Mutations within the selectivity filter of the NMDA receptor-channel influence voltage dependent block by 5-hydroxytryptamine.

BACKGROUND AND PURPOSE: Voltage-dependent block by Mg2+ is a cardinal feature of NMDA receptors which acts as a coincidence detector to prevent the receptor from over-activation. Inhibition of NMDA receptor currents by 5-hydroxytryptamine (5-HT) indicated that 5-HT, similar to Mg2+, binds within the membrane electric field. In the present study, we assessed whether point mutations of critical asparagine residues located within the selectivity filter of NR1 and NR2A subunits of NMDA receptor-channel affect voltage-dependent block by 5-HT. EXPERIMENTAL APPROACH: The mode of action of 5-HT and Mg2+ on wild-type and mutated NMDA receptor-channels expressed in Xenopus oocytes was investigated using the two-electrode voltage clamp recording technique. KEY RESULTS: The mutation within the NR1 subunit NR1(N0S or N0Q) strongly reduced the voltage dependent block by 5-HT and increased the IC50. The corresponding mutations within the NR2 subunits NR2A(N0Q or N+1Q) reduced the block by 5-HT to a lesser extent. This is in contrast to the block produced by external Mg2+ where a substitution at the NR2A(N0) and NR2A(N+1) sites but not at the NR1(N0) site significantly reduced Mg2+ block. CONCLUSION AND IMPLICATIONS: The block of NMDA receptor-channels by 5-HT depends on the NR1-subunit asparagine residue and to a lesser extent on the NR2A-subunit asparagine residues. These data suggest that the interaction of 5-HT with functionally important residues in a narrow constriction of the pore of the NMDA receptor-channel provides a significant barrier to ionic fluxes through the open channel due to energetic factors governed by chemical properties of the binding site and the electric field.

Site-directed spin-labeling analysis of reconstituted Mscl in the closed state.

The mechanosensitive channel from Escherichia coli (Eco-MscL) responds to membrane lateral tension by opening a large, water-filled pore that serves as an osmotic safety valve. In an attempt to understand the structural dynamics of MscL in the closed state and under physiological conditions, we have performed a systematic site-directed spin labeling study of this channel reconstituted in a membrane bilayer. Structural information was derived from an analysis of probe mobility, residue accessibility to O(2) or NiEdda and overall intersubunit proximity. For the majority of the residues studied, mobility and accessibility data showed a remarkable agreement with the Mycobacterium tuberculosis crystal structure, clearly identifying residues facing the large water-filled vestibule at the extracellular face of the molecule, the narrowest point along the permeation pathway (residues 21-26 of Eco-MscL), and the lipid-exposed residues in the peripheral transmembrane segments (TM2). Overall, the present dataset demonstrates that the transmembrane regions of the MscL crystal structure (obtained in detergent and at low pH) are, in general, an accurate representation of its structure in a membrane bilayer under physiological conditions. However, significant differences between the EPR data and the crystal structure were found toward the COOH-terminal end of TM2.

Structural and functional differences between two homologous mechanosensitive channels of Methanococcus jannaschii.

We report the molecular cloning and characterization of MscMJLR, a second type of mechanosensitive (MS) channel found in the archaeon Methanococcus jannaschii. MscMJLR is structurally very similar to MscMJ, the MS channel of M.jannaschii that was identified and cloned first by using the TM1 domain of Escherichia coli MscL as a genetic probe. Although it shares 44% amino acid sequence identity and similar cation selectivity with MscMJ, MscMJLR exhibits other major functional differences. The conductance of MscMJLR of approximately 2 nS is approximately 7-fold larger than the conductance of MscMJ and rectifies with voltage. The channel requires approximately 18 kT for activation, which is three times the amount of energy required to activate MscMJ, but is comparable to the activation energy of Eco-MSCL: Our study indicates that a multiplicity of conductance-wise and energetically well-tuned MS channels in microbial cell membranes may provide for cell survival by the sequential opening of the channels upon challenge with different osmotic cues.

Molecular identification of a mechanosensitive channel in archaea.

The TM1 domain of the large conductance mechanosensitive (MS) channel of Escherichia coli was used as a genetic probe to search the genomic database of the archaeon Methanoccoccus jannashii for MscL homologs. We report that the hypothetical protein MJ0170 of M. jannashii exhibited 38.5% sequence identity with the TM1 domain of Eco-MscL. Moreover, MJ0170 was found to be a conserved homolog of MscS, the second type of E. coli MS channel encoded by the yggB gene. Furthermore, we identified a cluster of charged residues KIKEE in the C-terminus of MJ0170 that strikingly resembled the charged C-terminal amino acid cluster present in Eco-MscL (RKKEE). We cloned and expressed MJ0170 in E. coli, which when reconstituted into liposomes or expressed in the cell membrane of giant E. coli spheroplasts, exhibited similar activity to the bacterial MS channels. Our study suggests that the M. jannashii MS channel and its homologs evolved as a result of gene duplication of the ancestral MscL-like molecule with the TM1 domain remaining the most conserved structural motif among prokaryotic MS channels.

Mechanosensitive channel of Thermoplasma, the cell wall-less archaea: cloning and molecular characterization.

By using a functional approach of reconstituting detergent-solubilized membrane proteins into liposomes and following their function in patch-clamp experiments, we identified a novel mechanosensitive (MS) channel in the thermophilic cell wall-less archaeon Thermoplasma volcanium. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the enriched protein fractions revealed a band of approx 15 kDa comparable to MscL, the bacterial MS channel of large conductance. 20 N-terminal residues determined by protein microsequencing, matched the sequence to an unknown open reading frame in the genome of a related species Thermoplasma acidophilum. The protein encoded by the T. acidophilum gene was cloned and expressed in Escherichia coli and reconstituted into liposomes. When examined for function, the reconstituted protein exhibited properties typical of an MS ion channel: 1) activation by negative pressure applied to the patch-clamp pipet, 2) blockage by gadolinium, and 3) activation by the anionic amphipath trinitrophenol. In analogy to the nomenclature used for bacterial MS channels, the MS channel of T acidophilum was termed MscTA. Secondary structural analysis indicated that similar to MscL, the T. acidophilum MS protein may have two transmembrane domains, suggesting that MS channels of thermophilic Archaea belong to a family of structurally related MscL-like ion channels with two membrane-spanning regions. When the mscTA gene was expressed in the mscL- knockout strain and the MscTA protein reconstituted into liposomes, the gating of MscTA was characterized by very brief openings of variable conductance. In contrast, when the mscTA gene was expressed in the wild-type mscL+ strain of E. coli, the gating properties of the channel resembled MscL. However, the channel had reduced conductance and differed from MscL in its kinetics and in the free energy of activation, suggesting that MscTA and MscL can form functional complexes and/or modulate each other activity. Similar to MscL, MscTA exhibited an increase in activity in liposomes made of phospholipids having shorter acyl chain, suggesting a role of hydrophobic mismatch in the function of prokaryotic MS channels.

Mechanosensitive channels in archaea.

The ubiquity of mechanosensitive (MS) channels triggered a search for their functional homologues in Archaea, the third domain of the phylogenetic tree. Two types of MS channels have been identified in the cell membranes of Haloferax volcanii using the patch clamp technique. Recently MS channels were identified and cloned from two archaeal species occupying different environmental habitats. These studies demonstrate that archaeal MS channels share structural and functional homology with bacterial MS channels. The mechanical force transmitted via the lipid bilayer alone activates all to date known prokaryotic MS channels. This implies the existence of a common gating mechanism for bacterial as well as archaeal MS channels according to the bilayer model. Based on recent evidence that the bilayer model also applies to eukaryotic MS channels, mechanosensory transduction probably originated along with the appearance of the first life forms according to simple biophysical principles. In support of this hypothesis the phylogenetic analysis revealed that prokaryotic MS channels of large and small conductance originated from a common ancestral molecule resembling the bacterial MscL channel protein. Furthemore, bacterial and archaeal MS channels share common structural motifs with eukaryotic channels of diverse function indicating the importance of identified structures to the gating mechanism of this family of channels. The comparative approach used throughout this review should contribute towards understanding of the evolution and molecular basis of mechanosensory transduction in general.

Mechanosensitive ion channels of the archaeon Haloferax volcanii.

Mechanosensitive (MS) ion channels have been documented in a variety of cells belonging to Eukarya and Eubacteria. We report the novel finding of two types of MS ion channels in the cell membrane of the halophilic archaeon Haloferax volcanii, a member of the Archaea that comprise the third phylogenetic domain. The two channels, MscA1 and MscA2, differed in their kinetic properties with MscA1 exhibiting more frequent open-closed transitions than MscA2. Both channels have large conductances that rectify between -40 mV and +40 mV where the conductance of MscA1 ranged from 380 to 680 picosiemens, whereas MscA2 ranged from 850 to 490 picosiemens. Both channels were blocked by submillimolar gadolinium. In addition, the channels of either membrane vesicles or detergent-solubilized membrane proteins remained functional upon reconstitution into artificial liposomes, a result that indicates that these channels are activated by mechanical force transmitted via the lipid bilayer alone. Subsequently a 37-kDa protein corresponding to the MscA1 channel activity was purified. With the possible functional similarity to bacterial MS channels, our finding of MS channels in Archaea emphasizes the ubiquity and importance of these channels in all domains of the evolutionary tree.

Dexamethasone suppresses release of soluble TNF receptors by human monocytes concurrently with TNF-alpha suppression.

Glucocorticoids suppress many monocyte functions, including endotoxin-stimulated release of TNF-alpha. Monocytes also release soluble receptors for TNF (sTNF-R), which can modulate TNF bioactivity. We therefore examined the effects of the glucocorticoid, dexamethasone, on the release of soluble forms of the 55 kDa and 75 kDa receptors for TNF (sTNF-R55 and sTNF-R75) by human monocytes and the human monocytic Mono Mac 6 cell line. Peripheral blood mononuclear cells (PBMC) spontaneously released 406 +/- 181 pg/10(6) cells of sTNF-R75 over 18 h in culture and Mono Mac 6 cells released 554 +/- 29 pg/10(6) cells. Lipopolysaccharide (LPS) exposure increased release of sTNF-R75 by 54 and 217%, respectively. Dexamethasone suppressed both spontaneous and LPS-stimulated release. The effect of dexamethasone was concentration dependent. At 1 mumol/L, dexamethasone suppressed the LPS-stimulated release of sTNF-R75 by 86% in PBMC and by 40% in Mono Mac 6 cells. Neither PBMC nor Mono Mac 6 cells released measurable amounts of sTNF-R55, but spontaneous release of sTNF-R55 from purified human monocytes (55 +/- 2 pg/10(6) cells over 18 h) was reduced by 45% in the presence of dexamethasone. Dexamethasone reduced bioactive TNF in PBMC cultures, as well as immunoassayable TNF-alpha, which indicates that suppression of TNF-alpha release was biologically more important than suppressed release of soluble inhibitors. Similar concurrent suppression of IL-1 beta and IL-1ra release occurred in PBMC and Mono Mac 6 cultures exposed to dexamethasone.

Cross-linking studies and membrane localization and assembly of radiolabelled large mechanosensitive ion channel (MscL) of Escherichia coli.

The gene encoding the large conductance mechanosensitive ion channel (MscL) of Escherichia coli and several deletion mutants of mscL were cloned under the control of the T7 RNA polymerase promoter. Transformation of these constructs into an E. coli strain carrying an inducible T7 RNA polymerase gene allowed the specific production and labelling of MscL with [35S]methionine. Preparation of membrane fractions of E. coli cells by sucrose gradient centrifugation indicated that the radiolabelled MscL was present in the inner cytoplasmic membrane in agreement with results of several studies. However, treatment of the labelled cells and cell membrane vesicles with various cross-linkers resulted in the majority of labelled protein migrating as a monomer with a small proportion of molecules (approximately 25%) migrating as dimers and higher order multimers. This result is in contrast with a finding of a study suggesting that the channel exclusively forms hexamers in the cell membrane of E. coli (1) and therefore may have profound implication for the activation and/or "multimerization" of the channel by mechanical stress exerted to the membrane. In addition, from the specific activity of the radiolabelled protein and the amount of protein in the cytoplasmic membrane fraction we estimated the number of MscL ion channels expressed under these conditions to be approximately 50 channels per single bacterium.

Dexamethasone antagonizes IL-4 and IL-10-induced release of IL-1RA by monocytes but augments IL-4-, IL-10-, and TGF-beta-induced suppression of TNF-alpha release.

The activities of monocyte-derived tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta are potentially modified by IL-1RA and soluble receptors for TNF (sTNF-R), which are themselves monocyte products. IL-4, IL-10, TGF-beta, and glucocorticoids (GC) all suppress the lipopolysaccharide (LPS)-stimulated release of TNF-alpha and IL-1beta but vary in their effects on IL-1RA and sTNF-R. This raises the prospect of interactions between the cytokines and glucocorticoids, which may be antagonistic or additive on IL-1 and TNF activity. We, therefore, studied the interactions of the GC dexamethasone (Dex) with IL-4, IL-10, and transforming growth factor (TGF)-beta on the release of TNF-alpha and IL-1RA by human monocytes and the monocytic THP-1 cell line. Low concentration of Dex (10(-8)-10(-7)M) acted additively with low concentrations of IL-4 (0.01-1 ng/ml), IL-10 (0.01-0.1 U/ml), or TGF-beta (0.01-1 ng/ml) to profoundly suppress LPS-stimulated release of TNF-alpha by whole blood and, to a lesser degree, THP-1 cells. Dex also suppressed spontaneous release of IL-1RA from PBMC and THP-1 cells, whereas IL-4 and IL-10, but not TGF-beta, stimulated release. Dex antagonized the enhanced release in IL-4 and IL-10-stimulated cultures. The capacity to stimulate release of IL-1RA may contribute to the anti-inflammatory potential of IL-4 and IL-10 in monocyte/macrophage-mediated disease. GC, therefore, do not uniquely enhance the suppressive functions of IL-4 and IL-10 on monokine activity. The therapeutic benefit of combinations of GC and IL-4, IL-10 or TGF-beta in disease may depend on the roles of the individual monokines and antagonists in pathogenesis.