PIN: An associated protein inhibitor of neuronal nitric oxide synthase

Science

Volumn 274, Issue 5288, 1996, Pages 774-777

  Jaffrey, Samie R ^a   Snyder, Solomon H ^a  

^a JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

NITRIC OXIDE; NITRIC OXIDE SYNTHASE; NITRIC OXIDE SYNTHASE INHIBITOR;

ARTICLE; DIMERIZATION; ENZYME ACTIVITY; ENZYME INHIBITION; NEUROTRANSMISSION; PRIORITY JOURNAL; PROTEIN ANALYSIS; SEQUENCE ANALYSIS;

AMINO ACID SEQUENCE; ANIMALS; CARRIER PROTEINS; CELL LINE; CYCLIC GMP; DIMERIZATION; DROSOPHILA PROTEINS; ENZYME INHIBITORS; HUMANS; MOLECULAR SEQUENCE DATA; MOLECULAR WEIGHT; NEURONS; NITRIC OXIDE SYNTHASE; RATS; RECOMBINANT FUSION PROTEINS; SACCHAROMYCES CEREVISIAE; TRANSFECTION;

EID: 0029858301     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5288.774     Document Type: Article

References (42)

1. H. H. Schmidt and U. Walter, Cell 78, 919 (1994); S. R. Jaffrey and S. H. Snyder, Annu. Rev. Cell Dev. Biol. 11, 417 (1995).
2. H. H. Schmidt and U. Walter, Cell 78, 919 (1994); S. R. Jaffrey and S. H. Snyder, Annu. Rev. Cell Dev. Biol. 11, 417 (1995).
3. E. Bonfoco, D. Kraine, M. Ankarcrona, P. Nicotera, S. A. Lipton, Proc. Natl. Acad. Sci. U.S.A. 92, 7162 (1995); J. B. Mannick, K. Asano, K. Izumi, E. Kieff, J. S. Stamler, Cell 79, 1137 (1994).
4. E. Bonfoco, D. Kraine, M. Ankarcrona, P. Nicotera, S. A. Lipton, Proc. Natl. Acad. Sci. U.S.A. 92, 7162 (1995); J. B. Mannick, K. Asano, K. Izumi, E. Kieff, J. S. Stamler, Cell 79, 1137 (1994).
5. T. Wang, Z. Xie, B. Lu, Nature 374, 262 (1995).
6. S. Fields and O.-K. Song, ibid 340, 245 (1989).
7. P. M. Chevray and D. Nathans, Proc. Natl. Acad. Sci. U.S.A. 89, 5789 (1992).
8. Two-hybrid screens and parent vectors pPC97 and pPC86 were as described (5). Plasmid pBD-NOS(2-377) was constructed by insertion of a nNOS polymerase chain reaction (PCR) product encoding amino acids 2 to 377 into the Sal I-Bgl II sites of pPC97, resulting in an open reading frame encoding a Gal4 BD-NOS fusion protein. The nNOS fragment was constructed by PCR with the following primers: 5′-GACTAGTCGACTGAAGAGAACACGTTTGGG-3′ (coding strand) and 5′-TCTGCAGATCTCAGT-GGGCCTTGGAGCCAAA-3′ (noncoding strand). A rat hippocampal cDNA library in pPC86 [X.-J. Li et al., Nature 378, 398 (1995)] was amplified once in DH10B (Gibco BRL) (28) and transformed into yeast containing pBD-NOS(2-377). pAD-PIN was identified as a 0.5-kb clone that activated lacZ transcription and conferred histidine protorophy in the presence of pBD-NOS(2-377). Plasmids were sequenced by automated fluorescent sequencing. The PIN sequence has been deposited in GenBank (accession number U66461).
9. pPC97 derivatives containing fragments of RAFT were constructed by PCR and cloned into the Sal I and Sac I sites of pPC97. Truncated NOS fragments comprising amino acids 2 to 163 and 2 to 281 were generated by restriction of the initial NOS(2-377) PCR fragment with Nco I and Ava I, respectively, followed by blunt-end ligation into pPC97. Other truncated NOS fragments were prepared by PCR.
10. C. P. Pontig and C. Phillips, Trends Biochem. Sci. 20, 102 (1995).
11. J. E. Brenman, D. S. Chao, H. Xia, K. Aldape, D. S. Bredt, Cell 82, 743 (1995); J. E. Brenman et al., ibid. 84, 757 (1996).
12. J. E. Brenman, D. S. Chao, H. Xia, K. Aldape, D. S. Bredt, Cell 82, 743 (1995); J. E. Brenman et al., ibid. 84, 757 (1996).
13. S. R. Jeffrey and S. H. Snyder, unpublished observations.
14. A pBluescript plasmid containing the cDNA for PIN was obtained by screening a rat brain λZAPII cDNA library (Stratagene) with a probe derived from the Sal I-Not I insert in pAD-PIN. Library screening was performed according to the directions of the manufacturer.
15. M. Kozak, J. Biol. Chem. 266, 19867 (1991).
16. S. F. Altschul, W. Gish, W. Miller, E. W. Myers, D. J. Lipman, J. Mol. Biol. 215, 403 (1990).
17. R. Wilson et al., Nature 368, 32 (1994).
18. S. M. King and R. S. Patel-King, J. Biol. Chem. 270, 11445 (1995).
19. GenBank accession numbers for the referenced clones are as follows: N28047 (EST, S. mansoni), T01352 (EST, C. reinhardtii), T34147 (EST, human), and T88069 (A. thaliana). Sequences were aligned with BLAST (13), and percentage amino acid identity was determined. Ambiguous nucleotides from the ESTs that could not be translated were omitted from the analysis.
20. J. J. Siekierka, S. H. Hung, M. Poe, C. S. Lin, N. H. Sigal, J. Biol. Chem. 265, 21011 (1990).
21. N. Takahashi, T. Hayano, M. Suzuki, Nature 337, 473 (1989); G. Fischer, B. Wittmann-Liebold, K. Lang, T, Kiefhaber, F. X. Schmid, ibid., p. 476.
22. N. Takahashi, T. Hayano, M. Suzuki, Nature 337, 473 (1989); G. Fischer, B. Wittmann-Liebold, K. Lang, T, Kiefhaber, F. X. Schmid, ibid., p. 476.
23. A. Aitken et al., Trends Biochem. Sci. 17, 498 (1992).
24. The cDNA for PIN was excised from pAD-PIN with Sal I and Not I and cloned into those sites in pGEX-4T2 (Pharmacia). Fusion proteins were prepared in Escherichia coli BL21(DE3) (Novagen) with glutathione-agarose (Sigma) [D. B. Smith and K. S. Johnson, Gene 67, 31 (1988)], except that bacteria pellets were lysed in lysis buffer [50 mM tris-HCI (pH 7.7). 100 mM NaCl, and 2 mM EDTA], supernatants were adjusted to 1% Triton X-100, and protein was purified with elution buffer [50 mM tris-HCl (pH 7.7), 100 mM NaCl, 10 mM reduced glutathione, and 2 mM EDTA]. HEK 293 cells were transfected with plasmids for nNOS [D. S. Bredt et al., Nature 351, 714 (1991)], eNOS [S. Lamas, P. A. Marsden, G. K. Li, P. Tempst, T. Michel, Proc. Natl. Acad. Sci. U.S.A 89, 6348 (1992)]. and iNOS (C. J. Lowenstein, C. S. Glatt, D. S. Bredt, S. H. Snyder, ibid., p. 6711). Transfections were performed with 10 μg of each plasmid with the calcium phosphate method (28) After transfection, cells were sonicated in buffer A [50 mM tris-HCl (pH 7.7), 100 mM NaCl, 2 mM EDTA, and 1% Triton X-100] and cleared by centrifugation. This cellular lysate was incubated with GST or GST-PIN immobilized on glutathione-agarose and then washed extensively in HNTG buffer [20 mM Hepes (pH 7.4), 500 mM NaCl, 10% glycerol, and 0.1% Triton X-100], For assays testing PIN binding to immobilized NOS, 20 μg of bacterial lysate was added to 200 μg of transfected HEK 293 cell lysate and bound to 2′,5′-ADP-Sepharose 4B (Pharmacia) and subsequently washed and eluted with 10 mM NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) as described [D. S. Bredt and S. H. Snyder, ibid. 87, 682 (1990)]. The eluate was immunoblotted with a rabbit antibody to GST (NovaCastra, Burtingame, CA). For blot-overlay analysis, pGEX-4T2 was modified such that two sites for protein kinase A (PKA) encoded on complementary synthetic oligonucleotides (5′-AATTCGTCGTGCATCTGTTGAACTACGTCGAGCTTCAGTTGCG-3′, upper strand) were ligated into the Eco Rl-Sal I sites to generate plasmid pGEX4T-2K. Kinase reactions and blot overlays were performed as described [W. M. Kavanaugh and L. T. Williams, Science 266, 1862 (1994)].
25. The cDNA for PIN was excised from pAD-PIN with Sal I and Not I and cloned into those sites in pGEX-4T2 (Pharmacia). Fusion proteins were prepared in Escherichia coli BL21(DE3) (Novagen) with glutathione-agarose (Sigma) [D. B. Smith and K. S. Johnson, Gene 67, 31 (1988)], except that bacteria pellets were lysed in lysis buffer [50 mM tris-HCI (pH 7.7). 100 mM NaCl, and 2 mM EDTA], supernatants were adjusted to 1% Triton X-100, and protein was purified with elution buffer [50 mM tris-HCl (pH 7.7), 100 mM NaCl, 10 mM reduced glutathione, and 2 mM EDTA]. HEK 293 cells were transfected with plasmids for nNOS [D. S. Bredt et al., Nature 351, 714 (1991)], eNOS [S. Lamas, P. A. Marsden, G. K. Li, P. Tempst, T. Michel, Proc. Natl. Acad. Sci. U.S.A 89, 6348 (1992)]. and iNOS (C. J. Lowenstein, C. S. Glatt, D. S. Bredt, S. H. Snyder, ibid., p. 6711). Transfections were performed with 10 μg of each plasmid with the calcium phosphate method (28) After transfection, cells were sonicated in buffer A [50 mM tris-HCl (pH 7.7), 100 mM NaCl, 2 mM EDTA, and 1% Triton X-100] and cleared by centrifugation. This cellular lysate was incubated with GST or GST-PIN immobilized on glutathione-agarose and then washed extensively in HNTG buffer [20 mM Hepes (pH 7.4), 500 mM NaCl, 10% glycerol, and 0.1% Triton X-100], For assays testing PIN binding to immobilized NOS, 20 μg of bacterial lysate was added to 200 μg of transfected HEK 293 cell lysate and bound to 2′,5′-ADP-Sepharose 4B (Pharmacia) and subsequently washed and eluted with 10 mM NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) as described [D. S. Bredt and S. H. Snyder, ibid. 87, 682 (1990)]. The eluate was immunoblotted with a rabbit antibody to GST (NovaCastra, Burtingame, CA). For blot-overlay analysis, pGEX-4T2 was modified such that two sites for protein kinase A (PKA) encoded on complementary synthetic oligonucleotides (5′-AATTCGTCGTGCATCTGTTGAACTACGTCGAGCTTCAGTTGCG-3′, upper strand) were ligated into the Eco Rl-Sal I sites to generate plasmid pGEX4T-2K. Kinase reactions and blot overlays were performed as described [W. M. Kavanaugh and L. T. Williams, Science 266, 1862 (1994)].
26. The cDNA for PIN was excised from pAD-PIN with Sal I and Not I and cloned into those sites in pGEX-4T2 (Pharmacia). Fusion proteins were prepared in Escherichia coli BL21(DE3) (Novagen) with glutathione-agarose (Sigma) [D. B. Smith and K. S. Johnson, Gene 67, 31 (1988)], except that bacteria pellets were lysed in lysis buffer [50 mM tris-HCI (pH 7.7). 100 mM NaCl, and 2 mM EDTA], supernatants were adjusted to 1% Triton X-100, and protein was purified with elution buffer [50 mM tris-HCl (pH 7.7), 100 mM NaCl, 10 mM reduced glutathione, and 2 mM EDTA]. HEK 293 cells were transfected with plasmids for nNOS [D. S. Bredt et al., Nature 351, 714 (1991)], eNOS [S. Lamas, P. A. Marsden, G. K. Li, P. Tempst, T. Michel, Proc. Natl. Acad. Sci. U.S.A 89, 6348 (1992)]. and iNOS (C. J. Lowenstein, C. S. Glatt, D. S. Bredt, S. H. Snyder, ibid., p. 6711). Transfections were performed with 10 μg of each plasmid with the calcium phosphate method (28) After transfection, cells were sonicated in buffer A [50 mM tris-HCl (pH 7.7), 100 mM NaCl, 2 mM EDTA, and 1% Triton X-100] and cleared by centrifugation. This cellular lysate was incubated with GST or GST-PIN immobilized on glutathione-agarose and then washed extensively in HNTG buffer [20 mM Hepes (pH 7.4), 500 mM NaCl, 10% glycerol, and 0.1% Triton X-100], For assays testing PIN binding to immobilized NOS, 20 μg of bacterial lysate was added to 200 μg of transfected HEK 293 cell lysate and bound to 2′,5′-ADP-Sepharose 4B (Pharmacia) and subsequently washed and eluted with 10 mM NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) as described [D. S. Bredt and S. H. Snyder, ibid. 87, 682 (1990)]. The eluate was immunoblotted with a rabbit antibody to GST (NovaCastra, Burtingame, CA). For blot-overlay analysis, pGEX-4T2 was modified such that two sites for protein kinase A (PKA) encoded on complementary synthetic oligonucleotides (5′-AATTCGTCGTGCATCTGTTGAACTACGTCGAGCTTCAGTTGCG-3′, upper strand) were ligated into the Eco Rl-Sal I sites to generate plasmid pGEX4T-2K. Kinase reactions and blot overlays were performed as described [W. M. Kavanaugh and L. T. Williams, Science 266, 1862 (1994)].
27. The cDNA for PIN was excised from pAD-PIN with Sal I and Not I and cloned into those sites in pGEX-4T2 (Pharmacia). Fusion proteins were prepared in Escherichia coli BL21(DE3) (Novagen) with glutathione-agarose (Sigma) [D. B. Smith and K. S. Johnson, Gene 67, 31 (1988)], except that bacteria pellets were lysed in lysis buffer [50 mM tris-HCI (pH 7.7). 100 mM NaCl, and 2 mM EDTA], supernatants were adjusted to 1% Triton X-100, and protein was purified with elution buffer [50 mM tris-HCl (pH 7.7), 100 mM NaCl, 10 mM reduced glutathione, and 2 mM EDTA]. HEK 293 cells were transfected with plasmids for nNOS [D. S. Bredt et al., Nature 351, 714 (1991)], eNOS [S. Lamas, P. A. Marsden, G. K. Li, P. Tempst, T. Michel, Proc. Natl. Acad. Sci. U.S.A 89, 6348 (1992)]. and iNOS (C. J. Lowenstein, C. S. Glatt, D. S. Bredt, S. H. Snyder, ibid., p. 6711). Transfections were performed with 10 μg of each plasmid with the calcium phosphate method (28) After transfection, cells were sonicated in buffer A [50 mM tris-HCl (pH 7.7), 100 mM NaCl, 2 mM EDTA, and 1% Triton X-100] and cleared by centrifugation. This cellular lysate was incubated with GST or GST-PIN immobilized on glutathione-agarose and then washed extensively in HNTG buffer [20 mM Hepes (pH 7.4), 500 mM NaCl, 10% glycerol, and 0.1% Triton X-100], For assays testing PIN binding to immobilized NOS, 20 μg of bacterial lysate was added to 200 μg of transfected HEK 293 cell lysate and bound to 2′,5′-ADP-Sepharose 4B (Pharmacia) and subsequently washed and eluted with 10 mM NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) as described [D. S. Bredt and S. H. Snyder, ibid. 87, 682 (1990)]. The eluate was immunoblotted with a rabbit antibody to GST (NovaCastra, Burtingame, CA). For blot-overlay analysis, pGEX-4T2 was modified such that two sites for protein kinase A (PKA) encoded on complementary synthetic oligonucleotides (5′-AATTCGTCGTGCATCTGTTGAACTACGTCGAGCTTCAGTTGCG-3′, upper strand) were ligated into the Eco Rl-Sal I sites to generate plasmid pGEX4T-2K. Kinase reactions and blot overlays were performed as described [W. M. Kavanaugh and L. T. Williams, Science 266, 1862 (1994)].
28. The cDNA for PIN was excised from pAD-PIN with Sal I and Not I and cloned into those sites in pGEX-4T2 (Pharmacia). Fusion proteins were prepared in Escherichia coli BL21(DE3) (Novagen) with glutathione-agarose (Sigma) [D. B. Smith and K. S. Johnson, Gene 67, 31 (1988)], except that bacteria pellets were lysed in lysis buffer [50 mM tris-HCI (pH 7.7). 100 mM NaCl, and 2 mM EDTA], supernatants were adjusted to 1% Triton X-100, and protein was purified with elution buffer [50 mM tris-HCl (pH 7.7), 100 mM NaCl, 10 mM reduced glutathione, and 2 mM EDTA]. HEK 293 cells were transfected with plasmids for nNOS [D. S. Bredt et al., Nature 351, 714 (1991)], eNOS [S. Lamas, P. A. Marsden, G. K. Li, P. Tempst, T. Michel, Proc. Natl. Acad. Sci. U.S.A 89, 6348 (1992)]. and iNOS (C. J. Lowenstein, C. S. Glatt, D. S. Bredt, S. H. Snyder, ibid., p. 6711). Transfections were performed with 10 μg of each plasmid with the calcium phosphate method (28) After transfection, cells were sonicated in buffer A [50 mM tris-HCl (pH 7.7), 100 mM NaCl, 2 mM EDTA, and 1% Triton X-100] and cleared by centrifugation. This cellular lysate was incubated with GST or GST-PIN immobilized on glutathione-agarose and then washed extensively in HNTG buffer [20 mM Hepes (pH 7.4), 500 mM NaCl, 10% glycerol, and 0.1% Triton X-100], For assays testing PIN binding to immobilized NOS, 20 μg of bacterial lysate was added to 200 μg of transfected HEK 293 cell lysate and bound to 2′,5′-ADP-Sepharose 4B (Pharmacia) and subsequently washed and eluted with 10 mM NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) as described [D. S. Bredt and S. H. Snyder, ibid. 87, 682 (1990)]. The eluate was immunoblotted with a rabbit antibody to GST (NovaCastra, Burtingame, CA). For blot-overlay analysis, pGEX-4T2 was modified such that two sites for protein kinase A (PKA) encoded on complementary synthetic oligonucleotides (5′-AATTCGTCGTGCATCTGTTGAACTACGTCGAGCTTCAGTTGCG-3′, upper strand) were ligated into the Eco Rl-Sal I sites to generate plasmid pGEX4T-2K. Kinase reactions and blot overlays were performed as described [W. M. Kavanaugh and L. T. Williams, Science 266, 1862 (1994)].
29. The cDNA for PIN was excised from pAD-PIN with Sal I and Not I and cloned into those sites in pGEX-4T2 (Pharmacia). Fusion proteins were prepared in Escherichia coli BL21(DE3) (Novagen) with glutathione-agarose (Sigma) [D. B. Smith and K. S. Johnson, Gene 67, 31 (1988)], except that bacteria pellets were lysed in lysis buffer [50 mM tris-HCI (pH 7.7). 100 mM NaCl, and 2 mM EDTA], supernatants were adjusted to 1% Triton X-100, and protein was purified with elution buffer [50 mM tris-HCl (pH 7.7), 100 mM NaCl, 10 mM reduced glutathione, and 2 mM EDTA]. HEK 293 cells were transfected with plasmids for nNOS [D. S. Bredt et al., Nature 351, 714 (1991)], eNOS [S. Lamas, P. A. Marsden, G. K. Li, P. Tempst, T. Michel, Proc. Natl. Acad. Sci. U.S.A 89, 6348 (1992)]. and iNOS (C. J. Lowenstein, C. S. Glatt, D. S. Bredt, S. H. Snyder, ibid., p. 6711). Transfections were performed with 10 μg of each plasmid with the calcium phosphate method (28) After transfection, cells were sonicated in buffer A [50 mM tris-HCl (pH 7.7), 100 mM NaCl, 2 mM EDTA, and 1% Triton X-100] and cleared by centrifugation. This cellular lysate was incubated with GST or GST-PIN immobilized on glutathione-agarose and then washed extensively in HNTG buffer [20 mM Hepes (pH 7.4), 500 mM NaCl, 10% glycerol, and 0.1% Triton X-100], For assays testing PIN binding to immobilized NOS, 20 μg of bacterial lysate was added to 200 μg of transfected HEK 293 cell lysate and bound to 2′,5′-ADP-Sepharose 4B (Pharmacia) and subsequently washed and eluted with 10 mM NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) as described [D. S. Bredt and S. H. Snyder, ibid. 87, 682 (1990)]. The eluate was immunoblotted with a rabbit antibody to GST (NovaCastra, Burtingame, CA). For blot-overlay analysis, pGEX-4T2 was modified such that two sites for protein kinase A (PKA) encoded on complementary synthetic oligonucleotides (5′-AATTCGTCGTGCATCTGTTGAACTACGTCGAGCTTCAGTTGCG-3′, upper strand) were ligated into the Eco Rl-Sal I sites to generate plasmid pGEX4T-2K. Kinase reactions and blot overlays were performed as described [W. M. Kavanaugh and L. T. Williams, Science 266, 1862 (1994)].
30. Rat cerebella were homogenized in IP buffer [50 mM tris-HCl (pH 7.7), 100 mM NaCl] and clarified by centrifugation, Washes were performed with wash buffer 1 [50 mM tris-HCl (pH 7.7), 500 mM NaCl] and wash buffer 2 [50 mM tris-HCl (pH 7.7), 500 mM LiCl]. The rabbit polyclonal antibody to the hemagglutinin epitope (HA) was from BAbCO (Richmond, CA). A rabbit antiserum to PIN was generated to a hexahisitidine-tagged fusion protein containing the full-length PIN sequence (23). The antiserum was used in immunoblots at a dilution of 1:1000 and recognizes a band of the expected molecular size. Preincubation of the antiserum with GST-PIN confirmed the specificity of this antiserum (10).
31. 2-terminal Myc tag followed by a pentaglycine linker and the PIN insert.
32. 2, and 1% Triton X-100] for 16 hours at 37°C. The eluate was adjusted to 5 mM EGTA, 4 μM leupeptin, and 400 nM aprotinin. Dilutions were made with thrombin cleavage buffer adjusted in this manner. A GST-BIRK fusion consisting of amino acids 347 to 442 of BIRK2 [D. S. Bredt et al., Proc. Natl. Acad. Sci. U.S.A. 92, 6753 (1995)] was cleaved with thrombin as above and used as a control protein in NOS assays. A different preparation with a bacterially expressed hexahistidinetagged PIN fusion protein inhibited nNOS activity and dimerization in a similar concentration-dependent manner (10).
33. J. M. Hevel et al., J. Biol. Chem. 266, 289 (1991); H. H. H. W. Schmidt et al., Proc. Natl. Acad. Sci. U.S.A. 88, 365 (1991); D. J. Stuehr et al., ibid., p. 7773.
34. J. M. Hevel et al., J. Biol. Chem. 266, 289 (1991); H. H. H. W. Schmidt et al., Proc. Natl. Acad. Sci. U.S.A. 88, 365 (1991); D. J. Stuehr et al., ibid., p. 7773.
35. J. M. Hevel et al., J. Biol. Chem. 266, 289 (1991); H. H. H. W. Schmidt et al., Proc. Natl. Acad. Sci. U.S.A. 88, 365 (1991); D. J. Stuehr et al., ibid., p. 7773.
36. P. Watt et al., EMBO J. 14, 3687 (1995).
37. P. Klatt et al., J. Biol. Chem. 269, 13861 (1994).
38. T. Dick, K. Ray, H. K. Salz, W. Chia, Mol. Cell. Biol. 16, 1966 (1996).
39. J. Sambrcok, E. F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
40. 2.
41. J. Dinerman et al., Neuropharmacology 33, 1245 (1994).
42. We thank E. Fung for the yeast strain and plasmids; V. Dawson and T. Dawson for antibodies and advice; T. Michel for the eNOS plasmid; C. Lowenstein for the iNOS plasmid; A. Lanahan and P. Worley for the rat hippocampal cDNA library and the CMV expression vector; D. Sabatini for RAFT constructs; N. Cohen for BIRK constructs; A. Snowman and K. Collins for technical assistance; and D. Bredt, J. Huang, D. Sabatini, T. Schroer, S. Voglmaier, and R. Zakhary for helpful comments and suggestions. Supported by USPHS grant DA00266 and Research Scientist Award DA00074 (S.H.S.), and GM-07309 (S.R.J.).