Visualisation of calcium ion channels

2.6.1 Immunocytochemistry

Cells were plated onto either coverslips or glass-bottomed dishes (MatTek Corporation) which were coated with poly-L-lysine prior to transfection,

and cultured in a 5 % CO2 incubator at 37 °C. After 40-72 h expression, cells

were fixed with 4 % paraformaldehyde (PFA) in PBS, pH7.4 at room temperature for 10 min. For labelling the HA epitope on the cell surface in non- permeabilised conditions, the cells were incubated with primary antibody with 2.5% BSA and 10% goat serum in PBS at room temperature for 1 h for N2a

added with 2.5% BSA and 10% goat serum in PBS, and incubated for 1 h at room temperature. In experiments in which the D2R was activated, 100nM quinpirole (Quin, Sigma) was added to N2a cells in Krebs-Ringer solution with

HEPES (KRH) (in mM; 125 NaCl, 5 KCl, 1.1 MgCl2, 1.2 KH2PO4, 2 CaCl2, 6

Glucose, 25 HEPES, 1 NaHCO3) at 37°C for 30 min. 500 ng/ml pertussis toxin

(PTX, Life Technologies) was added to the cells in the culture media overnight. To label intracellular proteins, the cells were permeabilised with 0.2 % Triton X- 100 in PBS for 10 min. The primary and secondary antibodies in 2.5 % BSA and 10 % goat serum were added to the cells as above. Cell nuclei were stained with 0.5 µM 4’,6’-diamidino-2-phenylindole (DAPI) in PBS for 10 min. The coverslips were mounted onto glass slides using VECTASHIELD® mounting medium (Vector Laboratories).

2.6.2 Endocytosis assay

Transfected cells were plated onto glass-bottomed dishes (MatTek

Corporation), coated with poly-L-lysine, and cultured in a 5 % CO2 incubator at

37 °C. After 40 h expression, N2a cells in glass-bottomed dishes were washed

twice with KRH. The cells were incubated with 10 µg/ml α-bungarotoxin Alexa

Fluor® 488 conjugate (BTX-488) (Life Technologies) in 100 μl KRH for 30 min

at 17 °C. The unbound BTX-488 was removed by washing with KRH, and the labelled cells were returned to 37 °C for the kinetic assay. Endocytosis was terminated by fixing the cells with cold 4 % PFA in PBS. For endocytosis experiments in which the D2R was activated, 100 nM Quin was added to the cells in KRH buffer after the BTX-488 labelling stage. The cells were then

permeabilised and intracellular CaV2.2 and nuclei were labelled as described

above. The 13 mm coverslips were mounted onto dishes using VECTASHIELD® mounting medium.

2.6.3 Forward trafficking assay

Transfected cells were plated onto glass-bottomed dishes (MatTek

Corporation), coated with poly-L-lysine, and cultured in a 5 % CO2 incubator at

bungarotoxin (BTX) (Life technologies) for 30 min at 17 °C. The unbound BTX was washed off with KRH, and the cells were then incubated with 10 µg/ml BTX-488 in KRH at 37 °C. To terminate the reaction, cells were washed twice with cold KRH and then fixed with 4 % PFA in PBS at specified times for the kinetic assay. 200 ng/ml (0.713 μM) Brefeldin A (BFA, Sigma) in 0.4 % DMSO was added to the cells in FBS-free N2a cell culture medium for 4 h before and during the experiment in KRH buffer. The cells were then permeabilised and

intracellular CaV2.2 and nuclei were labelled as described above. The 13 mm

coverslips were mounted onto dishes using VECTASHIELD® mounting medium.

2.6.4 Confocal microscopy

All images were acquired using either an LSM 510 or LSM 780 Meta scanning confocal microscope (Zeiss), equipped with a Plan-Apochromat 63x/1.4 or 40x/1.3 DICII oil immersion objective lens, in 8- or 16-bit mode. The laser powers, gains and acquisition settings were kept constant for images that were used subsequently for quantification. The region of interest was determined by identifying cells with expression of a transfection marker or

intracellular staining of the protein of interest (e.g. GFP, CaV2.2 II-III loop

staining), without selecting for the cell surface immunostaining to avoid bias. In addition, on the LSM 780 confocal microscope, the region of interest was selected as described here, and the tile scan of 3 x 3 (9 tiles including the selected tile) was performed to further remove the bias in selecting cells with high expression. For cell surface expression analysis, images were taken with 0.7 μm optical section. For neurite expression analysis, images were taken with 0.9 μm optical sections in Z-stack, and presented as maximum projection. Confocal images were imported and analysed in ImageJ (National Institutes of Health). The membrane fluorescence was quantified using the freehand brush tool with a selection width of 0.66 μm, and tracing the membrane region manually. Neurite fluorescence was quantified using the freehand brush tool with a selection with of 2 μm and manually tracing the entire neurites using

mCherry expression as a marker. Lengths of neurites expressing GFP-CaV2.2

were quantified using the freehand brush tool with a selection with of 2 μm and manually tracing the neurites expressing GFP as a marker. The measured area

in μm2 _{was divided by the width of the selection (2 μm) to derive the length.}

Intracellular and whole-cell fluorescence was quantified using freehand selection, omitting the signal intensity from the nuclei. The background fluorescence intensity in each channel was also taken from the same image, and subtracted from the signal intensity. In the cell surface expression and

trafficking experiments, all cells chosen for analysis contained CaV2.2 II-III loop

immunostaining, confirming the CaV2.2 α subunit expression. The whole-cell

CaV2.2 expression levels were not significantly different in all experiments. For

the endocytosis assay, the normalized membrane fluorescent intensities were fitted to the single exponential decay equation (Equation 1), where x is time, y is intensity, y0 is the initial intensity, A is amplitude, and τ is the time constant.

(Equation 1)

For the forward trafficking assay, the membrane fluorescent intensities were fitted to the single exponential association equation (Equation 2), where x

is time, y is intensity, y0 is the initial intensity, A is amplitude, and τ is the time

constant.

(Equation 2)

All experiments were repeated n=3 to n=5, and approximately 30 to 50 cells (N2a) were analysed for each experiment. All data were presented as pooled in the resulting graphs, except for the trafficking rates and time constants, which are averages of separate experiments.