Differential K+ Channel Clustering Activity of PSD-95 and SAP97, Two Related Membrane-associated Putative Guanylate Kinases
Section snippets
Antibodies
The following K+ channel antibodies have been previously described: Kv1.2 (Sheng et al., 1994b); Kv1.3 (Klumpp et al., 1995); Kv1.4 (Sheng et al., 1992). Anti-PSD-95 antibodies were raised in guinea-pigs against a fusion protein which extends from residues 77 to 299 of PSD-95, and have been described in Kim et al. (1995). Anti-SAP97 mouse monoclonal antibodies used for the double immunofluorescence studies were a gift from Craig Garner, University of Alabama, Birmingham. Shaker antibodies were
Coclustering of Kv1.4 with PSD-95 or SAP97 dependent on Kv1.4 C-terminus
When expressed after transient transfection in COS-7 cells, the Shaker-type K+ channel subunit Kv1.4 distributes partly on the cell surface but accumulates largely intracellularly in a reticular pattern characteristic of the endoplasmic reticulum, whereas PSD-95 expressed alone in COS-7 cells is found diffusely distributed throughout the cell (Kim et al., 1995). Coexpression of Kv1.4 and PSD-95 in the same cell results in the dramatic redistribution of both proteins into irregularly shaped
Different channel clustering behaviors of PSD-95 and SAP97
PSD-95 and SAP97 appear to have indistinguishable binding specifities for K+ channel and NMDA receptor subunits, and are highly similar in primary structure, especially in the PDZ domains that mediate channel subunit binding (>90% identical in amino acid sequence). Not surprisingly, therefore, SAP97 behaves like PSD-95 in being able to specifically cocluster with Shaker-type subunit Kv1.4 in heterologous cells, in a manner dependent on the C-terminal -ETDV motif of Kv1.4. Unlike PSD-95,
Acknowledgements
We are grateful to Craig Garner (University of Alabama, Birmingham) for the gift of anti-SAP97 monoclonal antibodies, Jim Trimmer (SUNY Stony Brook) and Ken Rhodes (Wyeth Ayerst Research) for Kv1.1 antibodies, Gary Yellen (Massachusetts General Hospital) for the Shaker expression construct, Chris Miller (Brandeis University) for the anti-Shaker antibodies, and Kyung-Ok Cho (Baylor College) for the gift of SAP97 cDNA. We than Adam Rothschild for excellent technical assistance, and Elaine
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