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Proc Natl Acad Sci U S A
August 15, 1995;
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Molecular cloning and functional characterization of the Xenopus Ca(2+)-binding protein frequenin.
Abstract Frequenin was originally identified in Drosophila melanogaster as a Ca(2+)-binding protein facilitating transmitter release at the neuromuscular junction. We have cloned the Xenopus frequenin (Xfreq) by PCR using degenerate primers combined with low-stringency hybridization. The deduced protein has 70% identity with Drosophila frequenin and about 38-58% identity with other Ca(2+)-binding proteins. The most prominent features are the four EF-hands, Ca(2+)-binding motifs. Xfreq mRNA is abundant in the brain and virtually nondetectable from adult muscle. Western blot analysis indicated that Xfreq is highly concentrated in the adult brain and is absent from nonneural tissues such as heart and kidney. During development, the expression of the protein correlated well with the maturation of neuromuscular synapses. To determine the function of Xfreq at the developing neuromuscular junction, the recombinant protein was introduced into Xenopus embryonic spinal neurons by early blastomere injection. Synapses made by spinal neurons containing exogenous Xfreq exhibited a much higher synaptic efficacy. These results provide direct evidence that frequenin enhances transmitter release at the vertebrate neuromuscular synapse and suggest its potential role in synaptic development and plasticity.
FIG. 1. Comparison of the deduced frequenin amino acid sequence with sequences of closely related Ca2+-binding proteins. Boxed areas indicate
identical amino acids; shaded areas, conserved sequences. Four open bars on top of the sequences are the conserved Ca2+-binding domains
(EF-hands). The * indicates the first glycine residue potentially myristoylated. Xenopus (x.) frequenin has the highest similarity to Drosophila (d.)
frequenin (70% identity). It is also homologous to bovine (b.) neurocalcin (58% identity), rat (r.) hippocalcin (56%), rat visinin-like protein (vilip;
57%), bovine recoverin (46%), human (h.) recoverin (43%), mouse 23-kDa protein (m23kDprot; 43%), and chicken (c.) visinin (38%).
FIG. 2. Expression of Xfreq. (A) Detection of Xfreq mRNA (a
600-bp PCR fragment) by RT-PCR in adult brain but not in adult
muscle. Elongation factor la (a 268-bp PCR fragment) was used as an
internal control. (B) Tissue distribution of Xfreq protein detected by
Western blots. When a horseradish peroxidase method was used, the
rabbit polyclonal antibody (XF-3C, raised against purified recombinant
Xfreq) detected a single band of 24 kDa in adult brain. A very
weak signal was also seen in muscle, presumably reflecting nerve
endings innervating the muscle. (C) Expression of Xfreq protein
during early development. A sensitive chemiluminescence detection
method was used to detect Xfreq in embryos at indicated stages.
Protein from one embryo was loaded in each lane.
FIG. 3. Effect of Xfreq on SSCs. (A) Presence of exogenous Xfreq
in Xenopus embryos after early blastomere injection. The levels of
Xfreq in control and injected embryos at the indicated days after
injection were determined by Western blotting and chemoluminescence.
Protein from one embryo was loaded in each lane. Due to the
cloning strategy, the recombinant frequenin is 10 amino acids longer
at its NH2 terminus than the endogenous protein. (B) Examples of
SSCs recorded from myocytes innervated by a frequenin (-) neuron
(upper trace) and a frequenin (+) neuron (lower trace) in the same
1-day-old cultures. SSCs of various amplitudes were observed as
downward deflections (holding voltage Vh = -70 mV, filtered at 150
Hz). (C) Distribution of SSC amplitudes of frequenin (-) (0) and (+)
(0) synapses, plotted as cumulative frequency (the proportion of total
events with amplitudes smaller than a given amplitude). Curves
represent averaged amplitude distribution from 3 (-) and 4 (+)
synapses with more than 120 SSC events. For clarity, error bars (SE)
are shown only in some points. There is no difference between the two
histograms (Kolmogorov-Smimov test, P > 0.05).
FIG. 4. Effect of Xfreq on ESCs: Typical examples of ESCs
recorded from postsynaptic muscle cells innervated by frequenin (-)
(A) and (+) (B) neurons. ESCs were induced by stimulating neuronal
soma (arrowheads) and appear as regularly spaced large-amplitude
events amidst small randomly occurring SSCs (Left). Oscilloscope
pictures of five superimposed ESCs are shown on Right. Scale bars are
0.5 nA and 40 s (Left) and 2 nA and 10 ms (Right).
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