The Journal of Neuroscience, March 8, 2006, ():
A Common Ankyrin-G-Based Mechanism Retains KCNQ and NaV Channels at Electrically Active Domains of the Axon
J. Neurosci. Pan et al.
26: 2599
Supplemental data
Files in this Data Supplement:
- supplemental material
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Table S1. This Excel file contains a listing of data related to 21 invertebrate and vertebrate NaV and KCNQ clones, including channel names (as published or deposited), species, accession numbers, gene names (where available), complete deduced amino acid sequences, and references. The complete deduced sequences and literature citation data fields can be accessed by selection (i.e. mouse-click) of the particular cell.
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Figure S1. KCNQ3-C staining of granule cell layer ~5 mm diameter patches that may correspond to mossy fiber glomeruli. Ai, merged image showing distribution of KCNQ3-C immunoreactivity (green, arrows) is distinct from granule cell nuclei (stained blue, DAPI). KCNQ3-C also labels Purkinje cell nuclei (e.g., asterisk), a likely artifact reflecting imperfect specificity of the antibody. PC, Purkinje cell layer; GC, granule cell layer. Aii, NaV stain, showing labeling of AIS in PC and GC. Aiii, KCNQ3-C stain, showing labeling of both AIS and granule cell layer patches. Aiv, DAPI nuclear staining, showing that patches are in spaces not occupied by clusters of granule cell somata, which have scant cytoplasm. B, images as in A, but from ankyrin-G mutant mice. Patchy staining is still apparent, though less intense than in A. In Bii and Biii, labeling of two large granule cell layer AIS by NaV and KCNQ3-C antibodies is also apparent (see text). Scale bar, 20 μm.
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Figure S2. Full-length KCNQ2 and KCNQ3b subunit proteins co-expressed in HEK cells are primarily localized intracellularly. Optical sections, (obtained by deconvolution as described in Methods, and as shown also in Fig. 4A-G, part i), through 2 cells, stained for KCNQ2-N (left) or KCNQ3b-N (right). Unlike HA-NF fusion constructs (Fig. 4), the full-length channel subunits are inefficiently expressed at the membrane, resulting in a punctate and reticular, intracellular staining pattern. Scale bar, 10 μm.
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Figure S3. Fusion proteins possessing the KCNQ2 or KCNQ3 C3 domains are resistant to detergent extraction. A, representative hippocampal neuron, transfected with HA-NF-Q2C after 8 days in vitro, and subjected to detergent extraction prior to fixation (see Materials and Methods), followed by immunostaining for HA (red) and endogenous MAP2 (green), and costaining of nuclei with DAPI (blue), 7 days after transfection. Note that neurons at this point after transfection stained without prior detergent extraction showed a non-specific pattern of HA staining (Fig. 7A). Here, the fusion proteins localized to the AIS show detergent resistance (S3, Aii and iii), whereas the somatodendritic domain, is labeled by MAP2 (S3, Ai) but not for HA (Aii). B, lower magnification image showing 5 cells subjected to electroporation with HA-NF-Q3C at E18, then plated, cultured 22 days in vitro, and then immunostained (with detergent extraction prior to fixation) for Ankyrin-G (green) and HA. Cells 1,2 and 5 lack detectable nuclei (due to detergent extraction). For all 5 cells, AIS are detergent-resistant, as detected by intact ankyrin-G staining (Bi and iii). Cells 2,3 and 5 show immunostaining for HA-NF-Q3C proteins, which is restricted to AIS and co-localized with ankyrin-G immunostain. Scale bar: A, 10 μm, B, 20 μm.
