Ml. Michaelis et al., IMMUNOLOGICAL LOCALIZATION AND KINETIC CHARACTERIZATION OF A NA+ CA2+EXCHANGER IN NEURONAL AND NONNEURONAL CELLS/, Brain research, 661(1-2), 1994, pp. 104-116
The plasma membrane Na+/Ca2+ exchanger is believed to play a role in t
he regulation of Ca2+ fluxes in neurons, though the lack of specific i
nhibitors has limited the delineation of its precise contribution. We
recently reported the development of antibodies against a 36-kDa brain
synaptic membrane protein which immunoprecipitated exchanger activity
from solubilized membranes. In the present study we examined the kine
tics of the Na+/Ca2+ exchanger in primary neurons in culture, in a neu
ronal hybrid cell line (NCB-20), and in a fibroblast-like cell line (C
V-1) to see whether the level of exchanger activity correlated with th
e degree of immunostaining produced by our antibodies. The V-max was d
etermined for each cell type and found to be highest in primary neuron
s. Exchanger activity increased in primary neurons between days 1 and
6 in culture, but no such time-dependent change occurred in either of
the cell lines. Immunoblot analysis of the three cell types probed wit
h the anti-36-kDa protein antibodies revealed significantly greater im
munostaining in the primary neurons compared with the other two cell t
ypes. Intensity of staining of neurons also increased significantly be
tween days 1 and 6 in culture. Immunocytochemistry showed significant
labelling of the primary neurons on the neuritic processes and points
of contact between cells. The NCB-20 and CV-1 cells showed considerabl
y lower levels of immunoreactivity. The antibodies immunoextracted sim
ilar to 90% of the exchanger activity in the primary neurons and simil
ar to 70 and 50% of the activity in NCB-20 and CV-1 cells respectively
. Thus the expression of the 36-kDa protein appears to be closely asso
ciated with the Na+/Ca2+ exchanger in neuronal cells and, possibly to
a lesser extent, in non-neuronal cells.