The structure of hemocyanin II from the horseshoe crab, Limulus polyphemus. The amino acid sequence of the second largest cyanogen bromide fragment

Fourteen fragments have been isolated from hemocyanin component II of Limulus polyphemus by cleavage with CNBr. The amino acid sequence of the largest fragment, CNBr Ia has been reported (Yokota, E., and Riggs, A. F. (1984) J. Biol. Chem. 259, 4739-4749). The amino acid sequence of the 12 smaller fragments is reported in an accompanying paper (Moore, M. D., Behrens, P. Q., and Riggs, A. F. (1985) J. Biol. Chem. 261, 10511-10519). We have determined the amino acid sequence of the second largest fragment, CNBr Ib. The fragment contains 142 residues and has a molecular weight of 16,095.     

Structure of hemocyanin II from the horseshoe crab, Limulus polyphemus. Sequences of the overlapping peptides, ordering the CNBr fragments, and the complete amino acid sequence

The amino acid sequence of the largest fragment, CNBr Ia (203 residues) has been reported (Yokota, E., and Riggs, A. F. (1984) J. Biol. Chem. 259, 4739-4749). The amino acid sequences of the second largest fragment, CNBr Ib (142 residues), and of the 12 smaller fragments are reported in accompanying papers (Moore, M. D., Behrens, P. Q., and Riggs, A. F. (1986) J. Biol. Chem. 261, 10511-10519; Behrens, P. Q., Nakashima, H., and Riggs, A. F. (1986) J. Biol. Chem. 261, 10520-10525). The complete amino acid sequence of hemocyanin component II has been established by isolation and analysis of 13 methionine-containing peptides from either a tryptic digest or a Staphylococcus aureus strain V8 protease digest of whole carboxamidomethylated hemocyanin II. Hemocyanin II is composed of 628 residues and has a molecular weight with two copper atoms of 72,946.     

Characterization of the Taxol binding site on the microtubule. Identification of Arg(282) in beta-tubulin as the site of photoincorporation of a 7-benzophenone analogue of Taxol

Photoaffinity labeling methods have allowed a definition of the sites of interaction between Taxol and its cellular target, the microtubule, specifically beta-tubulin. Our previous studies have indicated that [(3)H]3'-(p-azidobenzamido)Taxol photolabels the N-terminal 31 amino acids of beta-tubulin (Rao, S., Krauss, N. E., Heerding, J. M., Swindell, C. S., Ringel, I., Orr, G. A., and Horwitz, S. B. (1994) J. Biol. Chem. 269, 3132-3134) and [(3)H]2-(m-azidobenzoyl)Taxol photolabels a peptide containing amino acid residues 217-233 of beta-tubulin (Rao, S., Orr, G. A., Chaudhary, A. G., Kingston, D. G. I., and Horwitz, S. B. (1995) J. Biol. Chem. 270, 20235-20238). The site of photoincorporation of a third photoaffinity analogue of Taxol, [(3)H]7-(benzoyldihydrocinnamoyl) Taxol, has been determined. This analogue stabilizes microtubules polymerized in the presence of GTP, but in contrast to Taxol, does not by itself enhance the polymerization of tubulin to its polymer form. CNBr digestion of [(3)H]7-(benzoyldihydrocinnamoyl)Taxol-labeled tubulin, with further arginine-specific cleavage by clostripain resulted in the isolation of a peptide containing amino acid residues 277-293. Amino acid sequence analysis indicated that the photoaffinity analogue cross-links to Arg(282) in beta-tubulin. Advances made by electron crystallography in understanding the structure of the tubulin dimer have allowed us to visualize the three sites of photoincorporation by molecular modeling. There is good agreement between the binding site of Taxol in beta-tubulin as determined by photoaffinity labeling and electron crystallography.     

Studies on adenosine triphosphate transphosphorylases. Amino acid sequence of rabbit muscle ATP-AMP transphosphorylase

The total amino acid sequence of rabbit muscle adenylate kinase has been determined, and the single polypeptide chain of 194 amino acid residues starts with N-acetylmethionine and ends with leucyllysine at its carboxyl terminus, in agreement with the earlier data on its amino acid composition [Mahowald, T. A., Noltmann, E. A., & Kuby, S. A. (1962) J. Biol. Chem. 237, 1138-1145] and its carboxyl-terminus sequence [Olson, O. E., & Kuby, S. A. (1964) J. Biol. Chem. 239, 460-467]. Elucidation of the primary structure was based on tryptic and chymotryptic cleavages of the performic acid oxidized protein, cyanogen bromide cleavages of the 14C-labeled S-carboxymethylated protein at its five methionine sites (followed by maleylation of peptide fragments), and tryptic cleavages at its 12 arginine sites of the maleylated 14C-labeled S-carboxymethylated protein. Calf muscle myokinase, whose sequence has also been established, differs primarily from the rabbit muscle myokinase's sequence in the following: His-30 is replaced by Gln-30; Lys-56 is replaced by Met-56; Ala-84 and Asp 85 are replaced by Val-84 and Asn-85. A comparison of the four muscle-type adenylate kinases, whose covalent structures have now been determined, viz., rabbit, calf, porcine, and human [for the latter two sequences see Heil, A., Müller, G., Noda, L., Pinder, T., Schirmer, H., Schirmer, I., & Von Zabern, I. (1974) Eur. J. Biochem. 43, 131-144, and Von Zabern, I., Wittmann-Liebold, B., Untucht-Grau, R., Schirmer, R. H., & Pai, E. F. (1976) Eur. J. Biochem. 68, 281-290], demonstrates an extraordinary degree of homology.(ABSTRACT TRUNCATED AT 250 WORDS)     

The amino acid sequences of chains a, b, and c that form the trimer subunit of the extracellular hemoglobin from Lumbricus terrestris

The extracellular hemoglobin of Lumbricus terrestris comprises four major heme-containing chains, a, b, c, and d in equal proportions. We have determined the amino acid sequences of chains a, b, and c which form a disulfide-linked trimer. Chains a, b, and c have 151, 145, and 153 residues and calculated molecular weights of 17,525, 16,254, and 17,289, respectively. The sequence of chain b, reported previously (Garlick, R. L., and Riggs, A. F. (1982) J. Biol. Chem. 287, 9005-9015) has been completely redetermined and found to contain 12 fewer residues than originally reported. Chains a and c both contain unusual, highly polar NH2-terminal extensions of 7 residues before the A helix. These segments must be close together because they are joined by a disulfide bond. We suggest that this structure, with seven negatively charged groups, may be part of a functionally important Ca2+-binding site in the trimer. Comparison of the sequences of chains a, b, and c with those of chain d (Shishikura, F., Snow, J. W., Gotoh, T., Vinogradov, S. N., and Walz, D. A. (1987) J. Biol. Chem. 262, 3123-3131) and the four chains of the hemoglobin of Tylorrhynchus heterochaetus (Suzuki, T., and Gotoh, T. (1986) J. Biol. Chem. 261, 9257-9267) shows that the number and positions of the cysteinyl residues are all conserved. This suggests that the extracellular hemoglobins from both the Oligochaeta and Polychaeta have the same number and configuration of disulfide bonds within the molecule. Phylogenetic analysis suggests that gene duplication first generated an intracellular hemoglobin branch and an extracellular hemoglobin branch. DNA coding for a signal peptide would have been acquired by the extracellular globin gene after this event. At least two further gene duplications are required to account for the present four polypeptide chains.     

Identification and primary structure of the cardiolipin-binding domain of mitochondrial creatine kinase

It was recently shown that the mitochondrial isozyme of heart creatine kinase binds to cardiolipin on the outer half of the inner membrane [Müller, M., et al. (1985) J. Biol. Chem. 260, 3839-3843]. The enzyme has now been extracted and purified to homogeneity from rat heart mitochondria, and cleaved with CNBr. The fragments have been separated on an FPLC system using a Mono Q HR 5/5 column. Only one of these binds to cardiolipin-containing liposomes and has thus been identified as the cardiolipin-binding domain of the enzyme. Its amino acid sequence has been determined. The fragment contains 25 amino acids and corresponds to the N-terminal region of the protein. The binding of the fragment of cardiolipin-containing liposomes was inhibited by adriamycin. Another and larger CNBr fragment could be specifically labelled with periodate-oxidized (di-aldehyde) ATP and has thus been identified as the ATP-binding domain. Chemical modification of the basic amino acids Lys and Arg of the enzyme abolished its binding to cardiolipin.     

Isolation and properties of a fragment of human serum albumin demonstrating the absence of a methionine residue from some of the albumin molecules.

1. A fragment designated D was isolated from human serum albumin degraded by CNBr. Its properties show that it is made up of the B and C fragments isolated by McMenamy et al. (1971) (J. Biol. Chem., 246, 4744-4750). 2. Reduction of fragment D gives rise to two chains, one of which consists of the second subfragment of reduced fragment B linked to fragment C by an amino acid different from methionine. It thus demonstrates the existence of albumin molecules from which the second methionine residue located between fragments B and C is missing.     

Amino acid sequence of the monomer subunit of the extracellular hemoglobin of Lumbricus terrestris

The giant extracellular hemoglobin (3,800 kDa) of the oligochaete Lumbricus terrestris consists of four subunits: a monomer (chain I), two subunits each of about 35 kDa (chains V and VI), and a disulfide-bonded trimer (50 kDa) of chains II, III, and IV. The complete amino acid sequence of chain I was determined: it consists of 142 amino acid residues and has a molecular weight of 16,750 including a heme group. Fifty-nine residues (42%) were found to be identical with those in the corresponding positions in Lumbricus chain II (Garlick, R. L., and Riggs, A. F. (1982) J. Biol. Chem. 257, 9005-9015); 45 (32%), 56 (40%), 44 (31%), and 45 (32%) residues were found to be in identical positions in the sequences of chains I, IIA, IIB, and IIC, respectively, of Tylorrhynchus heterochaetus hemoglobin (Suzuki, T., and Gotoh, T. (1986) J. Biol. Chem. 261, 9257-9267). When the sequences of all six annelid chains are compared, 18 invariant residues are found in the first 104 residues of the molecule; very little homology exists among the annelid chains in the carboxyl-terminal 38-residue region. Nine of the 18 invariant residues are also found in the human beta-globin chain.     

Purification and molecular cloning of succinyltransferase of the rat alpha-ketoglutarate dehydrogenase complex. Absence of a sequence motif of the putative E3 and/or E1 binding site

Full-length cDNA clones for succinyltransferase of the rat alpha-ketoglutarate dehydrogenase complex were isolated from rat heart cDNA libraries in lambda gt11. The cDNA clones were identified as those for rat succinyltransferase by the identity of their predicted amino acid sequence with the NH2-terminal amino acid sequence of rat succinyltransferase determined by protein chemical analysis and the known amino acid sequence of bovine succinyltransferase. The clone with the longest cDNA consisted of 2747 base pairs and coded for a leader peptide of 56 amino acid residues and a mature protein of 386 amino acid residues. The primary structure of rat succinyltransferase showed close similarity to Escherichia coli and Azotobacter vinelandii succinyltransferases, in the COOH-terminal part forming the lipoyl-binding domain and the NH2-terminal part forming the inner core-catalytic domain. However, the rat succinyltransferase did not contain a sequence motif that has been found as an E3- and/or E1-binding site in the dihydrolipoamide acyltransferases of three alpha-ketoacid dehydrogenase complexes (Hummel, K. B., Litwer, S., Bradford, A. P., Aitken, A., Danner, D. J., and Yeaman, S. J. (1988) J. Biol. Chem. 263, 6165-6168, Reed, L. J., and Hackert, M. L. (1990) J. Biol. Chem. 265, 8971-8974). The absence of this sequence was confirmed by direct sequencing of the polymerase chain reaction product of rat heart mRNA and by computer analysis. These results show that the rat succinyltransferase does not have the sequence motif of the putative E3- and/or E1-binding site.     

Rat liver glutathione S-transferases. Nucleotide sequence analysis of a Yb1 cDNA clone and prediction of the complete amino acid sequence of the Yb1 subunit

We have constructed a nearly full length cDNA clone, pGTA/C44, complementary to the rat liver glutathione S-transferase Yb1 mRNA. The nucleotide sequence of pGTA/C44 has been determined, and the complete amino acid sequence of the Yb1 subunit has been deduced. The cDNA clone contains an open reading frame of 654 nucleotides encoding a polypeptide comprising 218 amino acids with Mr = 25,919. The NH2-terminal sequence deduced from DNA sequence analysis of pGTA/C44 is in agreement with the first 19 amino acids determined for purified glutathione S-transferase A, a Yb1 homodimer, by Frey et al. (Frey, A. B., Friedberg, T., Oesch, F., and Kreibich, G. (1983) J. Biol. Chem. 258, 11321-11325). The DNA sequence of pGTA/C44 shares significant sequence homology with a cDNA clone, pGT55, which is complementary to a mouse liver glutathione S-transferase (Pearson, W. R., Windle, J. J., Morrow, J. F., Benson, A. M., and Talalay, P. (1983) J. Biol. Chem. 258, 2052-2062). We have also determined 37 nucleotides of the 5'-untranslated region and 348 nucleotides of the 3'-untranslated region of the Yb1 mRNA. The Yb1 mRNA and subunit do not share any sequence homology with the rat liver glutathione S-transferase Ya or Yc mRNAs or their corresponding subunits. These data provide the first direct evidence that the Yb1 subunit is derived from a gene or gene family which is distinct from the Ya-Yc gene family.     

Pyridoxal 5'-phosphate-dependent histidine decarboxylase. Nucleotide sequence of the hdc gene and the corresponding amino acid sequence

The nucleotide sequence of a 1.3-kilobase NaeI fragment from Morganella morganii AM-15 that contains the gene for histidine decarboxylase has been determined. The gene was initially identified among total chromosomal digests using a mixed sequence oligonucleotide probe corresponding to amino acids 11-16 of histidine decarboxylase and then cloned on a 5.5-kilobase PstI fragment. The structural gene contains 1131 nucleotides and encodes 377 amino acids with the sequence: (sequence: in text). The independently determined NH2-terminal sequence of this enzyme (Tanase, S., Guirard, B. M., and Snell, E. E. (1985) J. Biol. Chem. 260, 6738-6746) and the amino acid sequences of two tryptic peptides reported in the accompanying paper (Hayashi, H., Tanase, S., and Snell, E. E. (1986) J. Biol. Chem. 261, 11003-11009) are localized in the sequence presented here; the lysine that binds pyridoxal phosphate is situated at residue 232, whereas the serine that binds the adduct formed between pyridoxal phosphate and the inhibitor alpha-fluoromethylhistidine is positioned at residue 322.     

The primary structure of the basic isoform of Acanthamoeba profilin

Acanthamoeba profilin-II [Kaiser, D.A., Sato, M., Ebert, R. F. and Pollard, T.D. (1986) J. Cell. Biol. 102, 221-226] was digested with trypsin or cleaved by 2-(2-nitrophenylsulphenyl)-3-methyl-3-bromoindolenine. The tryptic peptides were purified by reversed-phase-high-performance liquid chromatography and completely sequenced using automated gas-phase sequence analysis. The complete profilin-II sequence was deduced by ordering the tryptic peptides using the sequence information of the tryptophan-cleavage products. Acanthamoeba profilin-II was found to be homologous to the previously determined profilin-I sequence [Ampe, C., Vandekerckhove, J., Brenner, L., Tobacman, L. and Korn, E.D. (1985) J. Biol. Chem. 260, 834-840]. Like profilin-I, profilin-II consists of 125 amino acids, has a blocked NH2 terminus and a trimethyllysine residue at position 103. Profilin-II differs in at least 21 positions from one of the profilin-I isoforms. The amino acid exchanges are mainly concentrated in the middle part of the sequence. Profilin-II contains two more basic residues than profilin-I, which explains its higher isoelectric point.     

Identification of substrate recognition determinants for human ERK1 and ERK2 protein kinases

Two epidermal growth factor-stimulated protein kinases that correspond to ERK1 and ERK2 have been purified from human epidermoid carcinoma cells (Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276). A consensus primary sequence for substrates of ERK1 has been identified as -Pro-Leu-Ser/Thr-Pro- (Alvarez, E., Northwood, I. C., Gonzalez, F. A., Latour, D. A., Seth, A., Abate, C., Curran, T., and Davis, R. J. (1991) J. Biol. Chem. 266, 15277-15285). However, the structural determinants for substrate recognition are not understood. We performed a systematic analysis of the effect of point mutations in the primary sequence of peptide substrates on the rate of phosphorylation by ERK1 and ERK2. The results of this investigation demonstrate that the substrate specificities of the ERK1 and ERK2 protein kinases are very similar. We propose that the primary sequence of substrates for ERK1 and ERK2 protein kinases can be generalized as -Pro-Xaan-Ser/Thr-Pro- (where Xaa is a neutral or basic amino acid and n = 1 or 2).     

cDNA cloning and inducible expression during pregnancy of the mRNA for rabbit pulmonary prostaglandin omega-hydroxylase (cytochrome P-450p-2)

We have isolated cDNA clones of the mRNA for prostaglandin omega-hydroxylase (cytochrome P-450p-2) (Yamamoto, S., Kusunose, E., Ogita, K., Kaku, M., Ichihara, K., and Kusunose, M. (1984) J. Biochem. (Tokyo) 96, 593-603) in rabbit lung by using synthetic oligonucleotides as probes. The cDNA sequence contains an open reading frame of 1,470 nucleotides, the first 9 amino acids of which correspond to the residues 17-25 of cytochrome P-450p-2 determined from protein analysis. The predicted primary structure contains amino acid sequences of 23 tryptic fragments of cytochrome P-450p-2 and the deduced amino acid composition is in agreement with that determined from the purified protein. The complete polypeptide, including residues 1-16, contains 506 amino acids with a calculated molecular weight of 58,515. Cytochrome P-450p-2 shared 74% amino acid similarity with rat hepatic lauric acid omega-hydroxylase (cytochrome P-450LA omega) (Hardwick, J.P., Song, B.-J., Huberman, E., and Gonzalez, F. J. (1987) J. Biol. Chem. 262, 801-810), whereas it showed less than 25% similarity to other forms of cytochrome P-450, indicating that the two cytochrome P-450s constitute a unique cytochrome P-450 gene family. DNA blot analysis of the total genomic DNA of rabbits suggest the presence of several genes or gene-like DNA sequences which cross-hybridized with the cloned cDNA. RNA blot analysis showed that progesterone treatment increased the amount of mRNA hybridizable to the cDNA by about 100-fold in the lung of rabbits as compared with the basal level without the treatment. This high level of the mRNA was also observed in the lung of pregnant rabbits.     

The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. Peptic and chymotryptic peptides and the complete sequence.

Peptic and chymotryptic peptides were isolated form the NADP-specific glutamate dehydrogenase of Neurospora crassa and substantially sequenced. Out of 452 residues in the polypeptide chain, 265 were recovered in the peptic and 427 in the chymotryptic peptides. Together with the tryptic peptides [Wootton, J. C., Taylor, J. G., Jackson, A. A., Chambers, G. K. & Fincham, J. R. S. (1975) Biochem. J. 149, 749-755], these establish the complete sequence of the chain, including the acid and amide assignments, except for seven places where overlaps are inadequate. These remaining alignments are deduced from information on the CNBr fragments obtained in another laboratory [Blumenthal, K. M., Moon, K. & Smith, E. L. (1975), J. Biol. Chem. 250, 3644-3654]. Further information has been deposited as Supplementary Publication SUP 50054 (17 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem. J. (1975) 145, 5.     

Histidine decarboxylase of Lactobacillus 30a. Sequences of the overlapping peptides, the complete alpha chain, and prohistidine decarboxylase

The complete amino acid sequence of the alpha chain of histidine decarboxylase of Lactobacillus 30a has been established by isolation and analysis of the eight methionine-containing tryptic peptides of this chain. These peptides provide the overlaps required to order all nine peptides derived by complete cyanogen bromide cleavage of the alpha chain (Huynh, Q.K., Vaaler, G.L., Recsei, P.A., and Snell, E.E. (1984) J. Biol. Chem. 259, 2826-2832). Ordering of six of the latter peptides was confirmed by isolation and analysis of four peptides derived by incomplete cyanogen bromide cleavage. The alpha chain is composed of 226 residues and has a molecular weight of 24,892 calculated from the sequence. These results and the previously determined sequence of the beta chain (Vaaler, G.L., Recsei, P.A., Fox, J.L., and Snell, E.E. (1982) J. Biol. Chem. 257, 12770-12774) establish the complete amino acid sequence of the enzyme and of the pi chain of prohistidine decarboxylase. The latter is composed of 307 amino acids and has a calculated molecular weight of 33,731. Four segments of the pi chain sequence are repeated. The bond between Ser-81 and Ser-82 that is cleaved during proenzyme activation is in an uncharged portion of the sequence that is rich in serine and threonine residues and is predicted to be part of a beta sheet structure.     

Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora crassa. Isolation and sequences of several cyanogen bromide peptides from the NH2-terminal portion of the peptide chain

Previous studies of the amino acid sequence of the NAD-specific glutamate dehydrogenase of Neurospora crassa (EC 1.4.1.2) resulted in the assignments of peptides to four fragments, the longest being the COOH-terminal 669 residues of the protein. A further study of peptides derived by cyanogen bromide cleavage by different separation methods has yielded additional peptides that have provided new information concerning the sequence and has given overlaps of previously known sequences. This has permitted establishment of 313 residues in one sequence (fragment II). This is in addition to a sequence of 43 residues (fragment I) at the NH2-terminal end and a sequence of 669 residues (fragment III) previously established at the COOH-terminal end of the molecule. The present status of our knowledge of the overall sequence is given in the accompanying papers, together with some views regarding the conformation of the protein (Haberland, M.E., Chen, C.-W., and Smith, E.L. (1980) J. Biol. Chem. 255, 7993-8000, and Austen, B.M., Haberland, M.E., and Smith, E.L. (1980) J. Biol. Chem. 255, 8001-8004).     

Location of the inhibitory region of smooth muscle myosin light chain kinase

Proteolysis by trypsin of gizzard myosin light chain kinase (MLC kinase) in the absence of Ca2+-calmodulin produced a 64,000-dalton inactive fragment which was converted to a 61,000-dalton Ca2+-calmodulin-independent active fragment. This confirmed previous results (Ikebe, M., Stepinska, M., Kemp, B. E., Means, A. R., and Hartshorne, D. J. (1987) J. Biol. Chem. 262, 13828-13834). On the other hand, proteolysis of MLC kinase in the presence of Ca2+-calmodulin initially produced a 66,000-dalton Ca2+-calmodulin-dependent active fragment which was converted to a 61,000-dalton Ca2+-calmodulin-independent active fragment with further proteolysis. The amino acid sequences from the N terminus of the 66,000-dalton, 64,000-dalton, and 61,000-dalton fragments were determined. The sequence was not found in the reported partial amino acid sequence of MLC kinase (C-terminal 60% of whole sequence) (Guerriero, V., Jr., Russo, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381), and, therefore, the cleavage sites are in the remaining 40% N-terminal portion of the sequence of MLC kinase. The C terminus of these MLC kinase fragments was determined by employing the carboxypeptidases A, B, and Y digestion followed by the amino acid analysis of the released amino acids. As a result, it was concluded that the C terminus of the 66,000-dalton, 64,000-dalton, and 61,000-dalton MLC kinase fragments are arginine 522, lysine 490 and arginine 494, and lysine 473, respectively. These results show that the inhibitory domain is in the amino acid sequence of 474-490, and that the amino acid sequence 494-522 confers the calmodulin-dependent kinase activity.     

Lysophospholipids open the two-pore domain mechano-gated K(+) channels TREK-1 and TRAAK

The two-pore (2P) domain K(+) channels TREK-1 and TRAAK are opened by membrane stretch as well as arachidonic acid (AA) (Patel, A. J., Honoré, E., Maingret, F., Lesage, F., Fink, M., Duprat, F., and Lazdunski, M. (1998) EMBO J. 17, 4283-4290; Maingret, F., Patel, A. J., Lesage, F., Lazdunski, M., and Honoré, E. (1999) J. Biol. Chem. 274, 26691-26696; Maingret, F., Fosset, M., Lesage, F., Lazdunski, M. , and Honoré, E. (1999) J. Biol. Chem. 274, 1381-1387. We demonstrate that lysophospholipids (LPs) and platelet-activating factor also produce large specific and reversible activations of TREK-1 and TRAAK. LPs activation is a function of the size of the polar head and length of the acyl chain but is independent of the charge of the molecule. Bath application of lysophosphatidylcholine (LPC) immediately opens TREK-1 and TRAAK in the cell-attached patch configuration. In excised patches, LPC activation is lost, whereas AA still produces maximal opening. The carboxyl-terminal region of TREK-1, but not the amino terminus and the extracellular loop M1P1, is critically required for LPC activation. LPC activation is indirect and may possibly involve a cytosolic factor, whereas AA directly interacts with either the channel proteins or the bilayer and mimics stretch. Opening of TREK-1 and TRAAK by fatty acids and LPs may be an important switch in the regulation of synaptic function and may also play a protective role during ischemia and inflammation.