We quantified the diffusional mobility of the Ca²⁺-binding protein calretinin (CR) by two-photon fluorescence recovery after photobleaching (FRAP) in granule cells (GCs) in acute cerebellar slices from 21- to 24-day-old mice. CR was labeled with Alexa Fluor 488 reagent (Invitrogen) yielding a molecular ratio of 1:3 to 1:5. Dye-labeled CR (CR*, 80 mM pipette concentration) or, for control recordings, fluorescein-labeled dextrans (FDs, 10 or 40 kDa, 500 mM, Invitrogen) were dialyzed into GCs in the whole-cell patch-clamp configuration. After 10 min of whole-cell dialysis the dendritic morphology was fully visible with two-photon imaging. FRAP recordings were performed on 10–20 mm long dendritic segments devoid of varicosities and branch points. The analysis of the recovery phases showed that both FDs and CR* underwent anomalous subdiffusion with values for a (Feder et al., Biophys. J. 1996; Brown et al., Biophys. J. 1999) of ~ 0.64 to 0.68 that were not statistically different between CR* and FDs. However, the D(t)-value of CR* was unexpectedly low (3 mm²/s, interquartile range (IQR) 2 – 6 mm²/s) compared to 10 and 40 kDa FDs (12 mm²/s, IQR 8 – 18 mm²/s and 10 mm²/s, IQR 7 – 14 mm²/s, respectively), indicating transient binding of CR* to dendritic targets. CR's mobility was further retarded (2 mm²/s, IQR 1 – 4 mm²/s) by brief trains of action potentials (50–60 APs at ~100 Hz), which were associated with dendritic Ca²⁺ transients of ~42%DF/F peak amplitude, measured using 50 mM Oregon Green BAPTA-1 (Invitrogen). Notably, the mobility of FDs remained unchanged under these conditions. Taken together, our data reveal that anomalous subdiffusion governs the mobility of CR in smooth dendrites of GCs and that CR transiently interacts with one or more yet unidentified binding partner(s) in an activity-dependent manner. This interaction indicates a sensor function for CR.

Funded by the DFG (GraKo 1097 Interneuro, JE and HS).