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Inwardly rectifying K+ channels conduct more inward than outward current as a result of voltage-dependent block of the channel pore by intracellular Mg2+ and polyamines. We investigated the molecular mechanism and structural determinants of inward rectification and ion permeation in a strongly rectifying channel, IRK1. Block by Mg2+ and polyamines is found not to conform to one-to-one binding, suggesting...
In the bilaterally symmetrical vertebrate CNS, all developing axons must choose between remaining on the same side of the midline or growing across it. The mechanism underlying this axonal pathfinding is, however, poorly understood. Here we demonstrate that the ventral midline floor plate (FP) chemorepels two types of ipsilaterally projecting axons, one from the alar plate and another from the basal...
In nucleus basalis neurons, substance P (SP) causes a slow excitation, mediated through a pertussis toxininsensitive G protein, by suppressing an inward rectifier K+ channel. Here we report that SP applied outside the patch pipette inhibited the single-channel activity, recorded on-cell, of the inward rectifier. The PKC inhibitors staurosporine and PKC(19-36) suppressed this effect in whole-cell mode...
Currents mediated by a glutamate transporter cloned from human motor cortex were measured in Xenopus oocytes. In the absence of glutamate, voltage jumps induced Na+-dependent capacitive currents that were blocked by kainate, a competitive transport antagonist. The pre-steady-state currents can be described by an ordered binding model in which a voltage-dependent Na+-binding is followed by a voltage-independent...
Sympathetic neurons depend on nerve growth factor (NGF) for survival and die by apoptosis in its absence. We have investigated the pattern of expression of the Jun and Fos family of transcription factors in dying sympathetic neurons using antibodies specific for each family member. When sympathetic neurons are deprived of NGF, the level of c-Jun protein significantly increases, whereas the levels...
Members of the collapsin/semaphorin gene family have been proposed to act as growth cone guidance signals in vertebrates and invertebrates. To identify candidate molecules involved in axonal pathfinding during mouse embryogenesis, we isolated cDNAs encoding five new members of the semaphorin family (Sem A-Sem E). The murine semaphorin genes are differentially expressed in mesoderm and neuroectoderm...
In the neurological mutant mouse reeler, the histological organization of the neocortex develops abnormally and essentially results in an inversion of the relative positions of the cortical layers. The reeler mutation, therefore, provides an insight into the molecular mechanisms underlying the formation of the cortical layers. We have generated a monoclonal antibody (CR-50) that probes a distinct...
Two inward rectifier potassium channels, the G protein-dependent GIRK1 and the G protein-independent BIR10, display large differences in rectification and macroscopic kinetics. A chimeric channel was constructed in which the putative intracellular carboxy-terminal domain of the G protein-dependent channel replaced the corresponding domain of the G protein-independent channel. The chimeric channel...
Using the fluorescent membrane label FM 1-43, we have measured the release, reuptake, and repriming of synaptic vesicles in response to action potential stimulation of cultured hippocampal neurons. We find that ~90% of a recycling vesicle pool is released during 60 s of 10 Hz action potential firing, and that a single action potential releases approximately 0.5% of that pool. Our data also indicate...
Asymmetric cell divisions allow a sensory organ precursor (SOP) cell to generate a neuron and its support cells in the Drosophila PNS. We demonstrate a role of tramtrack (ttk), previously identified as a zinc finger-containing putative transcription factor, in the determination of different daughter cell fates. Both loss of function and overexpression of ttk affect the fates of the SOP progeny. Whereas...
The structure of the carboxyl half of the pore-forming region of Kv2.1 was studied by replacing each of 15 consecutive residues between positions 383 and 369 with a reporter cysteine residue. Extracellular application of charged, membrane-impermeant methanethiosulfonates irreversibly modified currents at four cysteine-substituted positions, K382, Y380,1379, and D378. Intracellular exposure to methanethiosulfonate...
To determine the kainate receptor subunits that are found in native kainate receptors, we have applied a multiplex PCR of cDNAs reverse transcribed from mRNA harvested from single cultured hippocampal neurons after electrophysiological recording. We found that all the cells showing rapidly desensitizing currents in response to kainate express the GIuR6 subunit mRNA, and that some of them also express...
Huntington's Disease (HD) is caused by expansion of a CAG repeat within a putative open reading frame of a recently identified gene, IT15. We have examined the expression of the gene's protein product using antibodies developed against the N-terminus and an internal epitope. Both antisera recognize a 350 kDa protein, the predicted size, indicating that the CAG repeat is translated into polyglutamine...
A novel heterologous expression system was used to examine the coupling of metabotropic glutamate receptors (mGluRs) to neuronal voltage-gated ion channels. Cytoplasmic injection of mGIuR2 cRNA into adult rat sympathetic neurons resulted in the expression of receptors that negatively coupled to N-typ ertussis toxin-sensitive pathway. Injection of mGIuR1α cRNA resulted in the expression of receptors...
Membrane fusion resulting in neurotransmitter secretion forms the basis of neural communication. Three multimeric complexes of the protein syntaxin are important in this process: syntaxin and n-sect; syntaxin, VAMP, and SNAP-25; and syntaxin, VAMP, SNAP-25, αSNAP, and NSF (20S complex). In this report, we demonstrate that unique, yet overlapping, domains of syntaxin are required to form these complexes...
Mutations in Cu/Zn superoxide dismutase (SOD1) cause a subset of cases of familial amyotrophic lateral sclerosis. Four linesof mice accumulating oneof these mutant proteins (G37R) develop severe, progressive motor neuron disease. At lower levels of mutant accumulation, pathology is restricted to lower motor neurons, whereas higher levels cause more severe abnormalities and affect a variety of other...
Plexin (previously referred to as 132) is a neuronal cell surface molecule that has been identified in Xenopus. cDNA cloning reveals that plexin has no homology to known neuronal cell surface molecules but possesses, in its extracellular segment, three internal repeats of cysteine clusters that are homologous to the cysteine-rich domain of the c-met proto-oncogene protein product. The exogenous plexin...
This study examines the mechanisms of spontaneous and induced [Ca2+]i spiking in nerve growth cones and the effect of spikes on growth cone migration. Over a 10-20 min observation period, 29% of DRG growth cones undergo spontaneous and transient elevations in physiological extracellular Ca2+ ((Ca2+)o; 2 mM), whereas 67% of growth cones exposed to 20 mM (Ca2+)o exhibit similar [Ca2+]i spikes. Spontaneous...
Expression of minK protein in Xenopus oocytes induces a slowly activating, voltage-dependent, potassium-selective current. Point mutations in minK that alter current gating kinetics, ion selectivity, pharmacology, and response to protein kinase C all support the notion that minK is a structural protein for a channel-type transporter. Yet, minK has just 130 amino acids and a single transmembrane domain...
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