Single-exponential Fits Research Articles

Thermotaxis in an apolar, non-neuronal animal.

Neuronal circuits are hallmarks of complex decision-making processes in the animal world. How animals without neurons process information and respond to environmental cues promises a new window into studying precursors of neuronal control and origin of the nervous system as we know it today. Robust decision making in animals, such as in chemotaxis or thermotaxis, often requires internal symmetry breaking (such as anterior-posterior (AP) axis) provided naturally by a given body plan of an animal. Here we report the discovery of robust thermotaxis behaviour in Trichoplax adhaerens, an early-divergent, enigmatic animal with no anterior-posterior symmetry breaking (apolar) and no known neurons or muscles. We present a quantitative and robust behavioural response assay in Placozoa, which presents an apolar flat geometry. By exposing T. adhaerens to a thermal gradient under a long-term imaging set-up, we observe robust thermotaxis that occurs over timescale of hours, independent of any circadian rhythms. We quantify that T. adhaerens can detect thermal gradients of at least 0.1°C cm-1. Positive thermotaxis is observed for a range of baseline temperatures from 17°C to 22.5°C, and distributions of momentary speeds for both thermotaxis and control conditions are well described by single exponential fits. Interestingly, the organism does not maintain a fixed orientation while performing thermotaxis. Using natural diversity in size of adult organisms (100 µm to a few millimetres), we find no apparent size-dependence in thermotaxis behaviour across an order of magnitude of organism size. Several transient receptor potential (TRP) family homologues have been previously reported to be conserved in metazoans, including in T. adhaerens. We discover naringenin, a known TRPM3 antagonist, inhibits thermotaxis in T. adhaerens. The discovery of robust thermotaxis in T. adhaerens provides a tractable handle to interrogate information processing in a brainless animal. Understanding how divergent marine animals process thermal cues is also critical due to rapid temperature rise in our oceans.

Fluorinated Bambusurils as Highly Effective and Selective Transmembrane Cl−/HCO3− Antiporters

Summary The exchange of chloride and bicarbonate across lipid bilayers is an important biological process. Synthetic molecules can act as mobile carriers for these anions, although most show little selectivity. Here we report on three bambus[6]uril macrocycles functionalized with fluorinated benzyl groups, which are able to exchange Cl− and HCO3− efficiently. Remarkably, rates for Cl−/NO3− exchange are two orders of magnitude lower. The higher rates of Cl−/HCO3− transport can be explained by the ability of the bambusurils to complex Cl− and HCO3− simultaneously, facilitating their exchange at the bilayer interface. Furthermore, the exceptionally high affinity and selectivity of these systems for NO3− appear to contribute to the poor Cl−/NO3− exchange. This work not only demonstrates the importance of anion binding characteristics on anion transport but also the potential relevance of bambusurils for anion transport applications considering the high rate observed for Cl−/HCO3− exchange.

A kinetic and mechanistic study of dinuclear Pt(II) 2,2′:6′,2″-terpyridine compounds bridged with polyethyleneglycol ether flexible linkers

A series of dinuclear Pt(II) complexes bridged with polyethyleneglycol ether of the type [ClPt(tpy)O(CH2CH2O)n(tpy)PtCl]Cl2 where n = 1 (Ptdteg), 2 (Ptdtdeg), 3 (Ptdtteg), 4 (Ptdttteg), and linker-free complex, (Ptdt) (where tpy = 2,2′:6′,2″-terpyridine), were synthesized and characterized to investigate the role of bridging polyethyleneglycol ether linker on the substitution reactivity of dinuclear Pt(II) complexes. Substitution reactions were studied using thiourea nucleophiles, viz. thiourea (TU), 1,3-dimethyl-2-thiourea (DMTU), 1,1,3,3-tetramethyl-2-thiourea (TMTU) under pseudo-first-order conditions as a function of concentration and temperature by conventional stopped-flow reaction analyzer. The reactions gave single exponential fits following the rate law kobs = k2[Nu]. Introduction of polyethyleneglycol ether linker decreases the electrophilicity of the platinum center and the whole complex. The results obtained indicate that the rate of substitution is controlled by both electronic and steric hindrance which increases with the length of the linker. Experimental results are supported by density functional theory calculations and structures obtained at B3LYP/LANL2DZ level. The order of the reactivity of the nucleophiles is TU > DMTU > TMTU. The magnitude and the size of the enthalpy of activation and entropy of activation support an associative mode of mechanism, where bond formation in the transition state is favored.

Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23-E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·Pi complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.

Propofol Binding to the Resting State of the Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Induces Structural Changes in the Inter- and Intrasubunit Transmembrane Domain (TMD) Cavities

General anesthetics exert many of their CNS actions by binding to and modulating membrane-embedded pentameric ligand-gated ion channels (pLGICs). The structural mechanisms underlying how anesthetics modulate pLGIC function remain largely unknown. GLIC, a prokaryotic pLGIC homologue, is inhibited by general anesthetics, suggesting anesthetics stabilize a closed channel state, but in anesthetic-bound GLIC crystal structures the channel appears open. Here, using functional GLIC channels expressed in oocytes, we examined whether propofol induces structural rearrangements in the GLIC transmembrane domain (TMD). Residues in the GLIC TMD that frame intrasubunit and intersubunit water-accessible cavities were individually mutated to cysteine. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive reagents in the absence and presence of propofol. Propofol slowed the rate of modification of L240C (intersubunit) and increased the rate of modification of T254C (intrasubunit), indicating that propofol binding induces structural rearrangements in these cavities that alter the local environment near these residues. Propofol acceleration of T254C modification suggests that in the resting state propofol does not bind in the TMD intrasubunit cavity as observed in the crystal structure of GLIC with bound propofol (Nury, H., Van Renterghem, C., Weng, Y., Tran, A., Baaden, M., Dufresne, V., Changeux, J. P., Sonner, J. M., Delarue, M., and Corringer, P. J. (2011) Nature 469, 428-431). In silico docking using a GLIC closed channel homology model suggests propofol binds to intersubunit sites in the TMD in the resting state. Propofol-induced motions in the intersubunit cavity were distinct from motions associated with channel activation, indicating propofol stabilizes a novel closed state.

Attenuation length and spectral response of Kuraray SCSF-78MJ scintillating fibres

Over three quarters of a million, 4-m long Kuraray double-clad SCSF-78MJ (blue–green) scintillating fibres have been used in the construction of the GlueX electromagnetic barrel calorimeter, as part of the Hall D experimental program at Jefferson Lab. The response and quality of a random sample of 4750 of these fibres have been evaluated by employing a 373-nm UV LED to stimulate the fibres along their length and reading out the light using a spectrophotometer and a photodiode in order to extract the spectral response and the attenuation length, respectively. Single exponential fits to the spectral response in the 100–280cm distance from the light source yielded an average bulk attenuation length and standard deviation of (387±26)cm. Double-exponential fits to the spectral response over the entire 4-m length also allowed the extraction of long and short wavelength components at (486±54)cm and (75±22)cm, respectively. Finally, diameter uniformity measurements were carried out. The quality assurance results confirmed that the fibres were of high quality and complied with GlueX specifications.

Functional Characterization of a Novel Aquaporin from Dictyostelium discoideum Amoebae Implies a Unique Gating Mechanism

The social amoeba Dictyostelium discoideum is a widely used model organism for studying basic functions of protozoan and metazoan cells, such as osmoregulation and cell motility. There is evidence from other species that cellular water channels, aquaporins (AQP), are central to both processes. Yet, data on D. discoideum AQPs is almost absent. Despite cloning of two putative D. discoideum AQPs, WacA, and AqpA, water permeability has not been shown. Further, WacA and AqpA are expressed at the late multicellular stage and in spores but not in amoebae. We cloned a novel AQP, AqpB, from amoeboidal D. discoideum cells. Wild-type AqpB was impermeable to water, glycerol, and urea when expressed in Xenopus laevis oocytes. Neither stepwise truncation of the N terminus nor selected point mutations activated the water channel. However, mutational truncation by 12 amino acids of an extraordinary long intracellular loop induced water permeability of AqpB, hinting at a novel gating mechanism. This AqpB mutant was inhibited by mercuric chloride, confirming the presence of a cysteine residue in the selectivity filter as predicted by our structure model. We detected AqpB by Western blot analysis in a glycosylated and a non-glycosylated form throughout all developmental stages. When expressed in D. discoideum amoebae, AqpB-GFP fusion constructs localized to vacuolar structures, to the plasma membrane, and to lamellipodia-like membrane protrusions. We conclude that the localization pattern in conjunction with channel gating may be indicative of AqpB functions in osmoregulation as well as cell motility of D. discoideum.

Chronic imaging of cortical sensory map dynamics using a genetically encoded calcium indicator

In vivo optical imaging can reveal the dynamics of large-scale cortical activity, but methods for chronic recording are limited. Here we present a technique for long-term investigation of cortical map dynamics using wide-field ratiometric fluorescence imaging of the genetically encoded calcium indicator (GECI) Yellow Cameleon 3.60. We find that wide-field GECI signals report sensory-evoked activity in anaesthetized mouse somatosensory cortex with high sensitivity and spatiotemporal precision, and furthermore, can be measured repeatedly in separate imaging sessions over multiple weeks. This method opens new possibilities for the longitudinal study of stability and plasticity of cortical sensory representations.

Specific mechanism of use-dependent channel block of calcium-permeable AMPA receptors provides activity-dependent inhibition of glutamatergic neurotransmission

This study examined the blocking action of the selective channel blocker of calcium-permeable (CP) AMPA receptors, N1-(1-phenylcyclohexyl)pentane-1,5-diaminium bromide (IEM-1925), on excitatory postsynaptic currents in rat neostriatal and cortical neurons and in fly neuromuscular junctions. In both preparations, the blocking of CP-AMPA receptor currents increased along with the stimulation frequency. The continuous presence of kainate, which activates AMPA receptors, in the external solution also caused an enhanced blocking effect. Likewise, decrease of the synaptic release by lowering calcium concentration resulted in significant reduction of the blocking action. The activity dependence of the block is explained using the guarded receptor model. The drug molecule can only bind if the channel is open. After the channel has closed, the drug molecule remains trapped inside. However, the trapped molecule slowly egresses from closed channels to the cytoplasm. The total block effect is determined by the equilibrium between accumulation of the drug in the open channels and relief from the closed channels. Therefore, the conditions that favour the open state result in enhanced inhibition. This significant finding reveals a new way to modulate CP-AMPAR-mediated transmission using a physiologically relevant approach. Moreover, it allows the involvement of CP-AMPARs in the physiological and pathological processes – such as high-frequency synaptic activity or increase of the steady-state glutamate concentration – to be examined.

Properties of the TRPML3 Channel Pore and Its Stable Expansion by the Varitint-Waddler-causing Mutation

TRPML3 is a H(+)-regulated Ca(2+) channel that shuttles between intracellular compartments and the plasma membrane. The A419P mutation causes the varitint-waddler phenotype as a result of gain-of-function (GOF). The mechanism by which A419P leads to GOF is not known. Here, we show that the TRPML3 pore is dynamic when conducting Ca(2+) to change its conductance and permeability, which appears to be mediated by trapping Ca(2+) within the pore. The pore properties can be restored by strong depolarization or by conducting Na(+) through the pore. The A419P mutation results in expanded channel pore with altered permeability that limits modulation of the pore by Ca(2+). This effect is specific for the A419P mutation and is not reproduced by other GOF mutations, including A419G, H283A, and proline mutations in the fifth transmembrane domain. These findings describe a novel mode of a transient receptor potential channel behavior and suggest that pore expansion by the A419P mutation may contribute to the varitint-waddler phenotype.

α5β1 Integrin Engagement Increases Large Conductance, Ca2+-activated K+ Channel Current and Ca2+ Sensitivity through c-src-mediated Channel Phosphorylation

Large conductance, calcium-activated K(+) (BK) channels are important regulators of cell excitability and recognized targets of intracellular kinases. BK channel modulation by tyrosine kinases, including focal adhesion kinase and c-src, suggests their potential involvement in integrin signaling. Recently, we found that fibronectin, an endogenous alpha5beta1 integrin ligand, enhances BK channel current through both Ca(2+)- and phosphorylation-dependent mechanisms in vascular smooth muscle. Here, we show that macroscopic currents from HEK 293 cells expressing murine BK channel alpha-subunits (mSlo) are acutely potentiated following alpha5beta1 integrin activation. The effect occurs in a Ca(2+)-dependent manner, 1-3 min after integrin engagement. After integrin activation, normalized conductance-voltage relations for mSlo are left-shifted at free Ca(2+) concentrations >or=1 microm. Overexpression of human c-src with mSlo, in the absence of integrin activation, leads to similar shifts in mSlo Ca(2+) sensitivity, whereas overexpression of catalytically inactive c-src blocks integrin-induced potentiation. However, neither integrin activation nor c-src overexpression potentiates current in BK channels containing a point mutation at Tyr-766. Biochemical tests confirmed the critical importance of residue Tyr-766 in integrin-induced channel phosphorylation. Thus, BK channel activity is enhanced by alpha5beta1 integrin activation, likely through an intracellular signaling pathway involving c-src phosphorylation of the channel alpha-subunit at Tyr-766. The net result is increased current amplitude, enhanced Ca(2+) sensitivity, and rate of activation of the BK channel, which would collectively promote smooth muscle hyperpolarization in response to integrin-extracellular matrix interactions.

Disease-causing Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator Determine the Functional Responses of Alveolar Macrophages

Alveolar macrophages (AMs) play a major role in host defense against microbial infections in the lung. To perform this function, these cells must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Recently, we demonstrated that murine alveolar macrophages employ the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel as a determinant in lysosomal acidification (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Lysosomes and phagosomes in murine cftr(-/-) AMs failed to acidify, and the cells were deficient in bacterial killing compared with wild type controls. Cystic fibrosis is caused by mutations in CFTR and is characterized by chronic lung infections. The information about relationships between the CFTR genotype and the disease phenotype is scarce both on the organismal and cellular level. The most common disease-causing mutation, DeltaF508, is found in 70% of patients with cystic fibrosis. The mutant protein fails to fold properly and is targeted for proteosomal degradation. G551D, the second most common mutation, causes loss of function of the protein at the plasma membrane. In this study, we have investigated the impact of CFTR DeltaF508 and G551D on a set of core intracellular functions, including organellar acidification, granule secretion, and microbicidal activity in the AM. Utilizing primary AMs from wild type, cftr(-/-), as well as mutant mice, we show a tight correlation between CFTR genotype and levels of lysosomal acidification, bacterial killing, and agonist-induced secretory responses, all of which would be expected to contribute to a significant impact on microbial clearance in the lung.

Membrane time constant during internal defibrillation strength shocks in intact heart: effects of Na+ and Ca2+ channel blockers.

We assessed defibrillation strength shock-induced changes of the membrane time constant (tau) and membrane potential (DeltaVm) in intact rabbit hearts after administration of lidocaine, a sodium (Na(+)) channel blocker, or nifedipine, a L-type calcium (Ca(2+)) channel blocker. We optically mapped anterior, epicardial, electrical activity during monophasic shocks (+/-100, +/-130, +/-160, +/-190, and +/-220 V; 150 microF; 8 ms) applied at 25%, 50%, and 75% of the action potential duration via a shock lead system in Langendorff-perfused hearts. The protocol was run twice for each heart under control and after lidocaine (15 microM, n = 6) or nifedipine (2 microM, n = 6) addition. tau in the virtual electrode area away from the shock lead was approximated with single-exponential fits from a total of 121,125 recordings. The same data set was used to calculate DeltaVm. We found (1) Under all conditions, there is inverse relationship between tau and DeltaVm with respect to changes of shock strength, regardless of shock polarity and phase of application: a stronger shock resulted in a larger DeltaVm, which corresponded to a smaller tau (faster cellular response); (2) Lidocaine did not cause appreciable changes in either tau or DeltaVm versus control, and (3) Nifedipine significantly increased both tau and DeltaVm in the virtual cathode area; in contrast, in the virtual anode area, this effect depended on the phase of shock application. tau and DeltaVm are inversely related. Na(+) channel blocker has minimal impact on either tau or DeltaVm. Ca(2+) blocker caused polarity and phase-dependent significant changes in tau and DeltaVm.

Molecular Basis of the Bohr Effect in Arthropod Hemocyanin

Flash photolysis and K-edge x-ray absorption spectroscopy (XAS) were used to investigate the functional and structural effects of pH on the oxygen affinity of three homologous arthropod hemocyanins (Hcs). Flash photolysis measurements showed that the well-characterized pH dependence of oxygen affinity (Bohr effect) is attributable to changes in the oxygen binding rate constant, k(on), rather than changes in k(off). In parallel, coordination geometry of copper in Hc was evaluated as a function of pH by XAS. It was found that the geometry of copper in the oxygenated protein is unchanged at all pH values investigated, while significant changes were observed for the deoxygenated protein as a function of pH. The interpretation of these changes was based on previously described correlations between spectral lineshape and coordination geometry obtained for model compounds of known structure (Blackburn, N. J., Strange, R. W., Reedijk, J., Volbeda, A., Farooq, A., Karlin, K. D., and Zubieta, J. (1989) Inorg. Chem., 28, 1349-1357). A pH-dependent change in the geometry of cuprous copper in the active site of deoxyHc, from pseudotetrahedral toward trigonal was assigned from the observed intensity dependence of the 1s --> 4p(z) transition in x-ray absorption near edge structure (XANES) spectra. The structural alteration correlated well with increase in oxygen affinity at alkaline pH determined in flash photolysis experiments. These results suggest that the oxygen binding rate in deoxyHc depends on the coordination geometry of Cu(I) and suggest a structural origin for the Bohr effect in arthropod Hcs.

Phospholamban Oligomerization, Quaternary Structure, and Sarco(endo)plasmic Reticulum Calcium ATPase Binding Measured by Fluorescence Resonance Energy Transfer in Living Cells

Phospholamban (PLB) oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase (SERCA) binding were quantified by fluorescence resonance energy transfer (FRET) in an intact cellular environment. FRET between cyan fluorescent protein-PLB and yellow fluorescent protein-PLB in AAV-293 cells showed hyperbolic dependence on protein concentration, with a maximum efficiency of 45.1 +/- 1.3%. The observed FRET corresponds to a probe separation distance of 58.7 +/- 0.5A(,) according to a computational model of intrapentameric FRET. This is consistent with models of the PLB pentamer in which cytoplasmic domains fan out from the central bundle of transmembrane helices. An I40A mutation of PLB did not alter pentamer conformation but increased the concentration of half-maximal FRET (K(D)) by >4-fold. This is consistent with the previous observation that this putatively monomeric mutant still oligomerizes in intact membranes but forms more dynamic pentamers than wild type PLB. PLB association with SERCA, measured by FRET between cyan fluorescent protein-SERCA and yellow fluorescent protein-PLB, was increased by the I40A mutation without any detectable change in probe separation distance. The data indicate that the regulatory complex conformation is not altered by the I40A mutation. A naturally occurring human mutation (L39Stop) greatly reduced PLB oligomerization and SERCA binding and caused mislocalization of PLB to the cytoplasm and nucleus. Overall, the data suggest that the PLB pentamer adopts a "pinwheel" shape in cell membranes, as opposed to a more compact "bellflower" conformation. I40A mutation decreases oligomerization and increases PLB binding to SERCA. Truncation of the transmembrane domain by L39Stop mutation prevents anchoring of the protein in the membrane, greatly reducing PLB binding to itself or its regulatory target, SERCA.

Temperature Dependence of Electron Transfer to the M-Side Bacteriopheophytin in Rhodobacter capsulatus Reaction Centers

Subpicosecond time-resolved absorption measurements at 77 K on two reaction center (RC) mutants of Rhodobacter capsulatus are reported. In the D(LL) mutant the D helix of the M subunit has been substituted with the D helix from the L subunit, and in the D(LL)-FY(L)F(M) mutant, three additional mutations are incorporated that facilitate electron transfer to the M side of the RC. In both cases the helix swap has been shown to yield isolated RCs that are devoid of the native bacteriopheophytin electron carrier HL (Chuang, J. I.; Boxer, S. G.; Holten, D.; Kirmaier, C. Biochemistry 2006, 45, 3845-3851). For D(LL), depending whether the detergent Deriphat 160-C or N-lauryl-N,N-dimethylamine-N-oxide (LDAO) is used to suspend the RCs, the excited state of the primary electron donor (P*) decays to the ground state with an average lifetime at 77 K of 330 or 170 ps, respectively; however, in both cases the time constant obtained from single-exponential fits varies markedly as a function of the probe wavelength. These findings on the D(LL) RC are most easily explained in terms of a heterogeneous population of RCs. Similarly, the complex results for D(LL)-FY(L)F(M) in Deriphat-glycerol glass at 77 K are most simply explained using a model that involves (minimally) two distinct populations of RCs with very different photochemistry. Within this framework, in 50% of the D(LL)-FY(L)F(M) RCs in Deriphat-glycerol glass at 77 K, P* deactivates to the ground state with a time constant of approximately 400 ps, similar to the deactivation of P* in the D(LL) mutant at 77 K. In the other 50% of D(LL)-FY(L)F(M) RCs, P* has a 35 ps lifetime and decays via electron transfer to the M branch, giving P+HM- in high yield (> or =80%). This result indicates that P* --> P(+)H(M)(-) is roughly a factor of 2 faster at 77 K than at 295 K. In alternative homogeneous models the rate of this M-side electron-transfer process is the same or up to 2-fold slower at low temperature. A 2-fold increase in rate with a reduction in temperature is the same behavior found for the overall L-side process P* --> P(+)H(L)(-) in wild-type RCs. Our results suggest that, as for electron transfer on the L side, the M-side electron-transfer reaction P* --> P(+)H(M)(-) is an activationless process.

Dwell Time Analysis of a Single-Molecule Mechanochemical Reaction

Force-clamp spectroscopy is a novel technique for studying mechanochemistry at the single-bond level. Single disulfide bond reduction events are accurately detected as stepwise increases in the length of polyproteins that contain disulfide bonds and that are stretched at a constant force with the cantilever of an atomic force microscope (AFM). The kinetics of this reaction has been measured from single-exponential fits to ensemble averages of the reduction events. However, exponential fits are notoriously ambiguous to use in cases of kinetic data showing multiple reaction pathways. Here we introduce a dwell time analysis technique, of widespread use in the single ion channel field, that we apply to the examination of the kinetics of reduction of disulfide bonds measured from single-molecule force-clamp spectroscopy traces. In this technique, exponentially distributed dwell time data is plotted as a histogram with a logarithmic time scale and a square root ordinate. The advantage of logarithmic histograms is that exponentially distributed dwell times appear as well-defined peaks in the distribution, greatly enhancing our ability to detect multiple kinetic pathways. We apply this technique to examine the distribution of dwell times of 4488 single disulfide bond reduction events measured in the presence of two very different kinds of reducing agents: tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) and the enzyme thioredoxin (TRX). A different clamping force is used for each reducing agent to obtain distributions of dwell times on a similar time scale. In the case of TCEP, the logarithmic histogram of dwell times showed a single peak, corresponding to a single reaction mechanism. By contrast, similar experiments done with TRX showed two well-separated peaks, marking two distinct modes of chemical reduction operating simultaneously. These experiments demonstrate that dwell time analysis techniques are a powerful approach to studying chemical reactions at the single-molecule level.

Structure/Function Characterization of μ-Conotoxin KIIIA, an Analgesic, Nearly Irreversible Blocker of Mammalian Neuronal Sodium Channels

Structure/Function Characterization of μ-Conotoxin KIIIA, an Analgesic, Nearly Irreversible Blocker of Mammalian Neuronal Sodium Channels

Calcium‐dependent inactivation of calcium channels in cochlear hair cells of the chicken

Voltage-gated calcium channels support both spontaneous and sound-evoked neurotransmitter release from ribbon synapses of cochlear hair cells. A variety of regulatory mechanisms must cooperate to ensure the appropriate level of activity in the restricted pool of synaptic calcium channels ( approximately 100) available to each synaptic ribbon. One potential feedback mechanism, calcium-dependent inactivation (CDI) of voltage-gated, L-type calcium channels, can be modulated by calmodulin-like calcium-binding proteins. CDI of voltage-gated calcium current was studied in hair cells of the chicken's basilar papilla (analogous to the mammalian cochlea) after blocking the predominant potassium conductances. For inactivating currents produced by 2.5 s steps to the peak of the current-voltage relation (1 mm EGTA internal calcium buffer), single exponential fits yielded an average decay time constant of 1.92 +/- 0.18 s (mean +/- s.e.m., n = 12) at 20-22 degrees C, while recovery occurred with a half-time of approximately 10 s. Inactivation produced no change in reversal potential, arguing that the observed relaxation did not result from alternative processes such as calcium accumulation or activation of residual potassium currents. Substitution of external calcium with barium greatly reduced inactivation, while inhibition of endoplasmic calcium pumps with t-benzohydroquinone (BHQ) or thapsigargin made inactivation occur faster and to a greater extent. Raising external calcium 10-fold (from 2 to 20 mm) increased peak current 3-fold, but did not alter the extent or time course of CDI. However, increasing levels of internal calcium buffer consistently reduced the rate and extent of inactivation. With 1 mm EGTA buffering and in 2 mm external calcium, the available pool of calcium channels was half-inactivated near the resting membrane potential (-50 mV). CDI may be further regulated by calmodulin-like calcium-binding proteins (CaBPs). mRNAs for several CaBPs are expressed in chicken cochlear tissue, and antibodies to CaBP4 label hair cells, but not supporting cells, equivalent to the pattern seen in mammalian cochlea. Thus, molecular mechanisms that underlie CDI appeared to be conserved across vertebrate species, may provide a means to adjust calcium channel open probability, and could serve to maintain the set-point for spontaneous release from the ribbon synapse.

Two Families of TARP Isoforms that Have Distinct Effects on the Kinetic Properties of AMPA Receptors and Synaptic Currents

Transmembrane AMPA receptor regulatory proteins (TARPs) are auxiliary AMPA receptor subunits that regulate both the trafficking and gating properties of AMPA receptors, and different TARP isoforms display distinct expression patterns in brain. Here, we compared the effects of four TARP isoforms on the kinetics of AMPA receptor currents. Each isoform slowed the deactivation of GluR1 currents, but the slowing was greatest with gamma-4 and gamma-8. Isoform-specific differences in desensitization were also observed that correlated with effects on deactivation. TARP isoforms also differentially modulated responses to trains of glutamate applications designed to mimic high-frequency presynaptic firing. Importantly, whereas both stargazin and gamma-4 rescued excitatory synaptic transmission in cerebellar granule cells from stargazer mice, the decay of miniature EPSCs was 2-fold slower in neurons expressing gamma-4. The results show that heterogeneity in the composition of AMPA receptor/TARP complexes contributes to synapse-specific differences in EPSC decays and frequency-dependent modulation of neurotransmission.