Bacterially expressed human calmodulin was labelled with dichloro-triazinyl-fluorescein (5-DTAF, Molecular Probes) as previously described for TA-Cl (Török & Trentham, 1994). Labelled calmodulin derivatives were separated by reverse phase HPLC and analysed by maldi-tof mass spectrometry. The major singly labelled product was Lys₇₅-labelled. Lys₇₅-DTAF-cal (FL-cal) activated phosphodiesterase with K_m 9.5 ± 1.4 nM (S.E.M.) compared with K_m 6.5 ± 0.7 nM (S.E.M.) for unlabelled calmodulin. The fluorescence of FL-calmodulin (exc. 494 nm, em. 525 nm at pH 7.2) was insensitive to Ca²⁺ and target peptide binding within 5 %. Primary cortical and hippocampal cell cultures were made from humanely killed neonatal rat pups (Wistar) and were allowed to mature for 8-10 days. Confocal imaging was carried out using a Leica SP system. Cells were characterised by stimulation of Ca²⁺ transients monitoring 4 µM membrane permeant fluo-4 AM ester loaded for 30 min at 22 °C. Ca²⁺ transients were compared with those evoked in cells into which fluo-4 and/or fluorescent calmodulin was administered by electroporation. Electroporation was carried out applying two opposite polarity 25 ms 105 V cm^-1 pulses at an estimated final fluo-4 concentration of 4 µM (Teruel & Meyer, 1997). Fluo-4 fluorescence intensity at rest was typically 1.5 ± 0.2-fold higher in the nucleus compared with the cytosol in neurones and 1.3 ± 0.2-fold in glia cells. Both intact and electroporated glia cells stimulated by 100 µM ATP, 100 µM 5-HT and 30 µM Glu responded with brief Ca²⁺ transients (t_1/2 rising, 3 s; t_1/2 decay, 3 s) and often produced oscillating patterns of [Ca²⁺].

Intact and electroporated cortical and hippocampal neurones gave prolonged Ca²⁺ transients when stimulated with 50 µM Glu (t_1/2 rising, < 1.5 s; t_1/2 decay, > 200 s). Electroporation thus did not significantly perturb the responsiveness of neurones or glia cells to stimulus. FL-cal was administered into hippocampal glia cells by electroporation (Teruel & Meyer, 1997) using two opposite polarity 25 ms 105 V cm^-1 pulses at an estimated final concentration of 2 µM. Relative fluorescence intensity in the nucleus compared with the cytosol of glia cells was 1.8-fold (see Table 2). The disribution of electroporated fluorescein-dextran (10 kDa) was similar to that of FL-calmodulin; the ratio of the fluorescence in the nucleus compared with that in the cytosol was 2.3 (see Table 1).

Wheat-germ agglutinin (WGA, estimated final concentration 2.5 µg ml^-1) co-electroporated with FL-calmodulin blocked the entry of FL-calmodulin into the nucleus while allowing diffusion of co-electroporated 10 kDa fluo-dextran (Forbes, 1992). FL-calmodulin is thus a faithful mimic of endogenous calmodulin and its presence in the nucleus requires facilitated transport through nuclear pore complexes in hippocampal glia cells.This work is supported by the Medical Research Council, The Wellcome Trust and the Royal Society.

Forbes, D.J. (1992). Annu. Rev. Cell Biol. 8, 495-528.

Teruel, M.N. & Meyer, T. (1997). Biophys. J. 73, 1785-1796.

Török, K. & Trentham, D.R. (1994). Biochemistry 33, 12807-12820.