In vivo assembly of the axon initial segment in motor neurons

International audienceThe axon initial segment (AIS) is responsible for both the modulation of action potentials and the maintenance of neuronal polarity. Yet, the molecular mechanisms controlling its assembly are incompletely understood. Our study in single electroporated motor neurons in mouse embryos revealed that AnkyrinG (AnkG), the AIS master organizer, is undetectable in bipolar migrating motor neurons, but is already expressed at the beginning of axonogenesis at E9.5 and initially distributed homogeneously along the entire growing axon. Then, from E11.5, a stage when AnkG is already apposed to the membrane, as observed by electron microscopy, the protein progressively becomes restricted to the proximal axon. Analysis on the global motor neurons population indicated that Neurofascin follows an identical spatio-temporal distribution, whereas sodium channels and beta 4-spectrin only appear along AnkG(+) segments at E11.5. Early patch-clamp recordings of individual motor neurons indicated that at E12.5 these nascent AISs are already able to generate spikes. Using knock-out mice, we demonstrated that neither beta 4-spectrin nor Neurofascin control the distal-to-proximal restriction of AnkG

Developmental Expression of Kv Potassium Channels at the Axon Initial Segment of Cultured Hippocampal Neurons

Axonal outgrowth and the formation of the axon initial segment (AIS) are early events in the acquisition of neuronal polarity. The AIS is characterized by a high concentration of voltage-dependent sodium and potassium channels. However, the specific ion channel subunits present and their precise localization in this axonal subdomain vary both during development and among the types of neurons, probably determining their firing characteristics in response to stimulation. Here, we characterize the developmental expression of different subfamilies of voltage-gated potassium channels in the AISs of cultured mouse hippocampal neurons, including subunits Kv1.2, Kv2.2 and Kv7.2. In contrast to the early appearance of voltage-gated sodium channels and the Kv7.2 subunit at the AIS, Kv1.2 and Kv2.2 subunits were tethered at the AIS only after 10 days in vitro. Interestingly, we observed different patterns of Kv1.2 and Kv2.2 subunit expression, with each confined to distinct neuronal populations. The accumulation of Kv1.2 and Kv2.2 subunits at the AIS was dependent on ankyrin G tethering, it was not affected by disruption of the actin cytoskeleton and it was resistant to detergent extraction, as described previously for other AIS proteins. This distribution of potassium channels in the AIS further emphasizes the heterogeneity of this structure in different neuronal populations, as proposed previously, and suggests corresponding differences in action potential regulation

Functional expression of the plant K+ channel KAT1 in insect cells

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Nav1.5 E1053K mutation causing Brugada syndrome blocks binding to ankyrin-G and expression of Nav1.5 on the surface of cardiomyocytes.

We identify a human mutation (E1053K) in the ankyrin-binding motif of Na(v)1.5 that is associated with Brugada syndrome, a fatal cardiac arrhythmia caused by altered function of Na(v)1.5. The E1053K mutation abolishes binding of Na(v)1.5 to ankyrin-G, and also prevents accumulation of Na(v)1.5 at cell surface sites in ventricular cardiomyocytes. Ankyrin-G and Na(v)1.5 are both localized at intercalated disc and T-tubule membranes in cardiomyocytes, and Na(v)1.5 coimmunoprecipitates with 190-kDa ankyrin-G from detergent-soluble lysates from rat heart. These data suggest that Na(v)1.5 associates with ankyrin-G and that ankyrin-G is required for Na(v)1.5 localization at excitable membranes in cardiomyocytes. Together with previous work in neurons, these results in cardiomyocytes suggest that ankyrin-G participates in a common pathway for localization of voltage-gated Na(v) channels at sites of function in multiple excitable cell types

Molecular determinants of the Arabidopsis AKT1 K+ channel ionic selectivity investigated by expression in yeast of randomly mutated channels

International audienceThe Avabidopsis thaliana K+ channel AKT1 was expressed in a yeast strain defective for K+ uptake at low K+ concentrations (<3 mM). Besides restoring K+ transport in this strain, AKT1 expression increased its tolerance to salt (NaCl or LiCl), whatever the external K+ concentration used (50 mu M, 5 mM, or 50 mM), We took advantage of the latter phenomenon for screening a library of channels randomly mutated in the region that shares homologies with the pore forming domain (the so-called P domain) of animal K+ channels (Shaker family). Cassette mutagenesis was performed using a degenerate oligonucleotide that was designed to ensure, theoretically, a single mutation per P cassette. The mean number of amino acid exchanges per cassette turned out to be 1.4, Mutant channels that conferred on the transformed cells a reduction in salt tolerance (increased Na+ content, decreased K+ content, and lower growth rate, as compared to control cells expressing the wild-type channel) were selected. By co-expressing them with the wild-type AKT1 cDNA, it was shown that the mutated polypeptides were expressed, stable and correctly targeted to the cell membrane where they formed channels with altered properties. Analysis of the mutation distribution in these channels suggests that the AKT1 P domain has a structure similar to that of animal Shaker channels (a strongly constrained central region lining the tunnel that includes the highly conserved consensus motif TXXTXGYGD, and flanking regions forming the outer mouth of the pare), with an additional selectivity filter located upstream from the tunnel and formed by residues present in the N-terminal flanking region

Identification and characterization of plant transporters using heterologous expression systems

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Identification and characterization of plant transporters using heterologous expression systems

In recent years major progress has been achieved in the understanding of transport processes in higher plants. The boom in the field of molecular plant physiology led to the identification and characterization of membrane transporters with transport activities for potassium, calcium, sugars, nitrate, ammonium, sulphate, phosphate, amino acids, peptides, and metal ions. Such progress was hardly feasible without heterologous expression of the isolated transport proteins. This review summarizes the different approaches in characterizing plant membrane transporters using heterologous expression systems. By presenting concrete examples, it outlines different cloning strategies, displays the methods used for (i) expression of transport proteins and detection of their function, (ii) biochemical analyses, (iii) explorations of the structure-function relationship through mutational analysis, and concludes with a discussion about the physiological relevance of the analyses in heterologous expression systems

Assessment of Oxygen Saturation in Retinal Vessels of Normal Subjects and Diabetic Patients with and without Retinopathy using Flow Oximetry System

Purpose: To assess oxygen saturation (StO2) in retinal vessels of normal subjects and diabetic patients with and without retinopathy using the modified version of the Flow Oximetry System (FOS) and a novel assessment software. Methods: The FOS and novel assessment software were used to determine StO2 levels in arteries and veins located between 1 and 2 mm from the margin of the optic disc and in the macular area. Results: Eighteen normal subjects, 15 diabetics without diabetic retinopathy (DM no DR), and 11 with non-proliferative diabetic retinopathy (NPDR) were included in final analysis. The mean [± standard deviation (SD)] StO2 in retinal arteries was 96.9%±3.8% in normal subjects; 97.4%±3.7% in DM no DR; and 98.4%±2.0% in NPDR. The mean venous StO2 was 57.5%±6.8% in normal subjects; 57.4%±7.5% in DM no DR; and 51.8%±6.8% in NPDR. The mean arterial and venous StO2 across the three groups were not statistically different (P=0.498 and P=0.071, respectively). The arterio-venous differences between the three study groups, however, were found to be statistically significant (P=0.015). Pairwise comparisons have demonstrated significant differences when comparing the A-V difference in the NPDR group to either normal subjects (P=0.02) or diabetic patients without DR (P=0.04). Conclusions: The arterio-venous difference was greater, and statistically significant, in patients with NPDR when compared to normal subjects and to patients with diabetes and no retinopathy. The mean venous StO2 was lower, but not statistically significant, in NPDR compared with diabetics without retinopathy and with normal subjects