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Background [K.sup.+] channels of the TASK family are believed to participate in sensory transduction by chemoreceptor (glomus) cells of the carotid body (CB). However, studies on the systemic CB-mediated ventilatory response to hypoxia and hypercapnia in TASK1- and/or TASK3-deficient mice have yielded conflicting results. We have characterized the glomus cell phenotype of TASK-null mice and studied the responses of individual cells to hypoxia and other chemical stimuli. CB morphology and glomus cell size were normal in wild-type as well as in [TASK1.sup.-/-] or double [TASK1/3.sup.-/-] mice. Patch-clamped TASK1/3-null glomus cells had significantly higher membrane resistance and less hyperpolarized resting potential than their wild-type counterpart. These electrical parameters were practically normal in [TASK1.sup.-/-] cells. Sensitivity of background currents to changes of extracellular pH was drastically diminished in TASK1/3-null cells. In contrast with these observations, responsiveness to hypoxia or hypercapnia of either [TASK1.sup.-/-] or double [TASK1/3.sup.-/-] cells, as estimated by the amperometric measurement of catecholamine release, was apparently normal. TASK1/3 knockout cells showed an enhanced secretory rate in basal (normoxic) conditions compatible with their increased excitability. Responsiveness to hypoxia of TASK1/3-null cells was maintained after pharmacological blockade of maxi-[K.sup.+] channels. These data in the TASK-null mouse model indicate that TASK3 channels contribute to the background [K.sup.+] current in glomus cells and to their sensitivity to external pH. They also suggest that, although TASK1 channels might be dispensable for [O.sub.2]/C[O.sub.2] sensing in mouse CB cells, TASK3 channels (or TASK1/3 heteromers) could mediate hypoxic depolarization of normal glomus cells. The ability of [TASK1/3.sup.-/-] glomus cells to maintain a powerful response to hypoxia even after blockade of maxi-[K.sup.+] channels, suggests the existence of multiple sensor and/or effector mechanisms, which could confer upon the cells a high adaptability to maintain their chemosensory function. doi/10.1085/jgp.200910302

The virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of many virulence factors, including type IV pill, which are required for colonization of host tissues and for twitching motility. Type IV pilus function is controlled in part by the Chp chemosensory system, which includes a histidine kinase, ChpA, and two CheY-like response regulators, PilG and PilH. How the Chp components interface with the type IV pilus motor proteins PilB, PilT, and PilU is unknown. We present genetic evidence confirming the role of ChpA, PilG, and PilB in the regulation of pilus extension and the role of PilH and PilT in regulating pilus retraction. Using informative double and triple mutants, we show that (i) ChpA, PilG, and PilB function upstream of PilH, PilT, and PilU; (ii) that PilH enhances PilT function; and (iii) that PilT and PilB retain some activity in the absence of signaling input from components of the Chp system. By site-directed mutagenesis, we demonstrate that the histidine kinase domain of ChpA and the phosphoacceptor sites of both PilG and PilH are required for type IV pilus function, suggesting that they form a phosphorelay system important in the regulation of pilus extension and retraction. Finally, we present evidence suggesting that pilA transcription is regulated by intracellular PilA levels. We show that PilA is a negative regulator of pill transcription in P. aeruginosa and that the Chp system functionally regulates pilA transcription by controlling PilA import and export. doi:10.1128/JB.01390-09

CheZ localizes to chemoreceptor patches by binding CheA-short (CheAs). Residues 70 to 134 of CheZ, constituting the apical loops and part of the dimerization domain, suffice for localization. Replacements of Tyr-118, Ile-119, Leu-123, Arg-124, and Leu-126 of CheA interfere with localization. These residues are exposed in the 'P1 domain of Che[A.sub.s].

Hypoxia activates chemoreceptor cells of the carotid body (CB) promoting an increase in their normoxic release of neurotransmitters. Catecholamine (CA) release rate parallels the intensity of hypoxia. Coupling of hypoxia to CA release requires cell depolarization, produced by inhibition of [O.sub.2]-regulated [K.sup.+] channels, and [Ca.sup.2+] entering the cells via voltage-operated channels. In rat chemoreceptor cells hypoxia inhibits large-conductance, calcium-sensitive K channels (maxiK) and a two-pore domain weakly inward rectifying [K.sup.+] channel (TWIK)-like acid-sensitive [K.sup.+] channel (TASK)-like channel, but the significance of maxiK is controversial. A proposal envisions maxiK contributing to set the membrane potential ([E.sub.m]) and the hypoxic response, but the proposal is denied by authors finding that maxiK inhibition does not depolarize chemoreceptor cells or alters intracellular [Ca.sup.2+] concentration or CA release in normoxia or hypoxia. We found that maxiK channel blockers (tetraethylammonium and iberiotoxin) did not modify CA release in rat chemoreceptor cells, in either normoxia or hypoxia, and iberiotoxin did not alter the [Ca.sup.2+] transients elicited by hypoxia. On the contrary, both maxiK blockers increased the responses elicited by dinitrophenol, a stimulus we demonstrate does not affect maxiK channels in isolated patches of rat chemoreceptor cells. We conclude that in rat chemoreceptor cells maxiK channels do not contribute to the genesis of the [E.sub.m], and that their full inhibition by hypoxia, preclude further inhibition by maxiK channel blockers. We suggest that full inhibition of this channel is required to generate the spiking behavior of the cells in acute hypoxia. hypoxia; catecholamine; iberiotoxin; intracellular calcium

The insect chemoreceptor superfamily comprises the olfactory receptor (Or) and gustatory receptor (Gr) multigene families. These families give insects the ability to smell and taste chemicals in the environment and are thus rich resources for linking molecular evolutionary and ecological processes. Although dramatic differences in family size among distant species and high divergence among paralogs have led to the belief that the two families evolve rapidly, a lack of evolutionary data over short time scales has frustrated efforts to identify the major forces shaping this evolution. Here, we investigate patterns of gene loss/gain, divergence, and polymorphism in the entire repertoire of ~130 chemoreceptor genes from five closely related species of Drosophila that share a common ancestor within the past 12 million years. We demonstrate that the overall evolution of the Or and Or families is nonneutral. We also show that selection regimes differ both between the two families as wholes and within each family among groups of genes with varying functions, patterns of expression, and phylogenetic histories. Finally, we find that the independent evolution of host specialization in Drosophila sechellia and D. erecta is associated with a fivefold acceleration of gene loss and increased rates of amino acid evolution at receptors that remain intact. Gene loss appears to primarily affect Grs that respond to bitter compounds while elevated [K.sub.a]/[K.sub.s] is most pronounced in the subset of Ors that are expressed in larvae. Our results provide strong evidence that the observed phenomena result from the invasion of a novel ecological niche and present a unique synthesis of molecular evolutionary analyses with ecological data.

C[O.sub.2] elicits a response from many insects, including mosquito vectors of diseases such as malaria and yellow fever, but the molecular basis of C[O.sub.2] detection is unknown in insects or other higher eukaryotes. Here we show that Gr21a and Gr63a, members of a large family of Drosophila seven-transmembrane-domain chemoreceptor genes, are coexpressed in chemosensory neurons of both the larva and the adult. The two genes confer C[O.sub.2] response when coexpressed in an in vivo expression system, the 'empty neuron system.' The response is highly specific for C[O.sub.2] and dependent on C[O.sub.2] concentration. The response shows an equivalent dependence on the dose of Gr21a and Gr63a. None of 39 other chemosensory receptors confers a comparable response to C[O.sub.2]. The identification of these receptors may now allow the identification of agents that block or activate them. Such agents could affect the responses of insect pests to the humans they seek. chemoreceptors | insect | Gr genes

The Aer and Tsr chemoreceptors in Escherichia coli govern tactic responses to oxygen and redox potential that are parts of an overall behaviour known as energy taxis. They are also proposed to mediate responses to rapidly utilized carbon sources, glycerol and succinate, via the energy taxis mechanism. In this study, the Aer and Tsr proteins were individually expressed in an 'all-transducer-knockout' strain of E. coli and taxis was analysed in gradients of various oxidizable carbon sources. In addition to the known response to glycerol and succinate, it was found that Aer directed taxis towards ribose, galactose, maltose, malate, proline and alanine as well as the phosphotransferase system (PTS) carbohydrates glucose, mannitol, mannose, sorbitol and fructose, but not to aspartate, glutamate, glycine and arabinose. Tsr directed taxis towards sugars (including those transported by the PTS), but not to organic acids or amino acids. When a mutated Aer protein unable to bind the FAD cofactor was expressed in the receptor-less strain, chemotaxis was not restored to any substrate. Aer appears to mediate responses to rapidly oxidizable substrates, whether or not they are effective growth substrates, whereas Tsr appears to mediate taxis to substrates that support maximal growth, whether or not they are rapidly oxidizable. This correlates with the hypothesis that Aer and Tsr sense redox and proton motive force, respectively. Taken together, the results demonstrate that Aer and Tsr mediate responses to a broad range of chemicals and their attractant repertoires overlap with those of specialized chemoreceptors, namely Trg (ribose, galactose) and Tar (maltose).

Olfactory receptor (OR) genes constitute the basis for the sense of smell and are encoded by the largest mammalian gene superfamily of >1,000 genes. In humans, >60% of these are pseudogenes. In contrast, the mouse OR repertoire, although of roughly equal size, contains only [approximately equal to] 20% pseudogenes. We asked whether the high fraction of nonfunctional OR genes is specific to humans or is a common feature of all primates. To this end, we have compared the sequences of 50 human OR coding regions, regardless of their functional annotations, to those of their putative orthologs in chimpanzees, gorillas, orangutans, and rhesus macaques. We found that humans have accumulated mutations that disrupt OR coding regions roughly 4-fold faster than any other species sampled. As a consequence, the fraction of OR pseudogenes in humans is almost twice as high as in the non-human primates, suggesting a human-specific process of OR gene disruption, likely due to a reduced chemosensory dependence relative to apes.

Research has been conducted on the chemoreceptor gene subset in Caenorhabditis elegans. The role of the daf-7 TGF-beta pathway in expression regulation of this subset has been invstigated and the results demonstrate that the expression of the individual chemoreceptor genes in C. elegans is subject to multiple regulation modes.

Research has been conducted on inflammatory cells. The influx of these cells to injury sites has been investigated in studying the cell ability to form chemotactic gradients, and the details are reported.

Research has been conducted on the M3 protein which alteres host antiviral inflammatory responses. The crystal structures of this protein alone and in complex with CC chemokine MPC-1 have been investigated and the details are reported.

Research has been conducted on the receptor methylation in Escherichia coli chemotaxis. The role of this receptor in adaptation has been investigated and the details are reported.

Research has been conducted on the spinal cord oligodendrocytes. The role of chemokine CXCL1 and its receptor CXCR2 in patterning the developing spinal cord has been investigated and the results indicate that the presumptive white mater population by oligodendrocyte precursors depends on localized CXCL1 expression.

The authors presents the topics which are going to be addressed at the national meeting of the American Chemical Society, August 18-22. These topics include comparative genomics, proteins engineered by molecular evolution, small molecule protein kinase inhibitors and glycosaminoglycan oligosaccharides' chemoenzymatic synthesis.

Research has been conducted on the gustatory receptors identified in the Drosophila genome searches. The hybridization and the transgene experiments have been carried out and the results of the experiments revealing the expression of these genes in gustatory and olfactory neurons of the lavrae and adult flies inducate that this gene family encodes both odorant and taste receptors.

Transmembrane signaling by bacterial chemoreceptors is thought to involve relative movement among the four transmembrane helices of the homodimer. We assayed that movement by measuring effects of ligand occupancy on rates of oxidative cross-linking between cysteines introduced into neighboring helices of the transmembrane domain of chemoreceptor Trg from Escherichia coli. Measurements were done on chemoreceptors in their native environment, intact cells that were motile and chemotactically responsive. Receptor occupancy did not appear to cause drastic rearrangement of the four-helix structure since, among 67 cysteine pairs tested, the same 19 exhibited oxidative cross-linking in the presence or absence of saturating chemoattractant. However, occupancy did cause subtle changes that were detected as effects on rates of cross-linking. Among the seven disulfides appropriate for measurements of initial rates of formation, ligand occupancy had significant and different effects on all three cross-links that connected the two helices within a subunit but had minimal effects on the four that spanned the packing interface between subunits. This constitutes direct evidence that the conformational change of transmembrane signaling involves significant movement within a subunit and minimal movement between subunits, a pattern deduced from several previous studies and now documented directly. Among possible modes of movement between the two helices of a subunit, axial sliding of one helix relative to the other was the conformational change that best accounted for the observed effects on cross-linking.

A gene encoding a putative methyl-accepting chemotaxis protein (MCP) in Desulfovibrio vulgaris Hildenborough was cloned and sequenced. The gene, designated as dcrA, contained an open reading frame coding for a 72,994 Da protein. Amino acid sequence analysis indicated that DcrA is homologous to MCPs of Escherichia coli and Salmonella typhimurium. Sequence comparisons among the three species revealed five conserved regions, corresponding to a hydrophobic domain which may play a role in signal transduction. Expression studies in E. coli showed that the dcrA promoter functions poorly in E. coli and the expressed gene inhibited host growth.

Experimental studies using alkaline phosphatase gene fusions help analyze the contribution of the small N-terminal cytoplasmic region to membrane insertion. Elimination of the positive charge of the domain by mutations reveals the vital role of the positive character of the N-terminal region in the establishment of Tsr membrane topology. Both normal insertion of the membrane-spanning sequence Tml and the activity of a novel amphiphathic sequence element influence the insertion of TM2, another membrane-spanning sequence. Orientation of the second transmembrane segment is determined by the partially redundant sequence information.

Details of a bitter taste receptor genes with relation to individual differences during taste perception are presented

Research has been conducted on CheZ phosphatase. The authors report that they have investigated the conditions required for CheZ phosphatase polar localization via the use of CheZ green fluorescent fusion protein, and that CheZ localization requires the truncated C-terminal portion of P1 domain in CheA-short.

Hempleman, S. C., T. A. Rodriguez, Y. A. Bhagat, and R. S. Begay. Benzolamide, acetazolamide, and signal transduction in avian intrapulmonary chemoreceptors. Am J Physiol Regulatory Integrative Comp Physiol 279: R1988-R1995, 2000.--Intrapulmonary chemoreceptors (IPC) are [CO.sub.2]-sensitive sensory neurons that innervate the lungs of birds, help control the rate and depth of breathing, and require carbonic anhydrase (CA) for normal function. We tested whether the CA enzyme is located intracellularly or extracellularly in IPC by comparing the effect of a CA inhibitor that is membrane permeable (iv acetazolamide) with one that is relatively membrane impermeable (iv benzolamide). Single cell extracellular recordings were made from vagal filaments in 16 anesthetized, unidirectionally ventilated mallards (Anas platyrhynchos). Without CA inhibition, action potential discharge rate was inversely proportional to inspired [PCO.sub.2] (-9.0 [+ or -] 0.8 [s.sup.-1] [multiplied by] [lnTorr.sup.-1]; means [+ or -] SE, n = 16) and exhibited phasic responses to rapid [PCO.sub.2] changes. Benzolamide (25 mg/kg iv) raised the discharge rate but did not alter tonic IPC [PCO.sub.2] response (-9.8 [+ or -] 1.6 [s.sup.-1] [multiplied by] [lnTorr.sup.-1], n = 8), and it modestly attenuated phasic responses. Acetazolamide (10 mg/kg iv) raised IPC discharge, significantly reduced tonic IPC [PCO.sub.2] response to -3.5 [+ or -] 3.6 [s.sup.-1] [multiplied by] [lnTorr.sup.-1] (n = 6), and severely attenuated phasic responses. Results were consistent with an intracellular site for CA that is less accessible to benzolamide. A model of IPC [CO.sub.2] transduction is proposed. carbonic anhydrase; action potentials; sensory neurons; carbon dioxide