cortex (Fig. la-d). In the hippocampus, the strongest binding occurred over the neuronal cell bodies of the dentate gyrus but substantial binding was also detected in neurones of the CA1 and CA3 regions. There was also a high density of silver grains in the cortex. No binding was detected in the substantia nigra (Fig. le), ventromedial hypothalamus, striatum or cerebellum (not shown). The sense probe did not bind to any brain region (Fig. lf). Total RNA was isolated from specific brain regions of 15-day-old rats using the one-step acid-phenol method [6]. Northern blotting under high stringency conditions with the antisense riboprobe gave a single strong 2.2 kb signal with total RNA from hippocampus and cortex but not with that from ventromedial hy-
354
S. Kenna et aL /Molecular Brain Research 24 (1994) 353-356
355
S. Kenna et al. /Molecular Brain Research 24 (1994) 353-356
VMH
2.2Kb
SN
HC
S
CX
CB
-----m--
Fig. 2. Northern blot analysis of RNA isolated from rat brain regions hybridized with an antisense riboprobe for ROMK1 under high stringency conditions: ventromedial hypothalamus (VMH); substantia nigra (SN); hippocampus (HC); striatum (S); cortex (CX); cerebellum (CB). The arrow on the left indicates the molecular weight of the hybridizing bands observed. Each lane contained 20/xg of total RNA which was electrophoresed through a 1% agarose gel in 20 mM MOPS, 5 mM sodium acetate pH 7.0, 1 mM EDTA and 0.63 M formaldehyde and subsequently transferred to a nylon membrane (Hybond N, Amersham). The membranes were prehybridised for 4 h at 45°C in 50% formamide, 5 X SET solution, 5 x Denhardts solution, 1% SDS, and 100 /~g/ml salmon sperm DNA. Hybridizations were carried out under the same conditions with the addition of the 32p-labelled antisense riboprobe for 16 h. The membranes were then washed in 3 exchanges of 0.1 x SET, 0.1% SDS at 65°C for 30 min each time and autoradiography was performed.
p o t h a l a m u s , s u b s t a n t i a nigra, c e r e b e l l u m o r s t r i a t u m (Fig. 2). N o signal was o b s e r v e d in any b r a i n r e g i o n using t h e sense r i b o p r o b e . T h e s e results suggest that a c h a n n e l c o r r e s p o n d i n g to R O M K 1 is specifically e x p r e s s e d in h i p p o c a m p u s and cortex. T o d e t e r m i n e the i d e n t i t y o f hybridizing species w e d e s i g n e d P C R p r i m e r s to r e c o g n i s e t h e 300 bp s e q u e n c e b e t w e e n the p o r e r e g i o n and the p u t a t i v e
A T P - b i n d i n g d o m a i n o f R O M K 1 . T h e 5' a m p l i f i c a t i o n primer (5'-TCAGCCTTTCTGTTTTCTA-3') corres p o n d e d to n u c l e o t i d e s 3 8 7 - 4 0 8 , while the 3' p r i m e r (5'-CTTGCCATATATGTGGCTGCC-3') corres p o n d e d to n u c l e o t i d e s 6 6 6 - 6 8 7 . T o t a l R N A p u r i f i e d f r o m h i p p o c a m p u s and c o r t e x was r e v e r s e t r a n s c r i b e d using 200 U o f c l o n e d M M L V r e v e r s e t r a n s c r i p t a s e ( B R L ) with 100 ng o l i g o - d T ( B o e h r i n g e r ) . T h e r e a c t i o n
Fig. 1. In situ hybridization of ROMK1 in rat brain using the antisense riboprobe (a-e) or the sense riboprobe (f). a: low-power ( x 40) sagittal section showing hippocampus, b: low-power (× 40) coronal section showing cortex, c: high-power (× 400) sagittal section showing hippocampus. d: high-power (x 400) coronal section showing cortex, e: low-power (x 40) coronal section at the level of substantia nigra under dark-field illumination, f: negative control with sense riboprobe showing sagittal section of hippocampus at low power ( x 40). 10/zm cryostat parasagittal and coronal sections from the brains of 15 day postnatal rats were thaw-mounted, dried and postfixed for 5 min in 4% paraformaldehyde in 0.1 M PBS using 0.02% DEPC-treated H20. Subsequently, slides were dehydrated and stored at -70°C. Hybridization was performed for 2 h at 45°C in buffer containing: 50% formamide, 10% dextran sulphate, 0.3 M NaC1, 20 mM Tris-HCl, 5 mM EDTA, 10 mM Na3PO4 (pH 8.0), 20 mM DTT, i X Denhardts solution and 0.5 mg/ml yeast tRNA. For each slide, 100/zl hybridization mix containing the appropriate [32p]rUTP-labelled sense or antisense riboprobe (5 × 106 dpm/ml) was used. The slides were washed twice in 1 x SSC at 50°C for 30 rain, dehydrated, dried, dipped in photographic emulsion (Kodak, NTB-2) and stored at 4°C overnight before being developed and counterstained with Cresyl violet. All sections are shown under dark-field illumination.
356
S. Kenna et al. /Molecular Brain Research 24 (1994) 353-356
was carried out in the presence of 1 x Taq buffer (Promega), 10/zM of each dNTP, 20 U of the RNase inhibitor RNasin (Promega), 2.5 mM MgCI 2 at 37°C for 30 min. cDNA was amplified by the addition of the appropriate amount of Taq buffer to give a final concentration of 1 x buffer, 50 pmol of each primer and 2 U of Taq DNA polymerase (Promega). Amplification was performed for 35 cycles, each cycle consisting of 92°C for 1 min, 57°C for 1 min, and 72°C for 2 min. The sample was purified through a Wizard PCR column (Promega) and ligated into Bluescript SK (Stratagene) and double-stranded sequencing performed in both directions using Sequenase version 2 (USB). Using this protocol, 300 bp cDNA amplification products were obtained. Subcloning and subsequent sequencing of these fragments revealed 100% identity with ROMK1 over this region, suggesting that hippocampus and cortex indeed express ROMK1. To exclude the possibility of genomic contamination, a second pair of primers was constructed to overlap an intron towards the 5' end of ROMK1. The primer pair was 5'-CAATGCAAGTAAATGTCATF-3' (sense) and 5'-GGCGCACTGT-FCTGTCACAA-3' (antisense) corresponding to nucleotides 38-57 and 592-611. These primers generated amplification products of the size expected for the spliced RNA, using RNA isolated from both hippocampus and cortex. Since ROMK1 possesses a putative ATP-binding site, it has been suggested that this channel may form (part of) a K-ATP channel [1]. Characteristically, classical K-ATP channels are blocked with high affinity by sulphonylureas [4] and their occurrence correlates with the presence of high affinity binding sites for these drugs [5], although it is not established whether the channel and the receptor are the same protein. The distribution of glibenclamide binding sites in the brains of 15 day old rats has been studied by Mourre et al. [7]. The highest level of sulphonylurea binding was observed in the substantia nigra, the cortex and the ventroposterior thalamic nucleus. In the hippocampus, CA2 was also clearly labelled, but labelling in the CA3 and the dentate gyrus was only just beginning to appear. The distribution of K-ATP channels in the brain has not been mapped. However, these channels have been identified at the single-channel level in dissociated neurones from the pars compacta [8] and slice
recordings from the pars reticulata region of the substantia nigra [9]. They have also been found in cultured neurones from hippocampus and cortex [10]. Expression of ROMKI in hippocampus and cortex would be consistent with the idea that ROMK1 codes for a neuronal K-ATP channel. However, there is a clear discrepancy between the presence of K-ATP channels and the high density of sulphonylurea binding in the substantia nigra and our finding that the level of ROMK1 mRNA expression in this region is undetectable in in situ hybridizations and Northern blots. This strongly suggests that ROMK1 is distinct from the nigral K-ATP channel. This work was supported by the Welcome Trust and the Medical Research Council (UK), whom we thank. S.K. and J.R. made equal contributions to the work. [1] Ho, K., Nichols, C.G., Lederer, W.J., Lytton, J., Vassilev, P.M., Kanazirska, M.V. and Hebert, S.C., Cloning and expression of an inwardly-rectifying ATP-regulated potassium channel, Nature, 362 (1993) 31-38. [2] Kubo, Y., Baldwin, T.J., Jan, Y.N. and Jan, L.Y., Primary structure and functional expression of a mouse inward rectifier potassium channel, Nature, 362 (1993) 127-133. [3] Kubo, Y., Reuveny, E., Slesinger, P.A., Jan, Y.N. and Jan, L.Y., Primary structure and functional expression of a rat G-proteincoupled muscarinic potassium channel, Nature, 364 (1993) 802806. [4] Ashcroft, F.M. and Ashcroft, S.J.H., Properties and functions of ATP-sensitive K-channels, Cell. Sign., 2 (1990) 197-214. [5] Ashcroft, S.J.H. and Ashcroft, F.M., The sulphonylurea receptor, Biochim. Biophys. Acta, 1175 (1992)45-49. [6] Chomczynski, P. and Sacchi, N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol chloroform extraction, Anal Biochem., 162 (1987) 156-159. [7] Mourre, C., Widmann, C. and Lazdunski, M., Sulphonylurea binding sites associated with ATP-regulated K channels in the central nervous system: autoradiographic analysis of their distribution and ontogenesis, and of their localisation in mutant mice cerebellum, Brain Res., 519 (1990) 29-43. [8] R6per, J., Hainsworth, A.H. and Ashcroft, F.M. ATP-sensitive K channels in guinea-pig isolated substantia nigra neurones are modulated by cellular metabolism, J. Physiol., 430 (1990) 130P. [9] Schwanstecher, C. and Panten, U., Tolbutamide-sensitive and diazoxide-sensitive K ÷ channels in neurones of substantia nigra pars reticulata, Naunyn-Schmiedeberg's Arch. Pharmacol., 348 (1993) 113-117. [10] Ohno-Shosaku, T. and Yamamoto, C., Identification of an ATP-sensitive K channel in rat cultured cortical neurones, Pfliigers Arch., 422 (1992) 260-266.
Molecular Brain Research24 (1994)353-356
Short Communication
Differential expression of the inwardly-rectifying K-channel ROMK1 in rat brain Susan Kenna b, Jochen R6per
a Kevin Ho c, Steven Hebert c, Stephen J.H. Ashcroft b Frances M. Ashcroft a,,
a University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK b Nuffield Departmertt of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK c Harvard Medical School, Department of Medicine, Renal Division, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 21115, USA
(Accepted 29 March 1994)
Abstract
We have investigated the distribution of the inwardly-rectifying K-channel ROMK1 in rat brain using Northern blotting and in situ hybridization. High levels of expression were found in cortex and hippocampus but not in substantia nigra, ventromedial hypothalamus, cerebellum or striatum. PCR primers generated against ROMK1 amplified a 300 bp cDNA fragment from cortex and hippocampus identical in sequence to ROMK1. Key words: ROMK1; ATP-sensitive K-channel; Rat brain
An inwardly-rectifying K-channel, ROMK1, has recently been cloned from the outer medulla of rat kidney [1]. This channel, together with IRK1 [2] and GIRK1 [3] belongs to a new family of inward-rectifier K-channels which have a molecular architecture distinct from that of voltage-gated K-channels. Since ROMK1 possesses a putative ATP-binding site, we have examined whether the distribution of this channel in the brain correlates with that found for ATP-sensitive K-channels [4] (K-ATP channel) or the high-affinity sulphonylurea receptors associated with K-ATP channels [5]. The distribution of ROMK1 was investigated using in situ hybridization and Northern blotting. The 5' end of ROMK1 (nt 0-522) was subcloned into pSPORT1 and used to generate riboprobes for hybridizations. The plasmid was linearised with the restriction enzyme XhoI and in vitro transcribed with the RNA polymerase SP6 for the antisense probe. For the sense
* Corresponding author. Fax: (44) 865-272469. 0169-328X/94/$07.00 © 1994ElsevierScienceB.V. All rights reserved SSDI 0169-328X(94)00071-L
probe, the plasmid was linearised with BamH1 and in vitro transcribed with RNA polymerase T7. In situ hybridization was performed on sagittal and coronal sections of rat brains (15 day postnatal) using the antisense riboprobe. Parallel experiments were carried out using the sense riboprobe to determine the level of background non-specific hybridization. A high density of specific binding was observed only in the hippocampus and cortex (Fig. la-d). In the hippocampus, the strongest binding occurred over the neuronal cell bodies of the dentate gyrus but substantial binding was also detected in neurones of the CA1 and CA3 regions. There was also a high density of silver grains in the cortex. No binding was detected in the substantia nigra (Fig. le), ventromedial hypothalamus, striatum or cerebellum (not shown). The sense probe did not bind to any brain region (Fig. lf). Total RNA was isolated from specific brain regions of 15-day-old rats using the one-step acid-phenol method [6]. Northern blotting under high stringency conditions with the antisense riboprobe gave a single strong 2.2 kb signal with total RNA from hippocampus and cortex but not with that from ventromedial hy-
354
S. Kenna et aL /Molecular Brain Research 24 (1994) 353-356
355
S. Kenna et al. /Molecular Brain Research 24 (1994) 353-356
VMH
2.2Kb
SN
HC
S
CX
CB
-----m--
Fig. 2. Northern blot analysis of RNA isolated from rat brain regions hybridized with an antisense riboprobe for ROMK1 under high stringency conditions: ventromedial hypothalamus (VMH); substantia nigra (SN); hippocampus (HC); striatum (S); cortex (CX); cerebellum (CB). The arrow on the left indicates the molecular weight of the hybridizing bands observed. Each lane contained 20/xg of total RNA which was electrophoresed through a 1% agarose gel in 20 mM MOPS, 5 mM sodium acetate pH 7.0, 1 mM EDTA and 0.63 M formaldehyde and subsequently transferred to a nylon membrane (Hybond N, Amersham). The membranes were prehybridised for 4 h at 45°C in 50% formamide, 5 X SET solution, 5 x Denhardts solution, 1% SDS, and 100 /~g/ml salmon sperm DNA. Hybridizations were carried out under the same conditions with the addition of the 32p-labelled antisense riboprobe for 16 h. The membranes were then washed in 3 exchanges of 0.1 x SET, 0.1% SDS at 65°C for 30 min each time and autoradiography was performed.
p o t h a l a m u s , s u b s t a n t i a nigra, c e r e b e l l u m o r s t r i a t u m (Fig. 2). N o signal was o b s e r v e d in any b r a i n r e g i o n using t h e sense r i b o p r o b e . T h e s e results suggest that a c h a n n e l c o r r e s p o n d i n g to R O M K 1 is specifically e x p r e s s e d in h i p p o c a m p u s and cortex. T o d e t e r m i n e the i d e n t i t y o f hybridizing species w e d e s i g n e d P C R p r i m e r s to r e c o g n i s e t h e 300 bp s e q u e n c e b e t w e e n the p o r e r e g i o n and the p u t a t i v e
A T P - b i n d i n g d o m a i n o f R O M K 1 . T h e 5' a m p l i f i c a t i o n primer (5'-TCAGCCTTTCTGTTTTCTA-3') corres p o n d e d to n u c l e o t i d e s 3 8 7 - 4 0 8 , while the 3' p r i m e r (5'-CTTGCCATATATGTGGCTGCC-3') corres p o n d e d to n u c l e o t i d e s 6 6 6 - 6 8 7 . T o t a l R N A p u r i f i e d f r o m h i p p o c a m p u s and c o r t e x was r e v e r s e t r a n s c r i b e d using 200 U o f c l o n e d M M L V r e v e r s e t r a n s c r i p t a s e ( B R L ) with 100 ng o l i g o - d T ( B o e h r i n g e r ) . T h e r e a c t i o n
Fig. 1. In situ hybridization of ROMK1 in rat brain using the antisense riboprobe (a-e) or the sense riboprobe (f). a: low-power ( x 40) sagittal section showing hippocampus, b: low-power (× 40) coronal section showing cortex, c: high-power (× 400) sagittal section showing hippocampus. d: high-power (x 400) coronal section showing cortex, e: low-power (x 40) coronal section at the level of substantia nigra under dark-field illumination, f: negative control with sense riboprobe showing sagittal section of hippocampus at low power ( x 40). 10/zm cryostat parasagittal and coronal sections from the brains of 15 day postnatal rats were thaw-mounted, dried and postfixed for 5 min in 4% paraformaldehyde in 0.1 M PBS using 0.02% DEPC-treated H20. Subsequently, slides were dehydrated and stored at -70°C. Hybridization was performed for 2 h at 45°C in buffer containing: 50% formamide, 10% dextran sulphate, 0.3 M NaC1, 20 mM Tris-HCl, 5 mM EDTA, 10 mM Na3PO4 (pH 8.0), 20 mM DTT, i X Denhardts solution and 0.5 mg/ml yeast tRNA. For each slide, 100/zl hybridization mix containing the appropriate [32p]rUTP-labelled sense or antisense riboprobe (5 × 106 dpm/ml) was used. The slides were washed twice in 1 x SSC at 50°C for 30 rain, dehydrated, dried, dipped in photographic emulsion (Kodak, NTB-2) and stored at 4°C overnight before being developed and counterstained with Cresyl violet. All sections are shown under dark-field illumination.
356
S. Kenna et al. /Molecular Brain Research 24 (1994) 353-356
was carried out in the presence of 1 x Taq buffer (Promega), 10/zM of each dNTP, 20 U of the RNase inhibitor RNasin (Promega), 2.5 mM MgCI 2 at 37°C for 30 min. cDNA was amplified by the addition of the appropriate amount of Taq buffer to give a final concentration of 1 x buffer, 50 pmol of each primer and 2 U of Taq DNA polymerase (Promega). Amplification was performed for 35 cycles, each cycle consisting of 92°C for 1 min, 57°C for 1 min, and 72°C for 2 min. The sample was purified through a Wizard PCR column (Promega) and ligated into Bluescript SK (Stratagene) and double-stranded sequencing performed in both directions using Sequenase version 2 (USB). Using this protocol, 300 bp cDNA amplification products were obtained. Subcloning and subsequent sequencing of these fragments revealed 100% identity with ROMK1 over this region, suggesting that hippocampus and cortex indeed express ROMK1. To exclude the possibility of genomic contamination, a second pair of primers was constructed to overlap an intron towards the 5' end of ROMK1. The primer pair was 5'-CAATGCAAGTAAATGTCATF-3' (sense) and 5'-GGCGCACTGT-FCTGTCACAA-3' (antisense) corresponding to nucleotides 38-57 and 592-611. These primers generated amplification products of the size expected for the spliced RNA, using RNA isolated from both hippocampus and cortex. Since ROMK1 possesses a putative ATP-binding site, it has been suggested that this channel may form (part of) a K-ATP channel [1]. Characteristically, classical K-ATP channels are blocked with high affinity by sulphonylureas [4] and their occurrence correlates with the presence of high affinity binding sites for these drugs [5], although it is not established whether the channel and the receptor are the same protein. The distribution of glibenclamide binding sites in the brains of 15 day old rats has been studied by Mourre et al. [7]. The highest level of sulphonylurea binding was observed in the substantia nigra, the cortex and the ventroposterior thalamic nucleus. In the hippocampus, CA2 was also clearly labelled, but labelling in the CA3 and the dentate gyrus was only just beginning to appear. The distribution of K-ATP channels in the brain has not been mapped. However, these channels have been identified at the single-channel level in dissociated neurones from the pars compacta [8] and slice
recordings from the pars reticulata region of the substantia nigra [9]. They have also been found in cultured neurones from hippocampus and cortex [10]. Expression of ROMKI in hippocampus and cortex would be consistent with the idea that ROMK1 codes for a neuronal K-ATP channel. However, there is a clear discrepancy between the presence of K-ATP channels and the high density of sulphonylurea binding in the substantia nigra and our finding that the level of ROMK1 mRNA expression in this region is undetectable in in situ hybridizations and Northern blots. This strongly suggests that ROMK1 is distinct from the nigral K-ATP channel. This work was supported by the Welcome Trust and the Medical Research Council (UK), whom we thank. S.K. and J.R. made equal contributions to the work. [1] Ho, K., Nichols, C.G., Lederer, W.J., Lytton, J., Vassilev, P.M., Kanazirska, M.V. and Hebert, S.C., Cloning and expression of an inwardly-rectifying ATP-regulated potassium channel, Nature, 362 (1993) 31-38. [2] Kubo, Y., Baldwin, T.J., Jan, Y.N. and Jan, L.Y., Primary structure and functional expression of a mouse inward rectifier potassium channel, Nature, 362 (1993) 127-133. [3] Kubo, Y., Reuveny, E., Slesinger, P.A., Jan, Y.N. and Jan, L.Y., Primary structure and functional expression of a rat G-proteincoupled muscarinic potassium channel, Nature, 364 (1993) 802806. [4] Ashcroft, F.M. and Ashcroft, S.J.H., Properties and functions of ATP-sensitive K-channels, Cell. Sign., 2 (1990) 197-214. [5] Ashcroft, S.J.H. and Ashcroft, F.M., The sulphonylurea receptor, Biochim. Biophys. Acta, 1175 (1992)45-49. [6] Chomczynski, P. and Sacchi, N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol chloroform extraction, Anal Biochem., 162 (1987) 156-159. [7] Mourre, C., Widmann, C. and Lazdunski, M., Sulphonylurea binding sites associated with ATP-regulated K channels in the central nervous system: autoradiographic analysis of their distribution and ontogenesis, and of their localisation in mutant mice cerebellum, Brain Res., 519 (1990) 29-43. [8] R6per, J., Hainsworth, A.H. and Ashcroft, F.M. ATP-sensitive K channels in guinea-pig isolated substantia nigra neurones are modulated by cellular metabolism, J. Physiol., 430 (1990) 130P. [9] Schwanstecher, C. and Panten, U., Tolbutamide-sensitive and diazoxide-sensitive K ÷ channels in neurones of substantia nigra pars reticulata, Naunyn-Schmiedeberg's Arch. Pharmacol., 348 (1993) 113-117. [10] Ohno-Shosaku, T. and Yamamoto, C., Identification of an ATP-sensitive K channel in rat cultured cortical neurones, Pfliigers Arch., 422 (1992) 260-266.