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PubMed2001

Attenuated LTP in hippocampal dentate gyrus neurons of mice deficient in the PAF receptor.

Platelet-activating factor (PAF), a bioactive lipid (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) derived from phospholipase A(2) and other pathways, has been implicated in neural plasticity and memory formation. Long-term potentiation (LTP) can be induced by the application of PAF and blocked by a PAF receptor (PAF-R) inhibitor in the hippocampal CA1 and dentate gyrus. To further investigate the role of PAF in synaptic plasticity, we compared LTP in dentate granule cells from hippocampal slices of adult mice deficient in the PAF-R and their age-matched wild-type littermates. Whole cell patch-clamp recordings were made in the current-clamp mode. LTP in the perforant path was induced by a high-frequency stimulation (HFS) and defined as >20% increase above baseline of the amplitude of excitatory postsynaptic potentials (EPSPs) from 26 to 30 min after HFS. HFS-induced enhancement of the EPSP amplitude was attenuated in cells from the PAF-R-deficient mice (163 +/- 14%, mean +/- SE; n = 32) when compared with that in wild-type mice (219 +/- 17%, n = 32). The incidence of LTP induction was also lower in the cells from the deficient mice (72%, 23 of 32 cells) than in the wild-type mice (91%, 29 of 32 cells). Using paired-pulse facilitation as a synaptic pathway discrimination, it appeared that there were differences in LTP magnitudes in the lateral perforant path but not in the medial perforant path between the two groups. BN52021 (5 microM), a PAF synaptosomal receptor antagonist, reduced LTP in the lateral path in the wild-type mice. However, neither BN52021, nor BN50730 (5 microM), a microsomal PAF-R antagonist, reduced LTP in the lateral perforant path in the receptor-deficient mice. These data provide evidence that PAF-R-deficient mice are a useful model to study LTP in the dentate gyrus and support the notion that PAF actively participates in hippocampal synaptic plasticity.

Animals↗

PubMed2001

Conformation of a peptide ligand bound to its G-protein coupled receptor.

Many peptide hormones elicit a wide array of physiological effects by binding to G-protein coupled receptors. We have determined the conformation of pituitary adenylate cyclase activating polypeptide, PACAP(1--21)NH(2), bound to a PACAP-specific receptor by NMR spectroscopy. Residues 3--7 form a unique beta-coil structure that is preceded by an N-terminal extended tail. This beta-coil creates a patch of hydrophobic residues that is important for receptor binding. In contrast, the C-terminal region (residues 8--21) forms an alpha-helix, similar to that in the micelle-bound PACAP. Thus, the conformational difference between PACAP in the receptor-bound and the micelle-bound states is limited to the N-terminal seven residues. This observation is consistent with the two-step ligand transportation model in which PACAP first binds to the membrane nonspecifically and then diffuses two-dimensionally in search of its receptor; a conformational change at the N-terminal region then allows specific interactions between the ligand and the receptor.

Amino Acid Sequence↗

PubMed1995

Exocytosis in peptidergic nerve terminals exhibits two calcium-sensitive phases during pulsatile calcium entry.

The link between electrical activity, Ca2+ entry through voltage-gated channels, and transmitter or hormone secretion is a central issue in neurobiology. In peptidergic nerve terminals of the mammalian neurohypophysis (NHP), secretion is elicited by patterned bursts of action potentials (APs). All parameters of the bursts are important to elicit efficient secretion, including AP frequency, AP broadening, burst duration, and interburst interval (Leng, 1988). We have studied Ca(2+)-secretion coupling of peptide-containing large dense-core vesicles (LDCV) in isolated rat NHP terminals. Ca2+ influx through voltage-gated Ca2+ channels was elicited and recorded by the whole-cell patch-clamp technique. Exocytosis was monitored on line with high temporal resolution by the capacitance detection technique (Neher and Marty, 1982). AP bursts were simulated by depolarizing pulse trains that mimic pulsatile submembrane Ca2+ elevations predicted for physiological stimuli. The characteristic capacitance response (delta Cm) to a train of depolarizing pulses was triphasic. It consisted of a threshold phase during which early pulses did not elicit secretion, a subsequent secretory phase during which Cm increases were coupled to depolarizing pulses, and a fatigued or inactivated state during which additional Ca2+ entry was ineffective. Both the threshold phase and secretory phase were correlated with the integrals of Ca2+ current. Ca2+ chelators affect both the threshold and secretory phase at submillimolar concentrations. Thus, a "shell" rather than "microdomain" model of Ca2+ elevation is appropriate for analyzing Ca(2+)-secretion coupling in NHP terminals (Nowycky and Pinter, 1993). We propose a two-step model, with a ca(2+)-dependent preparatory step followed by a final exocytotic step that is coupled to active Ca2+ influx. The results suggest that under physiological conditions, APs early in a burst prepare an NHP terminal for secretion, but later APs actually trigger exocytosis. Since NHP terminals do not possess a readily releasable pool of vesicles that require only a single Ca2+ step for exocytosis as seen in chromaffin cells (Neher and Zucker, 1993) and melanotrophs (Thomas et al, 1993a), Ca(2+)-secretion coupling mechanisms may be heterologous even within a single class of vesicles.

Action Potentials↗

PubMed1994

Cell coupling in the supporting cells of Corti's organ: sensitivity to intracellular H+ and Ca+2.

The input capacitance of cell pairs or small groups can be used to gauge the degree of electrical coupling via gap junctions (Santos-Sacchi, 1991). In order to estimate junctional sensitivity to intracellular Ca+2 and H+ concentration, the input capacitance of supporting cell syncytia of the organ of Corti was measured with the whole cell voltage clamp technique, while directly modifying the cation concentrations via the patch pipette. Typically, a pH below 6.5 was capable of uncoupling Hensen cells. On the other hand, pCa levels as low as 3 were ineffective.

Animals↗

PubMed1996

Flux coupling in a neuronal glutamate transporter.

Synaptic transmission is commonly terminated by diffusion and reuptake of neurotransmitter from the synaptic cleft. Glutamate reuptake prevents neurotoxicity and sets the lower limit for the concentration of extracellular glutamate, so it is important to understand the thermodynamics of this process. Here we use voltage clamping with a pH-sensitive fluorescent dye to monitor electrical currents and pH changes associated with flux of glutamate mediated by the human neuronal glutamate transporter EAAT3. In contrast to a previous model, we find that three sodium ions and one proton are cotransported with each glutamate ion into the cell, while one potassium ion is transported out of the cell. This coupling can support a transmembrane glutamate concentration gradient ([Glu]in/[Glu]out) exceeding 10(6) under equilibrium conditions, and would allow the transporter to continue removing glutamate over a wide range of ionic conditions.

ATP-Binding Cassette Transporters↗

PubMed1991

Ionic mechanisms involved in the flow- and pressure-sensor function of the endothelium.

Changes in hemodynamic forces (shear stress and pressure) modulate the tone of blood vessels. Recent evidence suggests that the endothelium plays an important role in these responses. Cascade bioassay experiments with various blood vessels demonstrated that increases in shear stress stimulated, and elevated transmural pressure suppressed the synthesis/release of endothelium-derived relaxing factor (EDRF). Studying the potential mechanisms of stimulus-response coupling in endothelial cells, it was shown that shear stress hyperpolarizes and pressure depolarizes the membrane of endothelial cells. Using whole-cell patch-clamping and various inhibitors of ion channels, it was demonstrated that modulation of the inward rectifying K(+)-channel may be the initial step in stimulus-response coupling: shear stress activates and pressure inactivates this channel, respectively. Since membrane potential is the main determinant of Ca(2+)-entry into endothelial cells, this still hypothetical mechanism may explain the opposing effects of shear stress (increased Ca(2+)-entry) and pressure (decreased Ca(2+)-entry) on the synthesis/release of EDRF.

Acetylcholine↗

PubMed1997

Drosophila serotonin transporters have voltage-dependent uptake coupled to a serotonin-gated ion channel.

Serotonin (5HT) transporters (SERTs) couple to existing ion gradients to transport 5HT into presynaptic terminals. In mammalian SERTs, the transport cycle is reported as electroneutral, with a translocation of zero net charge, and 5HT uptake is independent of membrane voltage. Yet mammalian SERTs exhibit 5HT-induced currents, and Drosophila SERTs (dSERTs) show voltage-dependent uptake. Thus, the relationship between uptake and current remains controversial; furthermore, the number of 5HT molecules translocated per ion channel event is unknown. To investigate this, we have used heterologous expression of cloned dSERTs to measure 5HT flux and dSERT currents concurrently under voltage clamp, and we have used fluctuation analysis to measure the size of the elementary ionic events in the same cells. RNA-injected Xenopus oocytes accumulate 5HT, and paroxetine or desipramine inhibit this uptake. RNA-injected oocytes also display paroxetine-sensitive 5HT-induced currents and 5HT-independent leak currents. Na replacement decreases the uptake and the induced currents. 5HT-induced current and 5HT uptake both increase at negative potentials, where 5HT carries approximately 5% of the induced current. Recently, several groups have reported similar phenomena for other transporters, in which transmitter-induced currents exceed the predictions of coupled transport. We now provide evidence that in dSERT, approximately 500 5HT molecules are translocated per channel opening, which, at -20 mV, carries approximately 10,000 electronic charges. These data support a model in which 500 SERT cycles occur for each 5HT-induced channel opening or a model in which 500 5HT molecules and 10,000 electronic charges pass through a common pore.

Animals↗

PubMed1997

Electrostatic effects on electron-transfer kinetics in the cytochrome f-plastocyanin complex.

In a complex of two electron-transfer proteins, their redox potentials can be shifted due to changes in the dielectric surroundings and the electrostatic potentials at each center caused by the charged residues of the partner. These effects are dependent on the geometry of the complex. Three different docking configurations (DCs) for intracomplex electron transfer between cytochrome f and plastocyanin were studied, defined by 1) close contact of the positively charged region of cytochrome f and the negatively charged regions of plastocyanin (DC1) and by (2, 3) close contact of the surface regions adjacent to the Fe and Cu redox centers (DC2 and DC3). The equilibrium energetics for electron transfer in DC1-DC3 are the same within approximately +/-0.1 kT. The lower reorganization energy for DC2 results in a slightly lower activation energy for this complex compared with DC1 and DC3. The long heme-copper distance (approximately 24 A) in the DC1 complex drastically decreases electronic coupling and makes this complex much less favorable for electron transfer than DC2 or DC3. DC1-like complexes can only serve as docking intermediates in the pathway toward formation of an electron-transfer-competent complex. Elimination of the four positive charges arising from the lysine residues in the positive patch of cytochrome f, as accomplished by mutagenesis, exerts a negligible effect (approximately 3 mV) on the redox potential difference between cyt f and PC.

Biophysical Phenomena↗

PubMed1997

Study of the inhibitor of the crayfish neuromuscular junction by presynaptic voltage control.

The inhibitor of the crayfish opener muscle was investigated by a presynaptic voltage control method. Two microelectrodes were inserted into the inhibitor and the amplitude and duration of presynaptic depolarization were controlled by a voltage-clamp amplifier. The inhibitory postsynaptic potential (IPSP) was measured from a muscle fiber located near the presynaptic voltage electrode. Nonlinear summation of IPSP amplitudes was corrected after chloride equilibrium potential was measured. With the use of 5-ms presynaptic pulses, the depolarization-release coupling (D-R) curve constructed from IPSP peak amplitudes (IPSPcor) had a threshold of about -35 mV and reached its maximal level at -5 to -10 mV. Depolarization beyond the maximum led to a suppression of neurotransmitter release. When transmitter release during a presynaptic pulse was completely suppressed, IPSPs activated by tail current could be identified with an average synaptic delay of 2.5 ms. Transmitter secretion triggered by a calcium current activated during the 5-ms pulses (IPSPon) was also measured on the rising phase of an IPSP, at 2.5 ms after the end of the 5-ms pulses. D-R coupling plots measured from IPSPon exhibited a more pronounced suppression than that obtained from IPSPcor. The effect of presynaptic pulse duration on the level of transmitter release was analyzed. Transmitter release increased with increasing duration and was nearly saturated by 20-ms pulses depolarized to 0 mV. The following conditions were identified as necessary to obtain a consistent D-R curve with a clear suppression: 1) small animals, 3.8 cm head to tail, 2) 15 degrees C, 3) 40 mM tetraethylammonium and 1 mM 4-aminopyridine, 4) an extracellular calcium concentration of < or = 10 mM. In addition, a consistent correlation was found among the branching pattern of the inhibitor, the placement of the presynaptic electrode, and the characteristics of the D-R curves. An ideal presynaptic electrode configuration involved placing the voltage electrode in a secondary branch, approximately 100 microns from the main branch point, and placing the current electrode at the branch point. Postsynaptically, optimal recordings were obtained from muscle fibers innervated by a single branch of the inhibitor that originated from a point near the presynaptic voltage electrode. A cable-release model was constructed to evaluate the relationship between the shape of the D-R coupling curves and the space constants of the presynaptic terminals. A comparison between the model and the D-R coupling curves suggested that the space constant of an inhibitor branch on a muscle fiber is > or = 8 times longer than its actual length. Therefore the upper limit estimate of the space constant of a typical preparation is approximately 3 mm. Results reported here outline morphological and physiological conditions needed to achieve optimal control of the presynaptic branch of the crayfish inhibitor. The cable-release model quantitatively defines the extent of presynaptic voltage control.

Animals↗

PubMed1997

Regulation of KATP channel activity by diazoxide and MgADP. Distinct functions of the two nucleotide binding folds of the sulfonylurea receptor.

KATP channels were reconstituted in COSm6 cells by coexpression of the sulfonylurea receptor SUR1 and the inward rectifier potassium channel Kir6.2. The role of the two nucleotide binding folds of SUR1 in regulation of KATP channel activity by nucleotides and diazoxide was investigated. Mutations in the linker region and the Walker B motif (Walker, J.E., M.J. Saraste, M.J. Runswick, and N.J. Gay. 1982. EMBO [Eur. Mol. Biol. Organ.] J. 1:945-951) of the second nucleotide binding fold, including G1479D, G1479R, G1485D, G1485R, Q1486H, and D1506A, all abolished stimulation by MgADP and diazoxide, with the exception of G1479R, which showed a small stimulatory response to diazoxide. Analogous mutations in the first nucleotide binding fold, including G827D, G827R, and Q834H, were still stimulated by diazoxide and MgADP, but with altered kinetics compared with the wild-type channel. None of the mutations altered the sensitivity of the channel to inhibition by ATP4-. We propose a model in which SUR1 sensitizes the KATP channel to ATP inhibition, and nucleotide hydrolysis at the nucleotide binding folds blocks this effect. MgADP and diazoxide are proposed to stabilize this desensitized state of the channel, and mutations at the nucleotide binding folds alter the response of channels to MgADP and diazoxide by altering nucleotide hydrolysis rates or the coupling of hydrolysis to channel activation.

ATP-Binding Cassette Transporters↗

PubMed2000

Neural mechanisms of visual feature grouping.

The present work relates compositions of visual scenes to signals in visual cortex and to cortical circuit models in order to understand neural mechanisms of perceptual feature grouping. It starts from the hypothesis that synchronization and decoupling of cortical gamma-activities (35-90 Hz) define the relations among visual objects. Here we concentrate on synchronization related to two basic visual situations, (1.) static retinal stimulation during ocular fixation, and (2.) transient stimulation during sudden luminance modulations or shifts in object position. For testing the synchronization hypothesis we investigated signal correlations of multiple micro-electrode recordings in visual cortex areas V1 and V2 of behaving monkeys. Static retinal stimuli induce gamma-activities that are loosely phase-coupled among neighboring neural populations of an object's cortical representation. This can explain why synchronization, measured by spectral coherence, is restricted to few millimeters cortex. Such patches of gamma-synchronization become de-coupled across the representation of an object's contour, and thereby can code figure-ground segregation. Transient stimuli evoke synchronized volleys of stimulus-locked activities that are typically non-rhythmic and include low frequency components in addition to those in the gamma-range. It is argued why stimulus-induced and stimulus-locked phase-coupled activations are both appropriate for supporting perceptual feature grouping. Clues for basic neural mechanisms participating in feature grouping are provided by our biologically motivated simulations of synchronization in cortical structures. (1.) Local populations generate gamma-oscillations via feedback inhibition during states of static retinal stimulation. (2.) Bidirectional facilitatory connections serve for phase-coupling among neighboring neural populations. (3.) Spike transmission delays, increasing with cortical distance, can explain the restriction of gamma-coherence to patches of few millimeters cortex. (4.) The size of synchronization patches in one visual area (e.g., V1) can define the size of classical receptive fields at the consecutive level of visual processing (V2) if Hebbian learning is operative. This may explain the increase in receptive field size at consecutive levels of visual processing. In conclusion, our results and those of others are supportive for the hypothesis that phase-coupled gamma-signals can code feature grouping and object continuity. However, convincing experimental proofs showing directly the dependence of perceptual grouping on cortical phase-coupling are still lacking.

Animals↗

PubMed1989

Patch clamp analysis of recovery of sodium channels from inactivation in mammalian myocardium in terms of a Markovian state model.

The kinetics of the recovery of Na channels from inactivation were investigated in ventricular cardiocytes of adult mice by a patch clamp technique. Single channel analysis showed an unaltered slope conductance of unitary currents during the recovery process to be 18.5 pS. The mean open time tau 0 and its monoexponential distribution remained also unchanged. The recovery time course of the apparent probability for a channel not to open coincided perfectly with the recovery time course determined from peak values of macroscopic currents which were recorded from cell-attached patches with up to 80 channels by averaging between 19 and 38 sweeps. Conclusively, the recovery was quantified by the time course of re-accumulation of the channels in a resting state. The initial recovery time course was sigmoidal. A serial Markov state model with three inactivated states in addition to the resting state was most appropriate for its description. The rate constants were assumed to be equal direction. A highly voltage-sensitive reaction towards the last inactivated state and a nearly voltage-independent reaction towards the resting state were obtained. Slow inactivation components were analyzed both during the recovery process (-100 mV ... -130 mV) after long preceding inactivation and at maintained depolarization (-50 mV ... -20 mV). For description, the model was extended by two further, serially coupled inactivated states. All four rate constants describing slow inactivation in the range of hundreds of milliseconds to seconds proved to be voltage-independent. Taken together, the results suggested the absorbing inactivated state I to be composed of five serially coupled inactivated states with only two transitions being steeply voltage-dependent.

Animals↗

PubMed2005

"Host tissue damage" signal ATP promotes non-directional migration and negatively regulates toll-like receptor signaling in human monocytes.

The activation of Toll-like receptors (TLRs) by lipopolysaccharide or other ligands evokes a proinflammatory immune response, which is not only capable of clearing invading pathogens but can also inflict damage to host tissues. It is therefore important to prevent an overshoot of the TLR-induced response where necessary, and here we show that extracellular ATP is capable of doing this in human monocytes. Using reverse transcription-PCR, we showed that monocytes express P2Y(1), P2Y(2), P2Y(4), P2Y(11), and P2Y(13) receptors, as well as several P2X receptors. To elucidate the function of these receptors, we first studied Ca(2+) signaling in single cells. ATP or UTP induced a biphasic increase in cytosolic Ca(2+), which corresponded to internal Ca(2+) release followed by activation of store-operated Ca(2+) entry. The evoked Ca(2+) signals stimulated Ca(2+)-activated K(+) channels, producing transient membrane hyperpolarization. In addition, ATP promoted cytoskeleton reorganization and cell migration; however, unlike chemoattractants, the migration was non-directional and further analysis showed that ATP did not activate Akt, essential for sensing gradients. When TLR2, TLR4, or TLR2/6 were stimulated with their respective ligands, ATPgammaS profoundly inhibited secretion of proinflammatory cytokines (tumor necrosis factor-alpha and monocyte chemoattractant protein-1) but increased the production of interleukin-10, an anti-inflammatory cytokine. In radioimmune assays, we found that ATP (or ATPgammaS) strongly increased cAMP levels, and, moreover, the TLR-response was inhibited by forskolin, whereas UTP neither increased cAMP nor inhibited the TLR-response. Thus, our data suggest that ATP promotes non-directional migration and, importantly, acts as a "host tissue damage" signal via the G(s) protein-coupled P2Y(11) receptor and increased cAMP to negatively regulate TLR signaling.

Adenosine Triphosphate↗

PubMed2005

Electrical synapses between dopaminergic neurons of the substantia nigra pars compacta.

Spatiotemporal properties of dopamine release play a major role both in striatal and nigral physiology because dopamine is released from nerve terminals and dendrites of nigrostriatal dopaminergic (DA) neurons. Pioneering work revealed gap junctional communication (assessed by dye-coupling experiments) between DA cells in the substantia nigra pars compacta (SNc). However, direct evidence of functional electrical synapses between DA neurons is still lacking. In this study, gap junctional communication between DA neurons was investigated in rat brain slices. Tracer coupling was observed in postnatal day 5 (P5) to P10 and P15-P25 rats. Dual whole-cell patch-clamp recordings revealed that 96% of DA neurons were coupled by electrical synapses in P7-P10 rats, and 20% were coupled in P17-P21 rats. These electrical synapses were mainly symmetrical and displayed strong low-pass filtering properties. When spontaneous firing activity was monitored, no significant synchronization was observed. Nevertheless, an efficient modulation of the spontaneous firing frequency of the postsynaptic cell occurred during injection of hyperpolarizing and depolarizing currents in the coupled presynaptic cell. Together, these observations demonstrate the existence of a fast communication between SNc DA neurons through electrical synapses.

Action Potentials↗

PubMed1997

Neural crest is involved in development of abnormal myocardial function.

Around 85% of embryos homozygous for the splotch (Sp2H) allele (Sp2H/Sp2H), a Pax3 mutation, develop persistent truncus arteriosus (PTA), a defect related to the cardiac neural crest. These embryos die by 14.5 days post coitum. In an investigation of the cause of lethality in these embryos, we used digital video imaging microscopy to examine beating embryonic hearts in situ at 13.5 dpc. The hearts of Sp2H/Sp2H embryos with PTA clearly showed poor function when compared with normal litter mates. Contractile force was examined in detergent-skinned ventricular muscle strips from Sp2H/Sp2H embryos at ages 12.5 and 13.5 dpc. There was no significant difference in the maximum force or in myosin content between Sp2H/Sp2H and control groups, indicating no significant dysfunction of the contractile apparatus in hearts from Sp2H/Sp2H embryos. Ca2+ transients were examined in enzymatically-dissociated ventricular myocytes and were significantly reduced in defective hearts, indicating that reduced cardiac function in Sp2H/Sp2H embryos with PTA was due to impaired excitation-contraction (EC) coupling. Ca2+ currents were examined using the perforated patch clamp technique. The magnitude of the Ca2+ current was found to be reduced by approximately 3.2-fold in Sp2H/Sp2H hearts with PTA compared to normal. Since the sarcoplasmic reticulum is sparse or absent in the embryonic heart, the impaired EC coupling was due to the reduction in Ca2+ current. These observations suggest that neural crest abnormalities result in a defect in EC coupling, causing depressed myocardial function and death in utero from cardiac failure. Interestingly, Sp2H/Sp2H hearts without PTA had normal EC coupling. These results indicated that impaired EC coupling was secondary to the Pax3 mutation. The findings in this report indicate an important role for the neural crest in the development of normal myocardial function, and represent the first demonstration of impaired excitation-contraction coupling in a genetically-defined embryonic mammalian model of a cardiac structural defect.

Animals↗

PubMed2004

Functional properties of electrical synapses between inhibitory interneurons of neocortical layer 4.

The existence of electrical synapses between GABAergic inhibitory interneurons in neocortex is well established, but their functional properties have not been described in detail. We made whole cell recordings from pairs of electrically coupled fast-spiking (FS) or low threshold-spiking (LTS) neurons, and filled some cells with biocytin for morphological reconstruction. Data were used to create compartmental cable models and to guide mathematical analysis. We analyzed the time course and amplitude of electrical postsynaptic potentials (ePSPs), the subthreshold events generated by presynaptic action potentials, in both FS and LTS neurons. The results imply that the generation of ePSPs is predominantly a linear process in both cell types for presynaptic firing of both single and repetitive spikes. Nonlinearities shape ePSPs near spike threshold, but our data suggest that the underlying synaptic current is still a linear process. Cell-to-cell electrical signaling on longer timescales also appears to be linear. Cable models of electrically coupled FS and LTS neurons imply that the analyzed electrical synapses are, on average, within 50 mum of the soma. Finally, we show that electrical coupling between 2 inhibitory cells promotes synchrony at all spiking frequencies. This contrasts with the effect of reciprocal inhibitory postsynaptic potentials (IPSPs) evoked by the same cells, which promote antisynchronous firing at frequencies less than about 100 Hz. Electrical coupling counteracts the antisynchronous behavior induced by IPSPs and facilitates spiking synchrony. Our results suggest that electrical synapses among inhibitory interneurons are most readily described as low-pass linear filters that promote firing synchrony.

Analysis of Variance↗

PubMed2006

High-density presynaptic transporters are required for glutamate removal from the first visual synapse.

Reliable synaptic transmission depends not only on the release machinery and the postsynaptic response mechanism but also on removal or degradation of transmitter from the synaptic cleft. Accumulating evidence indicates that postsynaptic and glial excitatory amino acid transporters (EAATs) contribute to glutamate removal. However, the role of presynaptic EAATs is unclear. Here, we show in the mouse retina that glutamate is removed from the synaptic cleft at the rod to rod bipolar cell (RBC) synapse by presynaptic EAATs rather than by postsynaptic or glial EAATs. The RBC currents evoked by electrical stimulation of rods decayed slowly after pharmacological blockade of EAATs. Recordings of the evoked RBC currents from EAAT subtype-deficient mice and the EAAT-coupled anion current reveal that functional EAATs are localized to rod terminals. Model simulations suggest that rod EAATs are densely packed near the release site and that rods are equipped with an almost self-sufficient glutamate recollecting system.

Animals↗

PubMed2001

Creation of a pluripotent ubiquitin-conjugating enzyme.

We describe the creation of a pluripotent ubiquitin-conjugating enzyme (E2) generated through a single amino acid substitution within the catalytic domain of RAD6 (UBC2). This RAD6 derivative carries out the stress-related function of UBC4 and the cell cycle function of CDC34 while maintaining its own DNA repair function. Furthermore, it carries out CDC34's function in the absence of the CDC34 carboxy-terminal extension. By using sequence and structural comparisons, the residues that define the unique functions of these three E2s were found on the E2 catalytic face partitioned to either side by a conserved divide. One of these patches corresponds to a binding site for both HECT and RING domain proteins, suggesting that a single substitution in the catalytic domain of RAD6 confers upon it the ability to interact with multiple ubiquitin protein ligases (E3s). Other amino acid substitutions made within the catalytic domain of RAD6 either caused loss of its DNA repair function or modified its ability to carry out multiple E2 functions. These observations suggest that while HECT and RING domain binding may generally be localized to a specific patch on the E2 surface, other regions of the functional E2 face also play a role in specificity. Finally, these data also indicate that RAD6 uses a different functional region than either UBC4 or CDC34, allowing it to acquire the functions of these E2s while maintaining its own. The pluripotent RAD6 derivative, coupled with sequence, structural, and phylogenetic data, suggests that E2s have diverged from a common multifunctional progenitor.

Amino Acid Sequence↗