Substrate translocation kinetics of excitatory amino acid carrier 1 probed with laser-pulse photolysis of a new photolabile precursor of D-aspartic acid

Here we report the synthesis and photochemical and biological characterization of a new photolabile precursor of D-aspartic acid, alpha-carboxynitrobenzyl-caged D-aspartate (alpha-CNB-caged D-aspartate), and its application for studying the molecular mechanism of the neuronal excitatory amino acid carrier 1 (EAAC1). Investigation of the photochemical properties of alpha-CNB-caged D-aspartate by transient absorption spectroscopy of the aci-nitro intermediate revealed that it photolyzes with a quantum yield of 0. 19 at pH 7.0. The major component of the aci-nitro intermediate (77% of the total absorbance) decays with a time constant of 26 s. This decay is slowed by only a factor of 2 when increasing the pH to 10. A minor component (21%) decays with a time constant of 410 s and is pH insensitive. The compound was tested with respect to its biological activity with the glutamate transporter EAAC1 expressed in HEK293 cells. Whole-cell current recordings from these cells in the presence and absence of alpha-CNB-caged D-aspartate demonstrated that the compound neither activates nor inhibits EAAC1. Upon photolysis, D-aspartate-mediated whole-cell currents were generated. In contrast to laser-pulse photolysis experiments with alpha-CNB-caged L-glutamate, only a minor and much slower transient current component was observed. These results indicate that the substrate translocation step, which is not rate-limiting for the overall turnover of the transporter with L-glutamate, becomes rate-limiting when D-aspartate is translocated. The results demonstrate that the new caged D-aspartate derivative is a useful tool for the investigation of the molecular mechanism of glutamate transporters and probably other aspartate translocating systems using rapid chemical kinetic techniques.     

Channel-opening kinetics of GluR6 kainate receptor

GluR6 is an ionotropic glutamate receptor subunit of the kainate subtype. It plays an essential role in synaptic plasticity and epilepsy. We expressed this recombinant receptor in HEK-293 cells and characterized the glutamate-induced channel-opening reaction, using a laser-pulse photolysis technique with the caged glutamate (gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate). This technique permits glutamate to be liberated photolytically from the caged glutamate with a time constant of approximately 30 micros. Prior to laser photolysis, the caged glutamate did not activate the GluR6 channel, nor did it inhibit or potentiate the glutamate response. At the transmembrane voltage of -60 mV, pH 7.4 and 22 degrees C, the channel-opening and -closing rate constants were determined to be (1.1 +/- 0. 4) x 10(4) and (4.2 +/- 0.2) x 10(2) s(-1), respectively. The intrinsic dissociation constant of glutamate and the channel-opening probability were found to be 450 +/- 200 microM and 0.96, respectively. These constants are derived from a minimal kinetic mechanism of the channel activation involving the binding of two glutamate molecules. This mechanism describes the time course of the open-channel form of the receptor as a function of glutamate concentration. On the basis of the channel-opening rate constants obtained, the shortest rise time (20-80% of the receptor current response) or the fastest time by which the GluR6Q channel can open is predicted to be 120 micros. The open-channel form of the receptor determines the transmembrane voltage change, which in turn controls synaptic signal transmission between two neurons. The comparison of the channel-opening kinetic rate constants between GluR6Q and GluR2Q(flip), reported in the companion paper, suggests that at a glutamate concentration of 100 microM, for instance, the integrated neuronal signal will be dominated by a slower GluR6Q receptor response, as compared to the GluR2Q(flip) component.     

Coumarin-caged glycine that can be photolyzed within 3 microseconds by visible light

The synthesis and characterization of a new photolabile precursor of glycine (coumarin-caged glycine) are reported. The new compound is suitable for rapid chemical kinetic investigations of the membrane-bound neurotransmitter receptor activated by glycine. Unlike previously used caging groups for glycine, this precursor can be photolyzed rapidly and efficiently in the visible wavelength region. This allows the use of a relatively inexpensive light source. The alpha-carboxyl group of glycine was covalently coupled to the 7-(diethylamino)coumarin (DECM) caging group. The caged compound has a major absorption band with a maximum at 390 nm (epsilon390 = 13,900 M-1 cm-1). Photolysis was performed at wavelengths of >or=400 nm (epsilon400 = 12,400 M-1 cm-1). Under physiological conditions, DECM-caged glycine is water soluble and stable. In the visible wavelength region, it photolyzes rapidly to release glycine with a half-life of approximately 2.5 micrometers and a quantum yield of 0.12 +/- 0.01. The experimental results demonstrated that neither DECM-caged glycine nor its byproduct inhibits or activates human alpha1 glycine receptors expressed on the surface of HEK 293 cells.     

Synthesis and photochemistry of a photolabile precursor of N-methyl-D-aspartate (NMDA) that is photolyzed in the microsecond time region and is suitable for chemical kinetic investigations of the NMDA receptor.

The amino acid L-glutamate is a major neurotransmitter at excitatory synapses within the central nervous system. Neuronal responses to glutamate are mediated by at least three receptor types, one of which is the NMDA subtype, named for its specific ligand N-methyl-D-aspartic acid. Neurotransmitter receptors are transmembrane proteins that can form ion channels upon binding a specific ligand and are involved in many physiological activities of the brain and in some neurological disorders. Elucidating the mechanisms of the formation of transmembrane receptor-channels and of receptor regulation and inhibition is necessary for understanding nervous system function and for designing potential therapeutic agents. This has been hampered by the lack of rapid reaction techniques suitable for investigating protein-mediated reactions on cell surfaces. Recently a laser-pulse photolysis technique was developed to study the chemical reactions of channel-forming receptor proteins in the microsecond-to-millisecond time region. To apply the technique to NMDA1 receptors a photolabile NMDA precursor (beta-DNB NMDA) was synthesized. In this precursor the side chain carboxylate was protected as a photosensitive 2,2'-dinitrobenzhydryl ester. Photolysis with 308 nm laser light generated free NMDA with a time constant of 4.2 +/- 0.1 microseconds at pH 7 and a photolysis quantum yield of 0.18 +/- 0.05. In rat hippocampal neurons the beta-DNB NMDA (250 microM) neither activated endogenously expressed receptors nor potentiated or inhibited the NMDA response. Equilibration of hippocampal neurons in the whole-cell current recording mode with 250 microM caged precursor followed by a pulse of 333 nm laser light resulted in a rapid current rise with a rate constant of 100 s-1 due to opening of NMDA-activated receptor-channels. The caged NMDA precursor described here now makes it possible to investigate the mechanism of NMDA receptors in the micro- to millisecond time region.     

Synthesis and photochemistry of photolabile N-glycine derivatives and effects of one on the glycine receptor.

Three photolabile precursors of glycine containing a photosensitive 2-nitrobenzyl moiety attached to the amino group have been synthesized. When exposed to ultraviolet radiation between 308 and 350 nm, the compounds photolyze to release glycine, an important inhibitory neurotransmitter in the central nervous system. The identification of glycine as a photolysis product was determined by two different methods: separation of the photolyzed sample by thin-layer chromatography followed by a reaction with ninhydrin, and recognition of derivatized glycine using the Waters pico-tag method in conjunction with high-performance liquid chromatography. The photolysis of these compounds at 22 degrees C has been investigated, and the rate of decay of a transient intermediate in the reaction, which is assumed to reflect product release, has been measured. For N-(alpha-carboxy-2-nitrobenzyl)glycine this decay rate was found to be 940 s-1 at pH 6.8 and 600 s-1 at pH 7.5. Additionally, this compound was found to exhibit biological activity upon photolysis; cultured mouse spinal cord cells containing neuronal glycine receptors were used to detect the glycine liberation. The approach adopted here is useful in demonstrating the utility of photolabile precursors of neurotransmitters that have the protecting group linked to the neurotransmitter through the amino group. The rapid photolysis of such compounds to release free neurotransmitter is valuable in gaining access to chemical kinetic studies of neurotransmitter receptors. Previously, such studies have been limited because the available methods for neurotransmitter delivery did not give a sufficiently high time resolution.     

Channel-opening kinetics of GluR2Q(flip) AMPA receptor: a laser-pulse photolysis study

AMPA receptors mediate fast excitatory neurotransmission in the central nervous system. GluR2 is an AMPA receptor subunit that controls some key heteromeric AMPA receptor properties, such as calcium permeability. The kinetic properties of GluR2, relevant to the time scale of its channel opening, however, are poorly understood. Here, to measure the channel-opening kinetics, we use a laser-pulse photolysis technique, which permits glutamate to be liberated photolytically from gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate (caged glutamate) with a time constant of approximately 30 micros. We show that GluR2Q(flip), an unedited and Ca(2+) permeable isoform, is by far the fastest ligand-gated channel with the channel-opening and -closing rate constants being (8.0 +/- 0.49) x 10(4) and (2.6 +/- 0.20) x 10(3) s(-1), respectively. Therefore, the shortest rise time (20-80% of the receptor current response) or the fastest observed time by which the GluR2Q(flip) channel can open is predicted to be 17 micros. The minimal kinetic mechanism for the channel opening is further consistent with the binding of two glutamate molecules with the channel-opening probability of 0.96. These results suggest that GluR2 is a temporally, highly efficient receptor to transduce the binding of chemical signals (i.e., glutamate) into an electrical impulse.     

How fast does the GluR1Qflip channel open?

Opening of a ligand-gated ion channel is the step at which the binding of a neurotransmitter is transduced into the electrical signal by allowing ions to flow through the transmembrane channel, thereby altering the postsynaptic membrane potential. We report the kinetics for the opening of the GluR1Qflip channel, an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit of the ionotropic glutamate receptors. Using a laser-pulse photolysis technique that permits glutamate to be liberated photolytically from gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate (caged glutamate) with a time constant of approximately 30 micros, we show that, after the binding of glutamate, the channel opened with a rate constant of (2.9 +/- 0.2) x 10(4) s(-1) and closed with a rate constant of (2.1 +/- 0.1) x 10(3) s(-1). The observed shortest rise time (20-80% of the receptor current response), i.e. the fastest time by which the GluR1Qflip channel can open, was predicted to be 35 micros. This value is three times shorter than those previously reported. The minimal kinetic mechanism for channel opening consists of binding of two glutamate molecules, with the channel-opening probability being 0.93 +/- 0.10. These findings identify GluR1Qflip as one of the temporally efficient receptors that transduce the binding of chemical signals (i.e. glutamate) into an electrical impulse.     

Synthesis, photochemistry, and biological activity of a caged photolabile acetylcholine receptor ligand

A biologically inert photolabile precursor of carbamoylcholine has been synthesized; it is photolyzed to carbamoylcholine, a well-characterized acetylcholine analogue, with a half-time of 40 microseconds at pH 7.0 and a quantum yield of 0.8. The compound, N-(alpha-carboxy-2-nitrobenzyl)carbamoylcholine, was synthesized from (2-nitrophenyl)glycine. The photolysis rates (of five compounds) and the biological activity (of two compounds) were determined, and both properties were found to depend on the nature of the substituents on the photolabile protecting group. Laser pulse photolysis at wavelengths between 308 and 355 nm was used to investigate the wavelength dependence, quantum yield, and rate of the photolysis reaction. Photolysis products were isolated by high-performance liquid chromatography and identified by chemical and spectroscopic analysis and by their ability to activate the nicotinic acetylcholine receptor. BC3H1 muscle cells containing those receptors and a cell-flow method were used in the biological assays. The approach described may be useful in the preparation and characterization of other photolabile precursors of neurotransmitters that contain amino groups. The importance of these rapidly photolyzed, inert precursors of neurotransmitters is in chemical kinetic investigations of the reactions involving diverse neuronal receptors; such studies have been hampered because the available techniques have an insufficient time resolution.     

Photolabile precursors of inositol phosphates. Preparation and properties of 1-(2-nitrophenyl)ethyl esters of myo-inositol 1,4,5-trisphosphate

1-(2-Nitrophenyl)ethyl esters of D-myo-inositol 1,4,5-trisphosphate (InsP3) have been synthesized and shown to have suitable properties for use as photolabile precursors of InsP3. Synthesis was accomplished by treatment of InsP3 with 1-(2-nitrophenyl)diazoethane in a CHCl3/water mixture. This resulted in esterification of each of the three phosphate residues in InsP3, the 1-phosphate being more reactive than the 4- or 5-phosphate. Singly esterified P-1, P-4, and P-5 esters, termed P-1, P-4, and P-5 caged InsP3, were isolated from the reaction mixture by anion-exchange HPLC and characterized by 500-MHz 1H NMR spectroscopy. Each of these caged InsP3 esters exists as a pair of diastereoisomers and was identified by examining the effects of pH and nitrophenyl ring current shielding on the chemical shifts of nonexchangeable inositol protons. 1H NMR spectra of InsP3 were analyzed for comparison. On photolysis the compounds released InsP3 with rate constants of 175 (P-1), 225 (P-4), and 280 s-1 (P-5) as determined by monitoring the aci-nitro decay reaction at pH 7.1, 0.2 M ionic strength, 21 degrees C. Quantum yields determined by steady-state near-UV photolysis were 0.65 +/- 0.08 for each compound. P-4 and P-5 caged InsP3 were the most promising biologically inactive InsP3 precursors since at concentrations up to 50 microM they did not release Ca2+ from smooth muscle sarcoplasmic reticulum (SR) and were not metabolized by vascular smooth muscle InsP3 5-phosphatase or bovine brain InsP3 3-kinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two-photon excitation and photolysis by pulsed laser illumination modelled by spatially non-uniform reactions with simultaneous diffusion

Two-photon absorption in the focus of a pulsed laser has the potential for localized photolysis of caged compounds, generating high concentrations of neurotransmitters, hormones and messengers. The concentrations of cage, intermediates and products in the femtolitre focal volume depend on reaction rates and diffusional exchange with the external volume. This problem of reaction with diffusion was analysed with analytical and numerical methods to determine simple relations between parameters useful in the design and interpretation of experiments. The diffraction-limited laser spot is approximated well by a sphere, radius A, in diffusional exchange with either an infinite uniform medium, representing extracellular photolysis, or within a non-permeable sphere, a "cell" of radius B, representing intracellular photolysis. Photolysis is modelled as sequential irreversible reactions, with either the excitation step alone, rate constant k(e), or with a subsequent "dark" reaction, rate constant k(p). For extracellular photolysis, steady-state depletion of a cage averaged in a spherical spot increases hyperbolically with k(e) with half-maximum depletion at k(e) = K0.5 = 2.5 D/A2, where D is the diffusion coefficient. With measured parameters for spot size A = 0.3 microm and diffusion D = 800 microm2/s, K0.5 = 22,200 s(-1). The optimal exposure for localized photolysis is the characteristic diffusion time tau = A2/D, 113 micros in this example, and is the time taken to reach 57% of steady state in the diffusion-limited case. In the two-step model, with excitation and "dark" reaction steps, rate constants both exceeding K0.5 are necessary to generate 50% of maximal product concentration in the illuminated volume. High concentrations of photolysis products depend particularly on a high excitation rate constant (k(e) > K0.5), and localization of the products requires fast dark reactions (k(p) > K0.5). If products diffuse faster than the cage, their steady-state concentrations are decreased, and concentration transients may occur. For localized intracellular photolysis, the duration of exposure that generates product concentration at the cell boundary, B, less than 10% of the spot concentration should be shorter than 0.043(B/A)3tau, and is determined by diffusion.     

Characterization of a new caged proton capable of inducing large pH jumps

A new caged proton, 1-(2-nitrophenyl)ethyl sulfate (caged sulfate), is characterized by infrared spectroscopy and compared with a known caged, proton 2-hydroxyphenyl 1-(2-nitrophenyl)ethyl phosphate (caged HPP). In contrast to caged HPP, caged sulfate can induce large pH jumps and protonate groups that have pK values as low as 2.2. The photolysis mechanism of caged sulfate is analogous to that of P(3)-[1-(2-nitrophenyl)ethyl] ATP (caged ATP), and the photolysis efficiency is similar. The utility of this new caged compound for biological studies was demonstrated by its ability to drive the acid-induced conformational change of metmyoglobin. This transition from the native conformation to a partially unfolded form takes place near pH 4 and was monitored by near-UV absorption spectroscopy.     

Photolabile protecting groups for an acetylcholine receptor ligand. Synthesis and photochemistry of a new class of o-nitrobenzyl derivatives and their effects on receptor function

Two compounds have been synthesized that feature a photosensitive o-nitrobenzyl moiety attached directly to the carbamate nitrogen of carbamoylcholine. The well-characterized acetylcholine analogue, carbamoylcholine, was released from these derivatives in response to laser light pulses at wavelengths between 300 and 355 nm. Photolysis products were isolated by high-performance liquid chromatography and identified by chemical and spectroscopic analysis. The yield of carbamoylcholine molecules per photon absorbed was 0.25. A short-lived photochromic intermediate in the photolysis reaction was detected by laser flash photolysis. A single laser flash induced an instantaneous increase in absorbance at 406 nm, followed by a first-order decay to products, with a half-time of 0.07 ms for one of the compounds [N-[1-(2-nitrophenyl)ethyl]carbamoylcholine iodide] in aqueous buffers at pH 7 and 23 degrees C. Decay rates and quantum yields depended on the nature of the substituent on the protecting group. Evidence is presented in support of the conclusion that the transient species is an aci-nitro intermediate that decays directly to carbamoylcholine and therefore determines its rate of release. The photosensitive carbamoylcholine derivatives activated the nicotinic acetylcholine receptor only after photolysis, as determined by 86Rb+ flux measurements with membrane vesicles prepared from Torpedo californica and Electrophorus electricus. Before photolysis, the compounds interacted weakly with the acetylcholine-binding sites as shown by competitive inhibition of acetylcholine-stimulated flux at high concentrations. The compounds did not induce receptor desensitization at a significant rate. The new compounds afford several major advantages over other photoactivatable acetylcholine analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular determinants of proton modulation of glycine receptors

Extracellular pH regulates glycine receptors through an unknown mechanism. Here we demonstrate that acidic pH remarkably inhibited glycine-activated whole-cell currents in recombinant glycine alpha1 and alpha1beta receptors transiently expressed in human embryonic kidney 293 cells. The proton effect was voltage-independent and pharmacologically competed with glycine receptor agonist glycine and antagonist strychnine. Using site-directed mutagenesis, we have identified an N-terminal domain that is essential for proton-induced inhibition of glycine current. In alpha1 homomers, removal of the hydroxyl group by mutation of residue Thr-112 to Ala or Phe abolished inhibition of glycine currents by acidification. In contrast, mutation of Thr-112 to another hydroxylated residue (Tyr) produced receptors that retained partial proton sensitivity. In alpha1beta heteromers, a single mutation of the beta subunit T135A, which is homologous to alpha1 Thr-112, reduced proton sensitivity, whereas the double mutation alpha1(T112A)beta(T135A) almost completely eliminated the proton sensitivity. In addition, the mutation alpha1 H109A greatly reduced sensitivity to protons in homomeric alpha1 receptors. The results demonstrate that extracellular pH can regulate the function of glycine alpha1 and alpha1beta receptors. An extracellular domain consisting of Thr-112 and His-109 at the alpha1 subunit and Thr-135 at the beta subunit plays a critical role in determining proton modulation of glycine receptor function.     

P2Y1 receptor activation by photolysis of caged ATP enhances neuronal network activity in the developing olfactory bulb

It has recently been shown that adenosine-5'-triphosphate (ATP) is released together with glutamate from sensory axons in the olfactory bulb, where it stimulates calcium signaling in glial cells, while responses in identified neurons to ATP have not been recorded in the olfactory bulb yet. We used photolysis of caged ATP to elicit a rapid rise in ATP and measured whole-cell current responses in mitral cells, the output neurons of the olfactory bulb, in acute mouse brain slices. Wide-field photolysis of caged ATP evoked an increase in synaptic inputs in mitral cells, indicating an ATP-dependent increase in network activity. The increase in synaptic activity was accompanied by calcium transients in the dendritic tuft of the mitral cell, as measured by confocal calcium imaging. The stimulating effect of ATP on the network activity could be mimicked by photo release of caged adenosine 5'-diphosphate, and was inhibited by the P2Y(1) receptor antagonist MRS 2179. Local photolysis of caged ATP in the glomerulus innervated by the dendritic tuft of the recorded mitral cell elicited currents similar to those evoked by wide-field illumination. The results indicate that activation of P2Y(1) receptors in the glomerulus can stimulate network activity in the olfactory bulb.     

Fluorescence polarization of skeletal muscle fibers labeled with rhodamine isomers on the myosin heavy chain.

Fluorescence polarization was used to examine orientational changes of Rhodamine probes in single, skinned muscle fibers from rabbit psoas muscle following either photolysis of caged nucleotides or rapid length changes. Fibers were extensively and predominantly labeled at SH1 (Cys-707) of the myosin heavy chain with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine. Results from spectroscopic experiments utilizing the two Rhodamine isomers were quite similar. Following photolysis of either caged ATP or caged ADP, probes promptly reoriented toward the muscle fiber axis. Changes in the fluorescence polarization signals with transients elicited by the photolysis of caged ATP in the presence of saturating Ca2+ greatly preceded active force generation. Photolysis of caged ADP caused only a small, rapid decrease in force but elicited changes in the fluorescence polarization signals with time course and amplitude similar to those following photolysis of caged ATP. Fluorescence polarization signals were virtually unchanged by rapid length steps in both rigor and active muscle fibers. These results indicate that structural changes monitored by Rhodamine probes at SH1 are not associated directly with the force-generating event of muscle contraction. However, the fluorescence polarization transients were slightly faster than the estimated rate of cross-bridge detachment following photolysis of caged ATP, suggesting that the observed structural changes at SH1 may be involved in the communication pathway between the nucleotide- and actin-binding sites of myosin.     

Studies of decarboxylation in photolysis of alpha-carboxy-2-nitrobenzyl (CNB) caged compounds

Photolysis of alpha-carboxy-2-nitrobenzyl (CNB) caged compounds, studied here by time-resolved IR and UV spectroscopy, involves at least two pathways. In one, a conventional 2-nitrobenzyl type rearrangement takes place to release the photoprotected species via rapid decay of an aci-nitro intermediate. The alpha-carboxylate moiety of the CNB group is retained and the final by-product from this pathway is 2-nitrosophenylglyoxylate. Direct measurements of product formation confirmed that release via this pathway is faster for CNB-caged compounds than for related caged compounds without an alpha-carboxylate substituent and a rationale for the faster release rate is proposed. In a second pathway, photodecarboxylation of the starting material occurs: this pathway leads only to a slow, minor release of the photoprotected species. The extent to which the latter pathway contributes is affected by the nature of buffer salts in the irradiated solution. It was more prominent in an amine-based buffer (MOPS) than in phosphate buffer.     

Excitatory signaling in bacterial probed by caged chemoeffectors.

Chemotactic excitation responses to caged ligand photorelease of rapidly swimming bacteria that reverse (Vibrio alginolyticus) or tumble (Escherichia coli and Salmonella typhimurium) have been measured by computer. Mutants were used to assess the effects of abnormal motility behavior upon signal processing times and test feasibility of kinetic analyses of the signaling pathway in intact bacteria. N-1-(2-Nitrophenyl)ethoxycarbonyl-L-serine and 2-hydroxyphenyl 1-(2-nitrophenyl) ethyl phosphate were synthesized. These compounds are a 'caged' serine and a 'caged' proton and on flash photolysis release serine and protons and attractant and repellent ligands, respectively, for Tsr, the serine receptor. The product quantum yield for serine was 0.65 (+/- 0.05) and the rate of serine release was proportional to [H+] near-neutrality with a rate constant of 17 s-1 at pH 7.0 and 21 degrees C. The product quantum yield for protons was calculated to be 0.095 on 308-nm irradiation but 0.29 (+/- 0.02) on 300-350-nm irradiation, with proton release occurring at > 10(5) s-1. The pH jumps produced were estimated using pH indicators, the pH-dependent decay of the chromophoric aci-nitro intermediate and bioassays. Receptor deletion mutants did not respond to photorelease of the caged ligands. Population responses occurred without measurable latency. Response times increased with decreased stimulus strength. Physiological or genetic perturbation of motor rotation bias leading to increased tumbling reduced response sensitivity but did not affect response times. Exceptions were found. A CheR-CheB mutant strain had normal motility, but reduced response. A CheZ mutant had tumbly motility, reduced sensitivity, and increased response time to attractant, but a normal repellent response. These observations are consistent with current ideas that motor interactions with a single parameter, namely phosphorylated CheY protein, dictate motor response to both attractant and repellent stimuli. Inverse motility motor mutants with extreme rotation bias exhibited the greatest reduction in response sensitivity but, nevertheless, had normal attractant response times. This implies that control of CheY phosphate concentration rather than motor reactions limits responses to attractants.     

Glucuronyl glycine,a novel N-terminus in a glycoprotein

Treatment of cutinase, an extracellular glycoprotein produced by Fusarium solani f. pisi, with NaB³H₄ at pH 7.0 generated labeled enzyme. Acid hydrolysis showed that all of the label was in an acidic carbohydrate which was identified as gulonic acid. The N-terminal amino group of the enzyme is blocked; the precursor of gulonic acid has a free reducing group and it is attached via a linkage resistant to β-elimination. Furthermore, pronase digestion of NaB³H₄-treated cutinase gave rise to a ninhydrin negative compound which contained the bulk of the ³H and this compound was identified as N-gulonyl glycine. These results strongly suggest that the amino group of glycine, the N-terminal amino acid of this enzyme, is in amide linkage with glucuronic acid.     

Synthesis and characterization of photolabile derivatives of serotonin for chemical kinetic investigations of the serotonin 5-HT(3) receptor

A series of photolabile o-nitrobenzyl derivatives of serotonin (caged serotonin) were synthesized: the amine-linked serotonin derivatives N-(2-nitrobenzyl) serotonin (Bz-5HT) and N-(alpha-carboxy-2-nitrobenzyl) serotonin (N-CNB-5HT), and O-alpha-carboxy-2-nitrobenzyl) serotonin (O-CNB-5HT), which has the caging group attached to the phenolic OH group. All the derivatives released free serotonin when excited by 308-nm or 337-nm laser pulses. The time constant of serotonin release from N-CNB-5HT was 1. 2 ms, with a quantum yield of 0.08. This is too slow for rapid chemical kinetic measurements. O-CNB-5HT is suitable for transient kinetic investigations of the serotonin 5-HT(3) receptor. It released serotonin with a time constant of 16 micros and a quantum yield of 0.03. The biological properties of O-CNB-5HT were evaluated, and the applicability of the compound for kinetic studies of the 5-HT(3) receptor was demonstrated. O-CNB-5HT does not activate the 5-HT(3) receptor by itself, nor does it modulate the response of a cell when co-applied with serotonin. When irradiated with a 337-nm laser pulse, O-CNB-5HT released free serotonin that evoked 5-HT(3) receptor-mediated whole-cell currents in NIE-115 mouse neuroblastoma cells.     

Mechanisms of homomeric alpha1 glycine receptor endocytosis

Little is known regarding the mechanism(s) by which glycine receptors are endocytosed. Here we examined the endocytosis of homomeric alpha1 glycine receptors expressed in HEK 293 cells using immunofluorescence/confocal microscopy and whole-cell patch-clamp recordings. Our studies demonstrate that constitutive endocytosis of glycine receptors is blocked by the dominant negative dynamin construct K44A and that intracellular dialysis with peptide P4, a dynamin/amphiphysin-disrupting peptide, increased whole-cell glycine-gated chloride currents. To examine whether receptor endocytosis could be regulated by PKC, experiments with the PKC activator PMA (phorbol 12-myristate 13-acetate) were performed. PMA, but not its inactive analogue PMM (phorbol 12-monomyristate), stimulated receptor endocytosis and inhibited glycine-gated chloride currents. Similar to constitutive endocytosis, PKC-stimulated endocytosis was blocked by dynamin K44A. Mutation of a putative AP2 adaptin dileucine motif (L314A, L315A) present in the receptor cytoplasmic loop blocked PMA-stimulated receptor endocytosis and also prevented PMA inhibition of glycine receptor currents. In patch-clamp experiments, intracellular dialysis of a 12-amino acid peptide corresponding to the region of the receptor containing the dileucine motif prevented PKC modulation of wild-type glycine receptors. Unlike PKC modulation of the receptor, constitutive endocytosis was not affected by mutation of this dileucine motif. These results demonstrate that PKC activation stimulates glycine receptor endocytosis, that both constitutive endocytosis and PKC-stimulated endocytosis are dynamin-dependent, and that PKC-stimulated endocytosis, but not constitutive endocytosis, occurs via the dileucine motif (L314A, L315A) within the cytoplasmic loop of the receptor.