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Figure 1. Noncanonical SH3 domain binding motifs in the intracellular C terminus of vertebrate BK channels. A) Schematic of a single murine BK channel, pore-forming 
-subunit. The noncanonical SH3 domain binding motifs (SBM1 and SBM2) in the proline-rich domain (PRD) between the two putative regulator of potassium conductance (RCK) domains of the C terminus are shown. An additional potential SH3 domain binding motif is also encoded by the murine alternatively spliced exon 22 (e22) at mammalian site of splicing C2. B) Representative overlay assay of a commercial glutathione S-transferase (GST)-SH3 domain fusion protein array (Panomics TranSignalTM SH3 domain array II) probed with a thioredoxin-fusion protein of the murine C terminus spanning amino acids V553 to H753, including (e22) or excluding (zero) the e22 alternatively spliced insert at site of splicing C2 (see Fig. 2A
). GST-SH3 domains are spotted in duplicate at the respective positions denoted by letter and number code. In this array, both e22 and zero fusion proteins interact with SH3 domains from mona/Gads (array position A2), CRKL (A5), and endophilin II (C8) but not with the GST control (D7). Note that under identical conditions, additional SH3 domains interact with the e22 but not zero fusion proteins. Thioredoxin (thio) showed no binding to any fusion protein. Membrane arrays were incubated with 30 µg ml–1 of the respective thioredoxin-fusion protein and interaction was detected by probing for the C-terminal –V5 epitope tag in the thioredoxin fusion protein using 1/1000 dilution of anti-V5 antibody (Ab). Immunoreactivity was detected by enhanced chemiluminescence (ECL).
