The predominant inhibitory neurotransmitter of the brain, GABA (gamma-aminobutyric acid), activates chloride-selective ion pores integral to the receptor complex. Subunits comprising the presumed hetero-pentameric GABA channel have been cloned, but little information is available on the domains important for activation. Rat wild-type or mutated alpha 1-, beta 2- and gamma 2-subunits (designated alpha, beta and gamma) were coexpressed in Xenopus oocytes and examined electrophysiologically. We report here the identification of two separate and homologous domains of the beta-subunit, each of which contributes a tyrosine and threonine essential for activation by GABA. Conservative substitution of each of these four amino acids dramatically decreased GABA channel sensitivity to activation by GABA and the GABA agonist muscimol. These substitutions, however, did not impair activation by the barbiturate pentobarbital, indicating these two different classes of agonists activate GABA channels through distinct mechanisms. We also present evidence suggesting that the two identified domains of the beta-subunit contribute a major component of the GABA receptor.

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GABA is the main inhibitory neurotransmitter in the mammalian brain. The postsynaptic GABA A receptor/pore complex is presumed to be a pentamer typically composed of a combination of ␣, ␤, and ␥ subunits, although the stoichiometry remains controversial. We probed the stoichiometry of the GABA A receptor by site-directed mutagenesis of a conserved leucine (to serine) in the putative second membrane-spanning domain of the rat ␣1(␣L263S), ␤2(␣L259S), and ␥2(␣L274S) subunit isoforms. Coexpression of wild-type and mutant subunits of each class (e.g., ␣ and ␣L263S), along with their wild-type counterparts (e.g., ␤ and ␥), in Xenopus laevis oocytes resulted in mixed populations of receptors with distinct GABA sensitivities. This is consistent with the interpretation that the leucine mutation increased the GABA sensitivity in proportion to the number of incorporated mutant subunits. The apparent number of incorporated subunits for each class (␣, ␤, and ␥) could then be determined from the number of components comprising the compound GABA dose-response relationships. Using this approach, we conclude that the recombinant ␣1␤2␥2 GABA A receptor is a pentamer composed of two ␣ subunits, two ␤ subunits, and one ␥ subunit.

SPOR domains are ϳ70 amino acids long and occur in >1,500 proteins identified by sequencing of bacterial genomes. The SPOR domains in the FtsN cell division proteins from Escherichia coli and Caulobacter crescentus have been shown to bind peptidoglycan. Besides FtsN, E. coli has three additional SPOR domain proteinsDamX, DedD, and RlpA. We show here that all three of these proteins localize to the septal ring in E. coli. The loss of DamX or DedD either alone or in combination with mutations in genes encoding other division proteins resulted in a variety of division phenotypes, demonstrating that DamX and DedD participate in cytokinesis. In contrast, RlpA mutants divided normally. Follow-up studies revealed that the SPOR domains themselves localize to the septal ring in vivo and bind peptidoglycan in vitro. Even SPOR domains from heterologous organisms, including Aquifex aeolicus, localized to septal rings when produced in E. coli and bound to purified E. coli peptidoglycan sacculi. We speculate that SPOR domains localize to the division site by binding preferentially to septal peptidoglycan. We further suggest that SPOR domain proteins are a common feature of the division apparatus in bacteria. DamX was characterized further and found to interact with multiple division proteins in a bacterial two-hybrid assay. One interaction partner is FtsQ, and several synthetic phenotypes suggest that DamX is a negative regulator of FtsQ function.

A conserved leucine residue in the midpoint of the second transmembrane domain (M2) of the ligand-activated ion channel family has been proposed to play an important role in receptor activation. In this study, we assessed the importance of this leucine in the activation of rat alpha 1 beta 2 gamma 2 GABA receptors expressed in Xenopus laevis oocytes by site-directed mutagenesis and two-electrode voltage clamp. The hydrophobic conserved M2 leucines in alpha1(L263), beta2(L259), and gamma 2(L274) subunits were mutated to the hydrophilic amino acid residue serine and coexpressed in all possible combinations with their wild-type and/or mutant counterparts. The mutation in any one subunit decreased the EC(50) and created spontaneous openings that were blocked by picrotoxin and, surprisingly, by the competitive antagonist bicuculline. The magnitudes of the shifts in GABA EC(50) and picrotoxin IC(50) as well as the degree of spontaneous openings were all correlated with the number of subunits carrying the leucine mutation. Simultaneous mutation of the GABA binding site (beta 2Y157S; increased the EC(50)) and the conserved M2 leucine (beta 2L259S; decreased the EC(50)) produced receptors with the predicted intermediate agonist sensitivity, indicating the two mutations affect binding and gating independently. The results are discussed in light of a proposed allosteric activation mechanism.

A universally conserved event in cell division is the formation of a cytokinetic ring at the future site of division. In the bacterium Escherichia coli, this ring is formed by the essential cell division protein FtsZ. We have used immunof luorescence microscopy to show that FtsZ assembles early in the division cycle, suggesting that constriction of the FtsZ ring is regulated and supporting the view that FtsZ serves as a bacterial cytoskeleton. Assembly of FtsZ rings was heterogeneously affected in an ftsI temperature-sensitive mutant grown at the nonpermissive temperature, some filaments displaying a striking defect in FtsZ assembly and others displaying little or no defect. By using low concentrations of the ␤-lactams cephalexin and piperacillin to specifically inhibit FtsI (PBP3), an enzyme that synthesizes peptidoglycan at the division septum, we show that FtsZ ring constriction requires the transpeptidase activity of FtsI. Unconstricted FtsZ rings are stably trapped at the midpoint of the cell for several generations after inactivation of FtsI, whereas partially constricted FtsZ rings are less effectively trapped. In addition, FtsZ rings are able to assemble in newborn cells in the presence of cephalexin, suggesting that newborn cells contain a site at which FtsZ can assemble (the nascent division site) and that the transpeptidase activity of FtsI is not required for assembly of FtsZ at these sites. However, aside from this first round of FtsZ ring assembly, very few additional FtsZ rings assemble in the presence of cephalexin, even after several generations of growth. One interpretation of these results is that the transpeptidase activity of FtsI is required, directly or indirectly, for the assembly of nascent division sites and thereby for future assembly of FtsZ rings.

FtsE and FtsX have homology to the ABC transporter superfamily of proteins and appear to be widely conserved among bacteria. Early work implicated FtsEX in cell division in Escherichia coli, but this was subsequently challenged, in part because the division defects in ftsEX mutants are often salt remedial. Strain RG60 has an ftsE::kan null mutation that is polar onto ftsX. RG60 is mildly filamentous when grown in standard Luria-Bertani medium (LB), which contains 1% NaCl, but upon shift to LB with no NaCl growth and division stop. We found that FtsN localizes to potential division sites, albeit poorly, in RG60 grown in LB with 1% NaCl. We also found that in wild-type E. coli both FtsE and FtsX localize to the division site. Localization of FtsX was studied in detail and appeared to require FtsZ, FtsA, and ZipA, but not the downstream division proteins FtsK, FtsQ, FtsL, and FtsI. Consistent with this, in media lacking salt, FtsA and ZipA localized independently of FtsEX, but the downstream proteins did not. Finally, in the absence of salt, cells depleted of FtsEX stopped dividing before any change in growth rate (mass increase) was apparent. We conclude that FtsEX participates directly in the process of cell division and is important for assembly or stability of the septal ring, especially in salt-free media.In Escherichia coli, the division septum forms via the coordinated inward growth of all three layers of the cell envelopethe cytoplasmic membrane, the peptidoglycan wall, and the outer membrane. To date, about a dozen E. coli genes are known to be specifically required for septation (3, 11). These genes share two important properties: (i) loss of function mutations result in the formation of long, aseptate filaments with regularly spaced nucleoids (the fts, or filamentation temperature-sensitive phenotype), and (ii) the proteins encoded by these genes localize to the division site. Because cell division genes are generally essential and because lesions in many housekeeping genes can affect cell division indirectly, there have not been any exhaustive screens for division mutants. Thus, it seems likely that more division genes remain to be described.A number of mutant hunts, starting with the pioneering work of Hirota and coworkers in the 1960s, suggested that there is an important cell division gene located at about 76 min on the E. coli chromosome (30). This locus was originally designated ftsE. One interesting property of ftsE mutants is that many are salt remedial, meaning that viability is restored by inclusion of NaCl in the growth medium. The amount of salt required for rescue is strain dependent, but generally in the range of 0.5%. Studies by Salmond and colleagues in the 1980s revealed that "ftsE" comprised two genes, which were then designated ftsE and ftsX (13). Moreover, the sequence of these genes revealed clear homology to ABC transporters; FtsE is the ATP-binding cassette (ABC) component, while FtsX is the membrane component. ABC transporters use energy from ATP to transport a wide variety of sub...

SummaryVibrio parahaemolyticus senses surfaces via impeded rotation of its polar flagellum. We have exploited this surface-sensing mechanism to trick the organism into thinking it is on a surface when it is growing in liquid. This facilitated studies of global gene expression in a way that avoided many of the complications of surface-to-liquid comparisons, and illuminated~70 genes that respond to surface sensing per se. Almost all are surface-induced (not repressed) and encode swarming motility proteins, virulence factors or sensory enzymes involved with chemoreception and c-di-GMP signalling. Follow-up studies were performed to place the surfaceresponsive genes in a regulatory hierarchy. Mapping the hierarchy revealed two surprises about LafK, a transcriptional activator that until now has been considered to be the master regulator for the lateral flagellar system. First, LafK controls a more diverse set of genes than previously appreciated. Second, some laf genes are not under LafK control, which means LafK is not the master regulator after all. Additional experiments motivated by the transcriptome analyses revealed that growth on a surface lowers c-di-GMP levels and enhances cytotoxicity. Thus, we demonstrate that V. parahaemolyticus can invoke a programme of gene control upon encountering a surface and the specific identities of the surfaceresponsive genes are pertinent to colonization and pathogenesis.