FIGURE 3.
Covalently linking two molecules of β-SH3 C113A dimerization-deficient mutant restores the endocytic capability. A, Western blot (WB) of a pulldown assay was done as described in the legend to Fig. 2 but instead used concatameric versions of β-SH3 and β-SH3 C113A. Lysate from cells expressing dynamin were incubated with either β-SH3 concatamer (lane 1) or β-SH3 C113A concatamer (lane 2) or with cobalt beads alone (lane 3). B, CMBI assay results from Xenopus oocytes expressing CaV1.2 alone and in combination with β-SH3 either as a concatamer or non-covalently linked. The bar graph shows N × q values from oocytes expressing the indicated channel-protein combinations; CaV1.2 alone (145. ± 8 pC, n = 9), CaV1.2+β-SH3 concatamer (15 ± 4 pC, n = 6), and CaV1.2+β-SH3 (39 ± 7 pC, n = 8). Average N × q values were significantly smaller in the presence of β-SH3 (p < 5.7 × 10−9) or β-SH3 concatamer (p < 2.4 × 10−8) compared with CaV1.2 alone. Values for N × q between monomer and concatamers were also different but to a smaller degree (p < 0.04). C, same as B, but from Xenopus oocytes expressing CaV1.2 alone and in combination with either a β-SH3 C113A concatamer or β-SH3 C113A. The bar graph shows average N × q values for the indicated channel-protein combinations; CaV1.2 alone (143 ± 18 pC, n = 11), CaV1.2+β-SH3 C113A concatamer (44 ± 5 pC, n = 9), or CaV1.2+β-SH3 C113A (136 ± 13 pC, n = 15). Average N × q values were significantly smaller for CaV1.2+β-SH3 C113A concatamer than for CaV1.2 alone (p < 4.2 × 10−6) or CaV1.2+β-SH3 C113A (p < 2.9 × 10−5) according to a two-tailed t test.