Axo-axonal coupling. a novel mechanism for ultrafast neuronal communication

We provide physiological, pharmacological, and structural evidence that axons of hippocampal principal cells are electrically coupled, with prepotentials or spikelets forming the physiological substrate of electrical coupling as observed in cell somata. Antidromic activation of neighboring axons induced somatic spikelet potentials in neurons of CA3, CA1, and dentate gyrus areas of rat hippocampal slices. Somatic invasion by these spikelets was dependent on the activation of fast Na(+) channels in the postjunctional neuron. Antidromically elicited spikelets were suppressed by gap junction blockers and low intracellular pH. Paired axo-somatic and somato-dendritic recordings revealed that the coupling potentials appeared in the axon before invading the soma and the dendrite. Using confocal laser scanning microscopy we found that putative axons of principal cells were dye coupled. Our data thus suggest that hippocampal neurons are coupled by axo-axonal junctions, providing a novel mechanism for very fast electrical communication.     

Involvement of a bifunctional,paired-like DNA-binding domain and a transpositional enhancer in Sleeping Beauty transposition

Sleeping Beauty (SB) is the most active Tc1/mariner-like transposon in vertebrate species. Each of the terminal inverted repeats (IRs) of SB contains two transposase-binding sites (DRs). This feature, termed the IR/DR structure, is conserved in a group of Tc1-like transposons. The DNA-binding region of SB transposase, similar to the paired domain of Pax proteins, consists of two helix-turn-helix subdomains (PAI + RED = PAIRED). The N-terminal PAI subdomain was found to play a dominant role in contacting the DRs. Transposase was able to bind to mutant sites retaining the 3' part of the DRs; thus, primary DNA binding is not sufficient to determine the specificity of the transposition reaction. The PAI subdomain was also found to bind to a transpositional enhancer-like sequence within the left IR of SB, and to mediate protein-protein interactions between transposase subunits. A tetrameric form of the transposase was detected in solution, consistent with an interaction between the IR/DR structure and a transposase tetramer. We propose a model in which the transpositional enhancer and the PAI subdomain stabilize complexes formed by a transposase tetramer bound at the IR/DR. These interactions may result in enhanced stability of synaptic complexes, which might explain the efficient transposition of Sleeping Beauty in vertebrate cells.     

A second human methionine sulfoxide reductase (hMSRB2) reducing methionine-R-sulfoxide displays a tissue expression pattern distinct from hMSRB1

Peptide methionine sulfoxide reductases are important enzymes in the defense against cellular oxidative stress as they reduce methionine sulfoxide, the product of methionine oxidation by physiologically relevant reactive oxygen species. Two distinct enzyme classes, MSRA and MSRB, have evolved for selectively reducing the two epimers, methionine-S-sulfoxide and methionine-R-sulfoxide. A new human MSR enzyme (hMSRB2) specifically reducing methionine-R-sulfoxide, which showed a conversion rate for peptide-bound methionine-S-sulfoxide similar to hMSRB1, was characterized with respect to its tissue expression. As previously found for hMSRB1, expression of hMSRB2 mRNA was weak in brain, but strong in heart and skeletal muscle. In contrast to hMSRB1, its expression was high in smooth muscle-containing organs (digestive system, bladder), lung and aorta, while hMSRB1 displayed a higher expression than hMSRB2 in liver and kidney.     

Inhibition of human ether à go-go potassium channels by Ca(2+)/calmodulin

Intracellular Ca(2+) inhibits voltage-gated potassium channels of the ether à go-go (EAG) family. To identify the underlying molecular mechanism, we expressed the human version hEAG1 in XENOPUS: oocytes. The channels lost Ca(2+) sensitivity when measured in cell-free membrane patches. However, Ca(2+) sensitivity could be restored by application of recombinant calmodulin (CaM). In the presence of CaM, half inhibition of hEAG1 channels was obtained in 100 nM Ca(2+). Overlay assays using labelled CaM and glutathione S-transferase (GST) fusion fragments of hEAG1 demonstrated direct binding of CaM to a C-terminal domain (hEAG1 amino acids 673-770). Point mutations within this section revealed a novel CaM-binding domain putatively forming an amphipathic helix with both sides being important for binding. The binding of CaM to hEAG1 is, in contrast to Ca(2+)-activated potassium channels, Ca(2+) dependent, with an apparent K(D) of 480 nM. Co-expression experiments of wild-type and mutant channels revealed that the binding of one CaM molecule per channel complex is sufficient for channel inhibition.     

Subunit composition of kainate receptors in hippocampal interneurons

Kainate receptor activation affects GABAergic inhibition in the hippocampus by mechanisms that are thought to involve the GluR5 subunit. We report that disruption of the GluR5 subunit gene does not cause the loss of functional KARs in CA1 interneurons, nor does it prevent kainate-induced inhibition of evoked GABAergic synaptic transmission onto CA1 pyramidal cells. However, KAR function is abolished in mice lacking both GluR5 and GluR6 subunits, indicating that KARs in CA1 stratum radiatum interneurons are heteromeric receptors composed of both subunits. In addition, we show the presence of presynaptic KARs comprising the GluR6 but not the GluR5 subunit that modulate synaptic transmission between inhibitory interneurons. The existence of two separate populations of KARs in hippocampal interneurons adds to the complexity of KAR localization and function.     

Voltage dependence of slow inactivation in Shaker potassium channels results from changes in relative K(+) and Na(+) permeabilities

Time constants of slow inactivation were investigated in NH(2)-terminal deleted Shaker potassium channels using macro-patch recordings from Xenopus oocytes. Slow inactivation is voltage insensitive in physiological solutions or in simple experimental solutions such as K(+)(o)//K(+)(i) or Na(+)(o)//K(+)(i). However, when [Na(+)](i) is increased while [K(+)](i) is reduced, voltage sensitivity appears in the slow inactivation rates at positive potentials. In such solutions, the I-V curves show a region of negative slope conductance between approximately 0 and +60 mV, with strongly increased outward current at more positive voltages, yielding an N-shaped curvature. These changes in peak outward currents are associated with marked changes in the dominant slow inactivation time constant from approximately 1.5 s at potentials less than approximately +60 mV to approximately 30 ms at more than +150 mV. Since slow inactivation in Shaker channels is extremely sensitive to the concentrations and species of permeant ions, more rapid entry into slow inactivated state(s) might indicate decreased K(+) permeation and increased Na(+) permeation at positive potentials. However, the N-shaped I-V curve becomes fully developed before the onset of significant slow inactivation, indicating that this N-shaped I-V does not arise from permeability changes associated with entry into slow inactivated states. Thus, changes in the relative contributions of K(+) and Na(+) ions to outward currents could arise either: (a) from depletions of [K(+)](i) sufficient to permit increased Na(+) permeation, or (b) from voltage-dependent changes in K(+) and Na(+) permeabilities. Our results rule out the first of these mechanisms. Furthermore, effects of changing [K(+)](i) and [K(+)](o) on ramp I-V waveforms suggest that applied potential directly affects relative permeation by K(+) and Na(+) ions. Therefore, we conclude that the voltage sensitivity of slow inactivation rates arises indirectly as a result of voltage-dependent changes in the ion occupancy of these channels, and demonstrate that simple barrier models can predict such voltage-dependent changes in relative permeabilities.     

Two exposed amino acid residues confer thermostability on a cold shock protein

Thermophilic organisms produce proteins of exceptional stability. To understand protein thermostability at the molecular level we studied a pair of cold shock proteins, one of mesophilic and one of thermophilic origin, by systematic mutagenesis. Although the two proteins differ in sequence at 12 positions, two surface-exposed residues are responsible for the increase in stability of the thermophilic protein (by 15.8 kJ mol-1 at 70 degrees C). 11.5 kJ mol-1 originate from a predominantly electrostatic contribution of Arg 3 and 5.2 kJ mol-1 from hydrophobic interactions of Leu 66 at the carboxy terminus. The mesophilic protein could be converted to a highly thermostable form by changing the Glu residues at positions 3 and 66 to Arg and Leu, respectively. The variation of surface residues may thus provide a simple and powerful approach for increasing the thermostability of a protein.     

Acceleration of P/C-type inactivation in voltage-gated K(+) channels by methionine oxidation

Oxidation of amino acid residues causes noticeable changes in gating of many ion channels. We found that P/C-type inactivation of Shaker potassium channels expressed in Xenopus oocytes is irreversibly accelerated by patch excision and that this effect was mimicked by application of the oxidant H(2)O(2), which is normally produced in cells by the dismutase action on the superoxide anion. The inactivation time course was also accelerated by high concentration of O(2). Substitution of a methionine residue located in the P-segment of the channel with a leucine largely eliminated the channel's sensitivity to patch excision, H(2)O(2), and high O(2). The results demonstrate that oxidation of methionine is an important regulator of P/C-type inactivation and that it may play a role in mediating the cellular responses to hypoxia/hyperoxia.     

The muO-conotoxin MrVIA inhibits voltage-gated sodium channels by associating with domain-3

Several families of peptide toxins from cone snails affect voltage-gated sodium (Na(V)) channels: mu-conotoxins block the pore, delta-conotoxins inhibit channel inactivation, and muO-conotoxins inhibit Na(V) channels by an unknown mechanism. The only currently known muO-conotoxins MrVIA and MrVIB from Conus marmoreus were applied to cloned rat skeletal muscle (Na(V)1.4) and brain (Na(V)1.2) sodium channels in mammalian cells. A systematic domain-swapping strategy identified the C-terminal pore loop of domain-3 as the major determinant for Na(V)1.4 being more potently blocked than Na(V)1.2 channels. muO-conotoxins therefore show an interaction pattern with Na(V) channels that is clearly different from the related mu- and delta-conotoxins, indicative of a distinct molecular mechanism of channel inhibition.     

Crystal structure analysis of an A-DNA fragment at 1.8 A resolution: d(GCCCGGGC).

Single crystals of the self-complementary octadeoxyribonucleotide d(GCCCGGGC) have been analysed by X-ray diffraction methods at a resolution of 1.8 A. The tetragonal unit cell of space group P4(3)2(1)2 has dimensions of a = 43.25 A and c = 24.61 A and contains eight strands of the oligonucleotide. The structure was refined by standard crystallographic techniques to an R factor of 17.1% using 1359 3 sigma structure factor observations. Two strands of the oligonucleotide are related by the crystallographic dyad axis to form a DNA helix in the A conformation. The d(GCCCGGGC) helix is characterized by a wide open major groove, a near perpendicular orientation of base pairs to the helix axis and an unusually small average helix twist angle of 31.3 degrees indicating a slightly underwound helix with 11.5 base pairs per turn. Extensive cross-strand stacking between guanine bases at the central cytosine-guanine step is made possible by a number of local conformational adjustments including a fully extended sugar-phosphate backbone of the central guanosine nucleotide.