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Presynaptic localization of sodium/calcium exchangers in neuromuscular preparations.
Luther PW
,
Yip RK
,
Bloch RJ
,
Ambesi A
,
Lindenmayer GE
,
Blaustein MP
.
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Calcium ions play a critical role in neurotransmitter release. The cytosolic Ca2+ concentration ([Ca2+]cyt) at nerve terminals must therefore be carefully controlled. Several different mechanisms, including a plasmalemmal Na/Ca exchanger, are involved in regulating [Ca2+]cyt. We employed immunofluorescence microscopy with polyclonal antiserum raised against dog cardiac sarcolemmal Na/Ca exchanger to determine the distribution of the exchanger in vertebrate neuromuscular preparations. Our data indicate that the Na/Ca exchanger is concentrated at the neuromuscular junctions of the rat diaphragm. The exchanger is also present in the nonjunctional sarcolemma, but at a much lower concentration than in the junctional regions. Denervation markedly lowers the concentration of the exchanger in the junctional regions; this implies that the Na/Ca exchanger is concentrated in the presynaptic nerve terminals. In Xenopus laevis nerve and muscle cell cocultures, high concentrations of the exchanger are observed along the neurites as well as at the nerve terminals. The high concentrations of Na/Ca exchanger at presynaptic nerve terminals in vertebrate neuromuscular preparations suggest that the exchanger may participate in the Ca-dependent regulation of neurotransmitter release. The Na/Ca exchanger is also abundant in developing neurites and growth cones, where it may also be important for Ca2+ homeostasis.
Figure 1. Immunoblot of proteins from rat and Xenopusti ssues incubated
with preimmune serum or anti-Na/Ca exchanger antiserum.
Particulate fractions containing plasma membrane (lanes 1-6, 40 pg
protein/lane; lane7 , 30 pg) from rat brain (lanes2 and 6), spinal cord
(lane3 ), sciatic nerve (lane4 ), and skeletal muscle (lanes1 and .5), and
Xenopusb rain (lane7 ), were probed with preimmune serum (fanes5
and 6) or NaKa exchangear ntiserum(l anesl- 4 and 7).
Figure 2. Immunofluorescent labeling
of NaKa exchanger in cryosectioned
rat diaphragm. A, Cross-se0
tioned myofibers were incubated with
anti-NaKa exchanger antisentm followed
by a fluorescein-conjugated secondary
antibody. 3, The same section
labeled with rhodamine-ol-bungarotoxin
to localize NMJs. Anti-exchanger labeling
is concentrated at the NMJs. C
and D, Similar to A and B, respectively,
but incubated with preimmune rabbit
serum; no labeling is seen in C. E and
F, Similar to A and B, respectively, but
from a diaphragm that had been denervated
for 5 weeks. Labeling in junctional
regions is greatly reduced. G,
Cross section of rat phrenic nerve incubated
with anti-Na/Ca exchanger antiserum,
followed by tluorescein-conjugated
secondary antibody. No labeling
is seen in the axolemma. H, Cross section
of nerve incubated with anti-spectrin
antibodies, followed by fluorescein-
conjugated secondary antibody. The
axonal membranes are labeled (arrows),
demonstrating that antibodies have access
to the cytoplasmic surface of the
axolemma in these preparations. Scale
bars: A, 20 pm for A-& G, 20 pm for
G and H.
Figure 3. Immunofluorescenlat belingo f Na/Cae xchangear t a synapsebe tweenc ulturedX enopucn erve andm usclec ells.A , Cellsl abeledw ith
affinity-purified anti-Na/Cae xchangear ntibodiesfo llowedb y a fluorescein-conjugatseedc ondarayn tibody.B , PostjunctionaAl Ch receptorsin the
samec ellsl abeledw ith rhodamine-conjugatead-b ungarotoxinC. , Differentiali nterferencec ontrastm icrographo f the samec ells.N ote that the
entire lengtho f the axon is labeledb y anti-Na/Cae xchangear ntibodiese, ven in the regionw heret he nerve andm usclec ellsa re not in contact
(arrowsin A and C), whereasc r-bungarotoxilna belsp erijunctiona(l arrowheadins B and C) asw ell asj unctionalA Ch receptora ggregateSs.c ale
bar, 5 pm.
Figure 4. Three pseudocolor images
from the data in Figure 3A-C, respectively,
superimposed to show the distribution
of the Na/Ca exchanger. Fluorescent
labeling for the Na/Ca exchanger
is shown in green, and for the
postjunctional ACh receptors, in red;
the nerve and muscle cells are shown
in blue. For clarity, the greenim agew as
subtracted from the red and blue images
to eliminate overlap of the colors.
Figure 5. Immunofluorescenlta beling
of Na/Ca exchangerin cultured
Xenopus neuronsA. -C, A neuronl abeled
with affinity-purified anti-Na/Ca
exchange(rA ) wasd isplacedfr om the
postjunctionarle gion( arrowi n B) duringt
issuep rocessingC., Differentiali nterferencec
ontrastm icrographo f the
samec ells.T he positiono f the neuron
is indicatedb v the arrowheads in A-C.
D, Neuronal growth cone labeled with
affinity-purified antibody. The label occursi
n patchesth at extendi nto the filopodia.
E , Similar,b ut sparserp, atches
labeledb y the affinity-purifieda ntibody
areo bserveod n the neuronasl omaT. he
labelinga ppearcso nfinedto the cells urface
(arrowheads); the large bright area
neart he nucleusis duet o nonspecific
labeling by the secondary antibody.
Scaleb ars,1 0g m (C for A-C).
