Inhibition of endocytosis with dynasore (80 μM) increased lateral membrane mobility of Ca²⁺ channels. (A) Trajectory maps illustrating the positions of individual Ca²⁺ channels labeled in the OPL with a primary antibody to the α₂δ₄ subunit and then tagged with a QD-conjugated secondary antibody. Channel positions were mapped every 30 ms for 12 s. Trajectory maps show the typical MSD observed in channels under control conditions during treatment with 20 mM KCl and with 20 mM KCl applied in the presence of dynasore. (B) MSD plotted as a function of measurement interval (30 ms/frame) for QDs in the OPL of retinal slices. The MSD was fit with a saturating curve as described in Materials and methods, attaining a plateau of L²/3 where L² is the membrane area in which channel movements are confined. With the 1.2 NA, 60× objective, QDs immobilized in vacuum grease yielded L = 0.087 ± 0.00133 µm (n = 16). Under control conditions, Ca²⁺ channels labeled with QDs yielded L = 0.227 ± 0.00178 µm (n = 18). A similar value was obtained in the presence of a control version of a myristoylated DIP (Myr-QPPASNPRVR-NH₂, 0.75 mM, L = 0.236 ± 0.00339 µm; n = 12). In the presence of dynasore (80 µM), L increased significantly (P < 0.0001, F test) to L = 0.385 ± 0.00579 µm (n = 11). L also increased significantly relative to the control peptide with application of a myristolated DIP (Myr-QVPSRPNRAP-NH₂, 0.75 mM, Tocris; L = 0.329 ± 0.00333 µm; n = 12; P < 0.0001, F test). (C) Application of 20 mM KCl alone yielded L = 0.28 ± 0.0042 µm (n = 10). Coapplication of KCl with dynasore significantly increased L to 0.50 ± 0.0046 µm (n = 15; P < 0.0001, F test). (D) QDs measured in isolated rods using a 1.45 NA, 60× objective. In Ca²⁺ channels of isolated rods, L averaged 0.20 ± 0.009 µm (n = 15) under control conditions. In the presence of 20 mM KCl, L = 0.199 ± 0.0012 µm (n = 16). Application of dynasore in the presence of 20 mM KCl significantly increased L to 0.266 ± 0.0027 µm (n = 12; P < 0.0001, F test). With immobilized QDs using a 1.45 NA, 60× objective, L = 0.079 ± 0.00129 (n = 10). Error bars represent ±SEM.