Electrophysiological properties of mouse and epitope-tagged human cardiac sodium channel Na v1.5 expressed in HEK293 cells

Background: The pore-forming subunit of the cardiac sodium channel, Na v1.5, has been previously found to be mutated in genetically determined arrhythmias. Na v1.5 associates with many proteins that regulate its function and cellular localisation. In order to identify more in situ Na v1.5 interacting proteins, genetically-modified mice with a high-affinity epitope in the sequence of Na v1.5 can be generated. Methods: In this short study, we (1) compared the biophysical properties of the sodium current (I Na) generated by the mouse Na v1.5 (mNa v1.5) and human Na v1.5 (hNa v1.5) constructs that were expressed in HEK293 cells, and (2) investigated the possible alterations of the biophysical properties of the human Na v1.5 construct that was modified with specific epitopes. Results: The biophysical properties of mNa v1.5 were similar to the human homolog. Addition of epitopes either up-stream of the N-terminus of hNa v1.5 or in the extracellular loop between the S5 and S6 transmembrane segments of domain 1, significantly decreased the amount of I Na and slightly altered its biophysical properties. Adding green fluorescent protein (GFP) to the N-terminus did not modify any of the measured biophysical properties of hNa v1.5. Conclusions: These findings have to be taken into account when planning to generate genetically-modified mouse models that harbour specific epitopes in the gene encoding mNa v1.5.

Modular organization of the cortical connections linking visual areas V1, V2 and V4 in Macaque monkeys

The macaque cortical visual system is hierarchically organized into two streams, the ventral stream for recognizing objects and the dorsal stream for analyzing spatial relationships. The ventral stream extends from striate cortex or area V1 to inferior temporal cortex (IT) through extra-striate areas V2 and V4. Between V1 and V2, the ventral stream consists of two roughly parallel sub-streams, one extending from the cytochrome oxidase (CO) rich blobs in V1 to the CO rich thin stripes in V2, the other extending from the interblobs in V1 to interstripes, in V2. The blob-dominated sub-stream is thought to analyze the surface features such as color, whereas the interblob-dominated one is thought to analyze the contour features such as shape. ^ In the current study, the organization of cortical pathways linking V2 thin stripe and interstripe compartments with area V4 was investigated using a combination of physiological and anatomical techniques. Different compartments of V2 were first characterized, in vivo, using optical recording of intrinsic cortical signals. These functionally derived maps of V2 stripe compartments were then used to guide iontophoretic injections of multiple, distinguishable, anterograde tracers into specific V2 compartments. The distribution of labeled axons was analyzed either in horizontal sections through the prelunate gyrus, or in tangentially sectioned portions of physically unfolded cortex containing the lunate sulcus, prelunate gyrus and superior temporal sulcus. When a V2 thin stripe and adjacent interstripe were injected with distinguishable tracers, a large primary and several secondary foci were observed in V4. The primary focus from the thin stripe injection was spatially segregated from the primary focus from the V2 interstripe injection, suggesting a retention of the pattern of compartmentation. ^ We examined the distribution of retrogradely labeled cells in V1 following the injections of tracers into V2 different compartments, in order to quantitate just how parallel the two sub-streams are from V1 to V2. Our results suggest that both blobs and interblobs project to thin stripes in V2, whereas only interblobs project to interstripes. This asymmetrical segregation argues against the original proposal of strict parallelism. (Abstract shortened by UMI.) ^

Ubiquitin-specific protease USP2-45 acts as a molecular switch to promote α2δ-1-induced downregulation of Ca v1.2 channels

Availability of voltage-gated calcium channels (Cav) at the plasma membrane is paramount to maintaining the calcium homeostasis of the cell. It is proposed that the ubiquitylation/de-ubiquitylation balance regulates the density of ion channels at the cell surface. Voltage-gated calcium channels Cav1.2 have been found to be ubiquitylated under basal conditions both in vitro and in vivo. In a previous study, we have shown that Cav1.2 channels are ubiquitylated by neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4-1) ubiquitin ligases, but the identity of the counterpart de-ubiquitylating enzyme remained to be elucidated. Regarding sodium and potassium channels, it has been reported that the action of the related isoform Nedd4-2 is counteracted by the ubiquitin-specific protease (USP) 2-45. In this study, we show that USP 2-45 also de-ubiquitylates Cav channels. We co-expressed USPs and Cav1.2 channels together with the accessory subunits β2 and α2δ-1, in tsA-201 and HEK-293 mammalian cell lines. Using whole-cell current recordings and surface biotinylation assays, we show that USP2-45 specifically decreases both the amplitude of Cav currents and the amount of Cav1.2 subunits inserted at the plasma membrane. Importantly, co-expression of the α2δ-1 accessory subunit is necessary to support the effect of USP2-45. We further show that USP2-45 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits. Remarkably, α2δ-1, but not Cav1.2 nor β2, co-precipitated with USP2-45. These results suggest that USP2-45 binding to α2δ-1 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits, in order to regulate the expression of Cav1.2 channels at the plasma membrane.

Global Lithological Map Database v1.0 (gridded to 0.5° spatial resolution)

Lithology describes the geochemical, mineralogical, and physical properties of rocks. It plays a key role in many processes at the Earth surface, especially the fluxes of matter to soils, ecosystems, rivers, and oceans. Understanding these processes at the global scale requires a high resolution description of lithology. A new high resolution global lithological map (GLiM) was assembled from existing regional geological maps translated into lithological information with the help of regional literature. The GLiM represents the rock types of the Earth surface using 1,235,400 polygons. The lithological classification consists of three levels. The first level contains 16 lithological classes comparable to previously applied definitions in global lithological maps. The additional two levels contain 12 and 14 subclasses, respectively, which describe more specific rock attributes. According to the GLiM, the Earth is covered by 64 % sediments (a third of which is carbonates), 13 % metamorphics, 7 % plutonics, and 6 % volcanics, and 10% are covered by water or ice. The high resolution of the GLiM allows observation of regional lithological distributions which often vary from the global average. The GLiM enables regional analysis of Earth surface processes at global scales.